Colposcopy

Comprehensive Textbook
and Atlas
Ralph J. Lellé
Volkmar Küppers

123
Colposcopy
Ralph J. Lellé • Volkmar Küppers

Colposcopy
Comprehensive Textbook and Atlas

With contributions by Widyorini Hanafy and John

France Rwegoshora
Ralph J. Lellé Volkmar Küppers
Department of Obstetrics & Gynaecology Düsseldorf, Germany
University of Münster
Münster, Germany

Translation from the German language edition: Kolposkopie in der Praxis by Ralph
J. Lellé, and Volkmar Küppers, © Springer-Verlag Berlin Heidelberg 2014. Published
by Springer-Verlag Berlin Heidelberg. All Rights Reserved.

ISBN 978-3-030-85386-0 ISBN 978-3-030-85388-4 (eBook)

[Link]

© Springer Nature Switzerland AG 2023

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We dedicate this book to our patients as well as our fellow
health care providers around the world who work hard to care
for the health of women, sometimes under difficult
circumstances.
May our efforts be another step forward towards the ultimate
goal: eliminating cancer of the cervix!
Preface

Cancer of the cervix uteri is a unique type of malignancy. On one hand, it is

the second most common cancer of women worldwide. On the other hand, it
is amenable to a broad range of preventive measures through secondary
screening. By identifying and removing precancerous lesions, incidence rates
of invasive cervical cancer have declined dramatically within the last 50 years.
Unfortunately, this is only true in so-called developed countries where people
possess the considerable financial and structural means to establish and main-
tain a screening program based on cytopathology and HPV testing.
With the introduction of HPV vaccination in 2006, worldwide elimination
of cervical cancer is on the horizon. However, for years to come, vaccination
efforts need to be complemented by effective secondary screening programs
which are to be adapted to local resources.
This book is intended to provide a comprehensive review of available
strategies for screening, triage, and treatment of cervical cancer and its pre-
cursors as well as other neoplastic and preneoplastic diseases of the lower
genital tract.
As visual diagnosis of many of these diseases is feasible, a comprehensive
collection of color illustrations is included. Furthermore, individual clinical
scenarios are discussed, including the management of patients within a low
resource environment.
May this publication be a contribution to the World Health Organization’s
global strategy for elimination of cervical cancer!

Münster, Germany Ralph J. Lellé

Düsseldorf, Germany Volkmar Küppers
November 2022

vii
Acknowledgements

We thank the numerous people who have contributed to this book.

Special thanks go to

– Ms. Alexandra Woltering for her invaluable assistance with the manu-
script’s creation
– Willi Kramer and Christiane Schliemann, Media Center of Münster
University, Germany, for the illustrations
– Rita Ruland, RN
– the entire team of Dr. Küppers, Düsseldorf, Germany
– Univ.-Prof. Dr. med. Gabriele Köhler, the former Director of the Gerhard
Domagk Institute of Pathology, Münster University, Germany
– Birgit Konert and Magdalena Marciniak, Cytological laboratory of the
Gerhard Domagk Institute for Pathology of Münster University, Germany
– Dr. Supriyatiningsih, [Link].,[Link], Obstetrics and Gynecology
Department, PKU Muhammadiyah Gamping UMY Teaching Hospital,
Faculty of Medicine and Health Sciences, Universitas Muhammadiyah
Yogyakarta, Indonesia
– Heinke Schimanowski-Thomsen, M.D., Matema Lutheran Hospital,
Tukuyu, Tanzania, East Africa

and of course our wives, children, and grandchildren.

ix
Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 The Origin of Colposcopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Acetic Acid Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3 Schiller’s Iodine Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2 Normal Anatomy of the Cervix . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1 General Principles of Cervical Anatomy . . . . . . . . . . . . . . . . . 5
2.2 Colposcopic Appearance of the Normal Cervix . . . . . . . . . . . 9
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3 Abnormal Findings of the Cervix . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.1.1 Leukoplakia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.1.2 Acetowhite Epithelium . . . . . . . . . . . . . . . . . . . . . . . . 16
3.1.3 Punctation and Mosaic . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.1.4 Abnormal Vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.1.5 True Erosion Versus Ulceration . . . . . . . . . . . . . . . . . . 20
3.1.6 Atrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.1.7 Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.2 Intraepithelial Neoplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.2.1 Low-Grade Dysplasia . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2.2 High-Grade Dysplasia . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.3 Carcinoma of the Cervix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.3.1 Epidemiology and Clinical Presentation . . . . . . . . . . . 41
3.3.2 Colposcopic Appearance . . . . . . . . . . . . . . . . . . . . . . . 44
3.4 Special Considerations on Cervical Adenocarcinoma
and Adenocarcinoma In Situ (AIS) . . . . . . . . . . . . . . . . . . . . . 52
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4 International Nomenclature of Colposcopy . . . . . . . . . . . . . . . . . 55
4.1 Cervix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.2 Vagina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.3 Vulva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
5 Indications for Colposcopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
5.1 General Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
5.2 Triage of Abnormal Cytological Findings . . . . . . . . . . . . . . . . 62

xi
xii Contents

5.3 Colposcopy as Triage of a Positive HPV Test Result . . . . . . . 62

5.4 Other Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.5 Contraindications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.6 Summary of Colposcopy Indications of the Cervix . . . . . . . . . 64
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
6 The Significance of Cytology, Biopsy, and HPV Testing . . . . . . . 67
6.1 Cytology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
6.1.1 Cytological Morphology . . . . . . . . . . . . . . . . . . . . . . . 68
6.1.2 Cytological Nomenclature . . . . . . . . . . . . . . . . . . . . . . 71
6.1.3 Cytology as a Screening Tool . . . . . . . . . . . . . . . . . . . 75
6.1.4 Liquid-Based Cytology . . . . . . . . . . . . . . . . . . . . . . . . 77
6.1.5 Adjunctive Cytological Tests . . . . . . . . . . . . . . . . . . . . 84
6.2 Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
6.2.1 Histological Morphology . . . . . . . . . . . . . . . . . . . . . . . 88
6.3 HPV Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
6.3.1 HPV Assays Suitable for Clinical Use . . . . . . . . . . . . . 95
6.3.2 Clinical Application of HPV Testing . . . . . . . . . . . . . . 98
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
7 Colposcopic Examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
7.1 History Taking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
7.2 Colposcopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
7.3 Handling of the Colposcope and Speculum . . . . . . . . . . . . . . . 110
7.4 Cytological Smear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
7.5 Tissue Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
7.5.1 Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
7.5.2 Endocervical Curettage . . . . . . . . . . . . . . . . . . . . . . . . 117
7.6 Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
7.7 Practical Colposcopic Examination Step by Step . . . . . . . . . . 119
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
8 Operative Colposcopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
8.1 Risks for Future Pregnancies . . . . . . . . . . . . . . . . . . . . . . . . . . 124
8.2 Surgical Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
8.2.1 Cold-Knife Conization . . . . . . . . . . . . . . . . . . . . . . . . . 125
8.2.2 Monopolar Loop Resection (LEEP) . . . . . . . . . . . . . . 126
8.2.3 Cryosurgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
8.2.4 CO2 Laser Vaporization . . . . . . . . . . . . . . . . . . . . . . . . 127
8.3 Assessment of Complete Removal of Cervical Dysplasia . . . . 128
8.3.1 Squamous Dysplasia . . . . . . . . . . . . . . . . . . . . . . . . . . 128
8.3.2 Adenocarcinoma In Situ (AIS) . . . . . . . . . . . . . . . . . . 129
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
9 Colposcopy of the Surgically Treated Cervix . . . . . . . . . . . . . . . . 133
9.1 State of Preservation of the Cervix . . . . . . . . . . . . . . . . . . . . . 134
9.2 Visibility of the Transformation Zone . . . . . . . . . . . . . . . . . . . 135
9.3 Stenosis of the Cervical Canal . . . . . . . . . . . . . . . . . . . . . . . . . 137
9.4 Colposcopic Diagnosis of Persistent
or Recurrent Cervical Dysplasia . . . . . . . . . . . . . . . . . . . . . . . 138
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Contents xiii

10 Colposcopy During Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

10.1 Pregnancy-Related Changes of the Cervix . . . . . . . . . . . . . . 144
10.2 Management of Cervical Dysplasia During Pregnancy . . . . . 153
10.3 Conization (LEEP) During Pregnancy . . . . . . . . . . . . . . . . . . 155
10.4 Management of HSIL After Delivery . . . . . . . . . . . . . . . . . . 156
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
11 Colposcopy of Radiation-Induced Changes . . . . . . . . . . . . . . . . . 157
12 Colposcopy of the Vagina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
12.1 Condylomata Acuminata . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
12.2 Adenoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
12.3 Vaginal Intraepithelial Neoplasia (VAIN) . . . . . . . . . . . . . . . 162
12.3.1 Pathogenesis of VAIN . . . . . . . . . . . . . . . . . . . . . . . . 162
12.3.2 VAIN with Existing Cervix with
or without Simultaneous CIN . . . . . . . . . . . . . . . . . . 164
12.3.3 VAIN Following Hysterectomy . . . . . . . . . . . . . . . . 164
12.3.4 VAIN and Immunosuppression . . . . . . . . . . . . . . . . . 165
12.3.5 Colposcopic Appearance of VAIN . . . . . . . . . . . . . . 165
12.3.6 Treatment Options for VAIN. . . . . . . . . . . . . . . . . . . 169
12.4 Vaginal Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
12.5 Vaginal Metastases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
13 Colposcopy of the Vulva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
13.1 Anatomy of the Vulva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
13.2 Altered Self-Perception of the Vulva . . . . . . . . . . . . . . . . . . . 176
13.3 Diagnosis of Vulvar Diseases . . . . . . . . . . . . . . . . . . . . . . . . . 178
13.4 Nomenclature of Vulvar Diseases . . . . . . . . . . . . . . . . . . . . . 179
13.5 Nonneoplastic Epithelial Changes of the Vulva . . . . . . . . . . . 180
13.5.1 Lichen Sclerosus . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
13.5.2 Lichen Ruber Planus . . . . . . . . . . . . . . . . . . . . . . . . . 186
13.6 Neoplastic Epithelial Changes of the Vulva . . . . . . . . . . . . . . 187
13.6.1 Condylomata Acuminata. . . . . . . . . . . . . . . . . . . . . . 187
13.6.2 Vulvar Intraepithelial Neoplasia
(VIN)/HSIL of the Vulva . . . . . . . . . . . . . . . . . . . . . 191
13.6.3 Paget’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
13.6.4 Carcinoma of the Vulva. . . . . . . . . . . . . . . . . . . . . . . 196
13.6.5 Malignant Melanoma . . . . . . . . . . . . . . . . . . . . . . . . 201
13.6.6 Follow-Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
14 The Immunocompromised Patient . . . . . . . . . . . . . . . . . . . . . . . . 205
14.1 The Relationship Between HIV and HPV . . . . . . . . . . . . . . . 205
14.2 Management of the Immunocompromised Patient . . . . . . . . 207
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
15 HPV Vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
15.1 Structure of the HP Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
15.2 Development of Prophylactic Vaccination . . . . . . . . . . . . . . . 210
15.3 Indications for HPV Vaccination . . . . . . . . . . . . . . . . . . . . . . 212
xiv Contents

15.3.1 HPV Vaccination for Children and Adolescents . . . . 212

15.3.2 HPV Vaccination for Men . . . . . . . . . . . . . . . . . . . . . 212
15.3.3 Catch-Up HPV Vaccination . . . . . . . . . . . . . . . . . . . 213
15.3.4 HPV Vaccination Following Treatment
for Cervical Dysplasia. . . . . . . . . . . . . . . . . . . . . . . . 213
15.4 Efficacy of HPV Vaccination . . . . . . . . . . . . . . . . . . . . . . . . . 214
15.4.1 Real-Life Effectiveness of HPV Vaccines. . . . . . . . . 214
15.4.2 HPV Vaccination Efficacy
in Glandular Lesions . . . . . . . . . . . . . . . . . . . . . . . . . 215
15.4.3 Prophylaxis of Condylomata
Acuminata through Gardasil® . . . . . . . . . . . . . . . . . . 216
15.4.4 Statistic Modeling of HPV Vaccination Impact . . . . 216
15.5 Adverse Reactions of HPV Vaccination
and the Risk of Serotype Replacement . . . . . . . . . . . . . . . . . 217
15.6 Impact of HPV Vaccination on Secondary Screening . . . . . . 217
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
16 Psychological Considerations of Screening and Triage . . . . . . . . 221
16.1 Psychological Effects of Screening for Cervical Cancer . . . . 221
16.2 Strategies to Reduce Psychological Distress . . . . . . . . . . . . . 222
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
17 Cervical Cancer Prevention, Diagnosis,
and Management Within a Low-Resource Environment . . . . . . 225
17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
17.2 Worldwide Cervical Cancer Statistics . . . . . . . . . . . . . . . . . . 226
17.3 Cervical Cancer Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . 233
17.3.1 Barriers to Cervical Cancer Prevention . . . . . . . . . . 233
17.3.2 Primary Prevention: HPV Vaccination . . . . . . . . . . . 234
17.3.3 Secondary Screening Strategies . . . . . . . . . . . . . . . . 236
17.3.4 Treatment of Preinvasive Disease . . . . . . . . . . . . . . . 260
17.4 Cervical Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
17.4.1 Visual Cervical Cancer Diagnosis . . . . . . . . . . . . . . 269
17.4.2 Pitfalls of Cervical Cancer Diagnosis . . . . . . . . . . . . 273
17.4.3 Management of Patients
with Invasive Cervical Carcinoma . . . . . . . . . . . . . . 275
17.5 Conclusions and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
17.6 Further Reading and Online Resources . . . . . . . . . . . . . . . . . 281
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
18 Clinical Scenarios for Colposcopy Training . . . . . . . . . . . . . . . . . 287
18.1 Cervical Dysplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
18.1.1 Overtreatment of LSIL/CIN1 . . . . . . . . . . . . . . . . . . 288
18.1.2 Cervix Before and After CO2
Laser Treatment for CIN2 . . . . . . . . . . . . . . . . . . . . . 290
18.1.3 Cervix Before and After LEEP for CIN2 . . . . . . . . . 292
18.1.4 Cervix Before and After LEEP for CIN2 . . . . . . . . . 294
18.1.5 Cervix Before and After LEEP for CIN3 . . . . . . . . . 296
18.1.6 Cervix Before and After LEEP for CIN3 . . . . . . . . . 298
Contents xv

18.1.7 Cervix Before and After LEEP for CIN3 . . . . . . . . . 300

18.1.8 Cervix Before and After LEEP for CIN3 . . . . . . . . . 302
18.1.9 Cervix Before and After LEEP and CO2
Laser Treatment for CIN3 . . . . . . . . . . . . . . . . . . . . . 305
18.1.10 Cervix Before and After LEEP and CO2
Laser Treatment for CIN3 . . . . . . . . . . . . . . . . . . . . . 307
18.1.11 Cervix Before and After LEEP and CO2
Laser Treatment for CIN3 . . . . . . . . . . . . . . . . . . . . . 309
18.1.12 Cervix Before and After LEEP and CO2
Laser Treatment for CIN3 . . . . . . . . . . . . . . . . . . . . . 311
18.1.13 Cervix Before and After LEEP and CO2
Laser Treatment for CIN3 . . . . . . . . . . . . . . . . . . . . . 313
18.1.14 CIN3 Presenting with Prominent Vessels . . . . . . . . . 315
18.1.15 Diagnostic Surgery Revealing CIN3 . . . . . . . . . . . . . 316
18.1.16 CIN3 Look-Alike . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
18.1.17 Intracervical CIN3 . . . . . . . . . . . . . . . . . . . . . . . . . . 318
18.1.18 CIN3 and HIV Infection . . . . . . . . . . . . . . . . . . . . . . 319
18.1.19 CIN3 and HIV Infection . . . . . . . . . . . . . . . . . . . . . . 321
18.1.20 Ruling Out Cervical Dysplasia
Through Colposcopic Triage. . . . . . . . . . . . . . . . . . . 322
18.1.21 Delayed Colposcopic Diagnosis of HSIL . . . . . . . . . 324
18.1.22 Cervix After Three Surgical Interventions . . . . . . . . 326
18.2 Recurrent Cervical Dysplasia . . . . . . . . . . . . . . . . . . . . . . . . 328
18.2.1 HSIL Recurrence After Conization
During Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . 328
18.2.2 Suspected Recurrence After LEEP/CO2
Laser Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
18.2.3 Recurrence 16 Months After LEEP . . . . . . . . . . . . . 333
18.2.4 Recurrence After Positive Endocervical
Curettage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
18.2.5 Large Recurrence 4 Months After Conization . . . . . 336
18.2.6 Recurrence After Two Prior Conizations . . . . . . . . . 338
18.2.7 Atrophy and Intracervical Recurrence . . . . . . . . . . . 339
18.2.8 Cervical Stenosis After Two Conizations . . . . . . . . . 340
18.3 Vaginal Intraepithelial Neoplasia . . . . . . . . . . . . . . . . . . . . . . 341
18.3.1 VAIN After Hysterectomy for HSIL/CIN3 . . . . . . . . 341
18.3.2 VAIN3 and HSIL of the Vulva
in a Liver Transplant Patient . . . . . . . . . . . . . . . . . . . 343
18.3.3 VAIN3 Misdiagnosed as CIN3 . . . . . . . . . . . . . . . . . 345
18.4 Squamous Cell Carcinoma of the Cervix. . . . . . . . . . . . . . . . 347
18.4.1 Squamous Cell Carcinoma of the Cervix . . . . . . . . . 347
18.4.2 Squamous Cell Carcinoma of the Cervix . . . . . . . . . 348
18.4.3 Squamous Cell Carcinoma of the Cervix . . . . . . . . . 349
18.4.4 Hidden Cervical Carcinoma After
Prior Conization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350
18.4.5 Microcarcinoma of the Cervix . . . . . . . . . . . . . . . . . 351
xvi Contents

18.4.6 Long-Term Outcome of Cervical

Carcinoma Treated by Conization . . . . . . . . . . . . . . 353
18.5 Cervical Adenocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
18.5.1 Adenocarcinoma of the Cervix . . . . . . . . . . . . . . . . . 355
18.5.2 Adenosquamous Carcinoma of the Cervix . . . . . . . . 357
18.5.3 Villoglandular Carcinoma of the Cervix . . . . . . . . . . 359
18.6 Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
18.6.1 CIN3 at Tenth Week of Gestation
with Postpartum Diagnosis of Microcarcinoma . . . . 360
18.6.2 CIN3 at 14th Week of Gestation . . . . . . . . . . . . . . . . 362
18.6.3 CIN3 at 17th Week of Gestation
with 40 Months Colposcopic Follow-Up . . . . . . . . . 365
18.6.4 CIN3 at 29th Week of Gestation . . . . . . . . . . . . . . . . 369
18.6.5 Suspected Cervical Cancer
at Eighth Week of Gestation . . . . . . . . . . . . . . . . . . . 371
18.6.6 CIN3 Postpartum . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
18.6.7 Persistent CIN3 in Pregnancy and Postpartum . . . . . 376
18.6.8 Regression of CIN3 After Miscarriage
at 10 Weeks of Pregnancy . . . . . . . . . . . . . . . . . . . . . 379
18.7 Vulva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
18.7.1 Acetowhite Epithelium of the Vulva . . . . . . . . . . . . . 382
18.7.2 Paget’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
18.7.3 HSIL of the Vulva with Microcarcinoma
Mimicking Condyloma Acuminata . . . . . . . . . . . . . . 384
18.7.4 HSIL of the Vulva with Microcarcinoma . . . . . . . . . 385
18.7.5 HSIL of the Vulva Associated
with Vulvar Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . 386
18.7.6 Malignant Melanoma of the Vulva and Vagina . . . . . 387
18.7.7 Vulvar Manifestation of Primary Syphilis . . . . . . . . 388
18.7.8 Condylomata Acuminata of the Vulva
in the 33rd Week of Gestation. . . . . . . . . . . . . . . . . . 389
18.7.9 Lichen Planus with Complete Vulvar Synechia . . . . 390
18.7.10 Circumscribed HSIL of the Vulva . . . . . . . . . . . . . . . 392
18.7.11 Subclitoral HSIL . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
18.7.12 Periurethral HSIL . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
18.7.13 Long-Term Follow-Up of Recurrent
HSIL of the Vulva . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
18.7.14 Vulvar Carcinoma Associated
with Lichen Sclerosus . . . . . . . . . . . . . . . . . . . . . . . . 400
18.7.15 Vulvar Carcinoma Associated
with Lichen Sclerosus . . . . . . . . . . . . . . . . . . . . . . . . 404
18.7.16 Advanced Cancer of the Vulva . . . . . . . . . . . . . . . . . 406
18.7.17 Midline Carcinoma of the Vulva
in a 33-Year-Old Patient . . . . . . . . . . . . . . . . . . . . . . 408
18.7.18 Paget’s Disease of the Vulva . . . . . . . . . . . . . . . . . . . 410
18.8 Miscellaneous Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
18.8.1 Findings After Trachelectomy. . . . . . . . . . . . . . . . . . 412
18.8.2 Radiation-Induced Changes of the Vagina . . . . . . . . 414
18.8.3 Bicornuate and Bicollis Uterus . . . . . . . . . . . . . . . . . 415
Contents xvii

18.8.4 Colposcopic Triage of Irradiation-Induced

Tissue Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
18.8.5 Advanced Ovarian Carcinoma
Misdiagnosed as CIN . . . . . . . . . . . . . . . . . . . . . . . . 418
18.9 Scenarios Within a Low-Resource Environment . . . . . . . . . . 420
18.9.1 Recurrent Squamous Cell Carcinoma
of the Cervix 19 Months After Limited
Radical Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
18.9.2 Advanced-Stage Carcinoma of the Vulva
in a 29-Year-Old HIV-Positive Patient . . . . . . . . . . . 422
18.9.3 Advanced but Operable Adenocarcinoma
of the Cervix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
18.9.4 FIGO Stage IB1 Squamous Cell
Carcinoma of the Cervix . . . . . . . . . . . . . . . . . . . . . . 426
18.9.5 Fifteen Months of Treatment Delay
in a Patient with Cancer of the Cervix . . . . . . . . . . . 429
18.9.6 Hysterectomy for Suspected HSIL of the Cervix . . . 431
18.9.7 HSIL of the Cervix and VIN
in a 24-Year-Old HIV-Positive Patient . . . . . . . . . . . 433
18.9.8 HSIL of the Cervix Not Eligible
for Cryosurgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435
18.9.9 FIGO Stage IIIB Squamous
Cell Carcinoma of the Cervix . . . . . . . . . . . . . . . . . . 437
18.9.10 Operable FIGO Stage IIB Cancer of the Cervix . . . . 439
18.9.11 Large Cervical Condyloma
in an HIV-Positive Patient . . . . . . . . . . . . . . . . . . . . . 441
18.9.12 Choriocarcinoma Mimicking
Carcinoma of the Cervix . . . . . . . . . . . . . . . . . . . . . . 442
18.9.13 Radical Hysterectomy
and Pelvic Lymph Node Dissection
for FIGO Stage IB2 Carcinoma of the Cervix . . . . . 446
18.9.14 Large VIA-Positive Lesion Treated
by Loop Resection . . . . . . . . . . . . . . . . . . . . . . . . . . 448
18.9.15 Recurrent CIN3 and Condylomata
Acuminata of the Vulva
in a 51-Year-Old HIV-Positive Patient . . . . . . . . . . . 450
18.9.16 FIGO Stage IB1 Carcinoma
of the Cervix Treated by Radical
Hysterectomy and Bilateral Pelvic
Lymph Node Dissection . . . . . . . . . . . . . . . . . . . . . . 453
18.9.17 Myoma in Statu Nascendi 1 . . . . . . . . . . . . . . . . . . . 455
18.9.18 Myoma in Statu Nascendi 2 . . . . . . . . . . . . . . . . . . . 457
18.9.19 VIA+/VILI+ Patient Treated by LEEP . . . . . . . . . . . 458
18.9.20 Advanced Cervical Cancer
with Urinary Fistula Formation . . . . . . . . . . . . . . . . 460
18.9.21 Cervical Carcinoma Without Ulceration
or Exophytic Growth Pattern . . . . . . . . . . . . . . . . . . 461
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
Abbreviations

ACIS Adenocarcinoma in situ

ACOG American College of Obstetrics and Gynecology
ACS American Cancer Society
AGC Atypical glandular cells
AGCPC Arbeitsgemeinschaft für Zervixpathologie und Kolposkopie (=
German Federation of Cervical Pathology and Colposcopy)
AIN Anal intraepithelial neoplasia
AIS Adenocarcinoma in situ
ASCCP American Society of Colposcopy and Cervical Pathology
ASC-H Atypical squamous cells favor high grade
ASC-US Atypical squamous cells of undetermined significance
CI Confidence interval
CIN1 Cervical intraepithelial neoplasia grade 1
CIN2 Cervical intraepithelial neoplasia grade 2
CIN3 Cervical intraepithelial neoplasia grade 3
CIS Carcinoma in situ
CVLP Chimeric virus-like particle
DIMDI Deutsches Institut für Medizinische Dokumentation und
Information
ECC endocervical curettage
EFC European Federation of Colposcopy
FDA Food and Drug Administration
FIGO Federation Internationale de Gynécologie et Obstétrique
GTN Gestational trophoplastic neoplasia
HC2 Hybrid Capture 2 test
HGCGIN High-grade cervical glandular intraepithelial neoplasia
HLA Human leukocyte antigens
HPV Human papillomavirus
HSIL High-grade squamous intraepithelial lesion
IACR International Agency for Research on Cancer
ICSI Intracytoplasmic sperm injection
IFCPC International Federation of Cervical Pathology and Colposcopy
ISSVD International Society for the Study of Vulvovaginal Diseases
JHPIEGO Johns Hopkins Program for International Education in
Gynecology and Obstetrics
LBC Liquid-based cytology
LEEP Loop electrosurgical excision procedure

xix
xx Abbreviations

LLETZ Large loop excision of the transformation zone

LSIL Low-grade squamous intraepithelial lesion
MN III Münchner Nomenklatur III
N/C ratio Nuclear to cytoplasmic ratio
NILM Negative for intraepithelial lesion or malignancy
PCR Polymerase chain reaction
SGO Society of Gynecologic Oncology
SIL Squamous intraepithelial lesion
STD Sexually transmitted disease
STIKO Ständige Impfkommission
TCA Trichloroacetic acid
VAIN Vaginal intraepithelial neoplasia
VAIN 1 Vaginal intraepithelial neoplasia grade 1
VAIN 2 Vaginal intraepithelial neoplasia grade 2
VAIN 3 Vaginal intraepithelial neoplasia grade 3
VIA Visual inspection (of the cervix) with acetic acid
VIAM VIA with magnification
VILI Visual inspection with Lugol’s iodine
VIN Vulvar intraepithelial neoplasia
VLP Virus-like particle
 Introduction
1

Contents
1.1 The Origin of Colposcopy 1
1.2 Acetic Acid Test 3
1.3 Schiller’s Iodine Test 4
References 4

Colposcopy as a method of optically mag- Dr. Hans Hinselmann (1884–1959), then a senior
nifying the inspection of the external and physician at the Department of Gynecology of
internal genitalia was first described by Bonn University, Germany (Hinselmann 1925).
Hans Hinselmann from Bonn University, In his introduction, Hinselmann wrote1:
Germany, in 1925. While cytopathology Based on the needs for early diagnosis and the
and, more recently, HPV testing have etiology of cervical carcinoma, I was striving to
become the methods of choice for many improve the inspection of the cervix. (…) For
screening programs, colposcopy is the uni- this purpose, I have equipped the Leitz binocu-
lar magnifying device for dissection with a light
versally recognized procedure and gold source. Thus magnifications of 3.5 and more can
standard for triage of abnormal Pap smears be achieved with a long distance to the object
and/or positive HPV test results. and intense illumination of vagina and cervix.
[…] It allows us to study all diseases of the vulva,
the vestibulum, the vagina and the cervix in a
way which previously has not been possible.

Hinselmann called this device “colposcope”

1.1 The Origin of Colposcopy (Fig. 1.1). With the colposcope, it became possi-
ble for the first time to study the early stages of
In 1925, the journal “Münchner Medizinische cervical cancer and the characteristics of intraep-
Wochenschrift” (Munich Medical Weekly) ithelial changes. The colposcope also offered an
published the article “Verbesserung der opportunity to diagnose and treat preinvasive cer-
Inspektionsmöglichkeiten von Vulva, Vagina vical disease.
und Portio” (“Improving the possibilities of the
inspection of vulva, vagina, and cervix”) by Prof. 1
Authors’ translation.

© Springer Nature Switzerland AG 2023 1

R. J. Lellé, V. Küppers, Colposcopy, [Link]
2 1 Introduction

In a 2007 article for the American journal

“The Forward” titled “The deadly origins of a
life-saving procedure,”“2 the author Ruth Jolanda
Weinberger, a scientific researcher at the “Ludwig
Boltzmann Institute for Historical Social
Science” based in Vienna, Austria, states: “…
inasmuch as it is possible to reconstruct events
more than six decades after the fact, there
appears to be enough circumstantial evidence to
charge Hinselmann with at least passive knowl-
edge of the horrifically cruel medical experi-
ments conducted in Auschwitz.”
The connection between Hinselmann and
Auschwitz is based on the fact that one of his
residents at Hamburg Altona, Helmut Wirths, had
been a brother to Eduard Wirths, also a gynecolo-
gist by training and then the commanding physi-
cian at the Auschwitz concentration camp. Most
likely, a colposcope had been provided to
Auschwitz by Hinselmann to study the cervix of
female prisoners that were photographed and
subsequently removed completely. Those sam-
ples were sent to Hamburg to be studied by
Hinselmann and Helmut Wirths.
Weinberger states, “Following the fall of the
Third Reich, Hinselmann was found guilty by the
Allies of having conducted forced sterilizations of
Gypsy women at his gynecological clinic in
Hamburg. But what about his knowledge of the
medical experiments performed on Jewish women
in Auschwitz’s Block 10, perhaps the greatest vio-
lation of the Hippocratic Oath in modern his-
Fig. 1.1 The first colposcope as described by Hinselmann tory? Should judgment not also be passed on his
in 1925 (Hinselmann 1925) at least passive complicity in this most heinous of
crimes?”
Hinselmann could not foresee that “colpos- Thanks to the research conducted by Bruno
copy” as conceived by him would become an Halioua from France (Halioua 2010) and based
internationally recognized and practiced method on testimony from Adélaide Hautval (1906–
as well as a synonym for any kind of cervical 1988), a French physician who had been impris-
diagnosis. oned in Auschwitz (Halioua and Hauptmann
Hans Hinselmann left the University of Bonn 2015) and became a witness to crimes committed
in 1933 and went to Hamburg, Germany, where on women under the pretext of medical research,
he became head of the Altona Women’s Hospital.
Only recently, Hinselmann’s massive ethical mis-
demeanors during Nazi rule have been 2
[Link]
documented. life-saving-procedure/.
1.2 Acetic Acid Test 3

the dubious nature of Hinselmann’s actions are tion. Therefore, the realization that application of
now well known to the medical community. a 3–5% solution of acetic acid applied to the cer-
Subsequently, the historical circumstances vix will cause “acetowhite” staining together
have also been documented in Germany (Ebert with a whole range of other diagnostic clues has
and David 2014), and further studies are under revolutionized the visual diagnosis of dysplasia.
way (Hübner 2016). In the meantime, the As the acetic acid reaction of squamous epi-
“Deutsche Gesellschaft für Gynäkologie und thelium with HSIL changes will lead to a
Geburtshilfe” (German Society of Gynecology clearly visible “acetowhite” reaction, identifi-
and Obstetrics) has officially distanced itself cation of some of these characteristic changes
from its honorary member Hans Hinselmann. is feasible even without the colposcope. This is
The discussions and the competition between the basis for VIA (visual inspection (of the cer-
supporters of colposcopy as a primary screening vix) with acetic acid), thus providing an effec-
tool and supporters of cytopathology as estab- tive screening tool that can be used within a
lished by Papanicolaou had lasted for decades low-resource environment.
and are settled now. Cytopathology and colpos- It is unclear by whom and when acetic acid
copy do not compete but rather complement each application as an adjunct to colposcopy was con-
other. ceived. Initially, Hans Hinselmann, who intro-
Cytopathological examination is a simple duced colposcopy and the colposcope in 1925,
and fast procedure and has become the screen- did not use acetic acid. Instead, he and his
ing method of choice in most Western coun- German colleagues focused on “leukoplakia”
tries. In fact, it can be regarded as the gold (Hinselmann 1927b; Hinselmann 1927a; von
standard of cervical screening at which all other Franqué 1927), which is caused by hyperkeratini-
methods need to be measured such as HPV zation and is sometimes, but not always, associ-
screening, either alone or in combination with ated with cervical dysplasia or cancer (Fig. 1.2).
cytopathology. Subsequently, Hinselmann described the phe-
On the other hand, colposcopic examination is nomenon of mosaic formation as a colposcopic
the crucial method for further triage of abnormal sign of precancerous lesions, again without the
cytopathological findings. Thus, colposcopy is use of acetic acid (Hinselmann 1928a).
the most important requisite to decide whether to
observe or to treat cervical precancer. If the deci-
sion to treat has been made, the colposcope
guides the surgeon in order to ensure small vol-
ume resections, especially in younger women.

1.2 Acetic Acid Test

Inspection of the cervix with or without magnifi-

cation may identify cervical cancer. However,
identification of very early stage disease or the
differential diagnosis of precancerous lesions is
not possible, as the normal cervix is covered by Fig. 1.2 Colposcopic drawing of the cervix from a
24-year-old prostitute with a precancerous lesion. Large
mucus. Furthermore, epithelial changes due to
areas of leukoplakia are seen, which Hinselmann in 1927
high-grade squamous intraepithelial lesions are interpreted as the most significant colposcopic sign of a
not readily visible under colposcopic magnifica- precancerous condition (Hinselmann 1927b)
4 1 Introduction

1.3 Schiller’s Iodine Test Halioua B (2010) The participation of Hans Hinsel-
mann in medical experiments at Auschwitz. J Low
Genit Tract Dis 14(1):1–4. [Link]
Walter Schiller (1987–1960) from Vienna, LGT.0b013e3181af30ef
Austria, who later became Director of Halioua B, Hauptmann G (2015) Adélaïde Hautval (1906–
Laboratories in New York and Chicago, used 1988): une personnalité médicale exemplaire. Presse
Med 44(12):1290–1296. [Link]
iodine solution to perform an in vivo stain of the lpm.2015.05.012
cervical epithelium (Schiller 1928). He called Hinselmann H (1925) Verbesserung der Inspektions-
this test, which would later carry his name, möglichkeiten von Vulva, Vagina und Portio. Mmw
“Jodpinselung” or “brushing with iodine” 72:1733
Hinselmann H (1927a) Über die Methodik der Diag-
(Schiller 1929). Right from the beginning, he rec- nose der Portioleukoplakien. Zentralbl Gynaecol
ognized that abnormal epithelial cells do not 51(50):3162–3163
store a sufficient amount of glycogen and do not Hinselmann H (1927b) Zur Kenntnis der präcancerösen
react with the iodine. On the other hand, a posi- Veränderungen der Portio. Zentralbl Gynaecol
51:901–903
tive stain can be used to rule out cancer or pre- Hinselmann H (1928a) Schichtungskugeln in dem Epithel
cancer. In 1929, Schiller used his test as a guide der weißlichen Felder der Umwandlungszone der Por-
from where on the cervix to obtain cervical biop- tio. Zentralbl Gynaecol 52(20):1244–1247
sies or rather scrapings from the surface epithe- Hinselmann H (1928b) Zur Frage der Frühdiagnose des
Portiokarzioms anläßlich der Ausführungen von Ker-
lium (“Abschabung des Portioepithel”). Initially, mauner im Halban-Seitz Bd. IV, 1927. Zentralbl Gyn-
Schiller did not make use of Hinselmann’s colpo- aecol 52(3):168–169
scope. However, as early as 1928, Hinselmann Hübner J (2016) Kolposkopie ohne Menschlichkeit?!
suggested to combine all of these methods (Hans Hinselmann und die Versuche an Frauen in Auschwitz.
Geburtshilfe Frauenheilkd 76(03):A11. [Link]
Hinselmann 1928b). org/10.1055/s-0036-1571408
Today, Schiller’s test is an integral part of Schiller W (1928) Zur klinischen Frühdiagnose des Por-
colposcopic inspection and may even be used tiokarzinoms. Zentralbl Gynaecol 52(30):1886–1892
without colposcopic magnification as a screen- Schiller W (1929) Jodpinselung und Abschabung des Por-
tioepithels. Zentralbl Gynaecol 53(17):1056–1064
ing tool within a low-resource environment. von Franqué O (1927) Leukoplakie und präcanceröse
Veränderung des Plattenepithels. Zentralbl Gynaecol
51(15):898–899
References
Ebert A, David M (2014) Historische Behandlungs-
methode. Die Erfindung der Kolposkopie. Geburt-
shilfe Frauenheilkd 74(07):631–633. [Link]
org/10.1055/s-0034-1368424
 Normal Anatomy of the Cervix
2

Contents
2.1 General Principles of Cervical Anatomy 5
2.2 Colposcopic Appearance of the Normal Cervix 9
References 12

Understanding the transformations zone,

where squamous and glandular epithe-
lium meet, is the basis for understanding
colposcopic diagnosis. Squamous cell
carcinoma and its predecessors develop
within the transformation zone on the
basis of human papillomavirus infection.
Transformation and metaplasia cause a
variety of physiologic phenomena, which
need to be recognized by the colposcopists
and distinguished from pathologic findings.

Fig. 2.1 Uterus bicollis

2.1 General Principles
of Cervical Anatomy
types of epithelium meet and will later form the
The cervix develops through fusion of the distal so-called original squamocolumnar junction. At
part of the Müllerian ducts. If this fusion is the time the embryo is fully developed and the
incomplete, various malformations of the genital child is born, this junction is located within the
tract may result (Fig. 2.1). The Müllerian ducts cervical canal.
are lined by glandular epithelium. The vagina is Through colposcopy, only part of the cervix
derived from the urogenital plate and is covered can be visualized. This part or portion (“portio”)
by squamous epithelium. Eventually, these two of the cervix extends into the vagina and is called

© Springer Nature Switzerland AG 2023 5

R. J. Lellé, V. Küppers, Colposcopy, [Link]
6 2 Normal Anatomy of the Cervix

the “portio vaginalis uteri” (Figs. 2.2 and 2.3). as well as the overall anatomy of the cervix are
The portio vaginalis comprises only about quite variable.
30–50% of the entire length of the cervix, which The glandular epithelium of the cervix forms
is approximately 3 cm long. The length and size invaginations and crypts up to a depth of about
7 mm. This increases the surface of the secreting
epithelium. In a strict sense, the term “endocervi-
cal gland,” which is frequently used for histo-
pathological descriptions, does not apply, since
the so-called glands are merely a part of these
invaginations.
The location of the squamocolumnar junction
is variable. If this junction between glandular and
squamous epithelium is located in the periphery
of the outer cervix, and the process of transfor-
mation or metaplasia (see below) is still incom-
plete, the thin monolayer of columnar cells has a
bloodred appearance due to blood vessels within
the underlying stroma. This phenomenon is
called “ectopy” (Figs. 2.4, 2.5, and 2.6). The term
Fig. 2.2 Schematic drawing of vaginal and cervical epi- “erosion” is misleading and should be considered
thelium. Vagina and cervix are both lined by multilayer
squamous epithelium (yellow), which becomes a single- obsolete.
layered columnar epithelium within the endocervix (red)

Fig. 2.3 Colposcopic image of a cervix covered by squa- to the opening of the cervical canal, a tiny area of imma-
mous epithelium (left: acetic acid test, right: Schiller’s ture metaplasia can be seen, which is Lugol
test). The squamocolumnar junction is not visible. Close (iodine)-negative
2.1 General Principles of Cervical Anatomy 7

Squamous metaplasia is the decisive physi- epithelium. This mechanism occurs in several
ological mechanism and a basic prerequisite sites of the human body, for example, salivary
for the development of cervical intraepithelial glands, bronchi, stomach, or anus. In the case of
neoplasia or CIN. cervical metaplasia, transformation of mature
Metaplasia means that one type of cells/epi- glandular epithelium into mature squamous cell
thelium is transformed into another type of cells/ epithelium can be observed.
Metaplastic transformation is triggered when
the columnar epithelium is situated in the ecto-
cervix, i.e., outside the cervical canal. Obviously,
external influences such as mechanical irritation,
inflammation, and bacteria, a low pH, or estro-
genic stimulation may be triggers for metaplastic
transformation.
The process of metaplasia starts at the level of
so-called reserve cells. Reserve cells form a
single-layer epithelium located between the glan-
dular epithelium and the basement membrane.
These undifferentiated cells play a crucial role as
they are the primary target of HPV infection.
After transformation is completed, the new
metaplastic squamous epithelium is neither
Fig. 2.4 Schematic drawing of ectopy. The glandular colposcopically nor cytologically different
epithelium has not yet been modified by metaplasia and is from the original squamous epithelium.
visible on the ectocervix (on the right) However, on histopathological tissue sections,

Fig. 2.5 Ectopy before and after the application of acetic acid. The squamocolumnar junction is located on the ecto-
cervix. The glandular epithelium, which has not yet been modified by metaplasia, is bright red. At 6 o’clock, there is a
small area of immature metaplasia
8 2 Normal Anatomy of the Cervix

a high degree of maturity. Only the occasional

gland opening and/or Nabothian cyst will indi-
cate the metaplastic nature of the cells.
Identification of the newly formed squa-
mous epithelial border is critical for colpos-
copy diagnosis (Fig. 2.7). It is recognizable by

Fig. 2.6 Ectopy without metaplasia that encompasses the

entire ectocervix and exhibits a villous morphology

there will be residual glandular cells under-

neath the new squamous cell layer.
Colposcopically, small round openings of the
squamous surface are seen, which can secrete
cervical mucus. If the metaplastic squamous epi-
thelium does not leave gland openings, cysts will
form through continuous excretions of the glan-
dular epithelium, eventually leading to cysts of
variable sizes. These cysts are called “ovula
Nabothi” or Nabothian cysts, referring to Martin
Naboth (1675–1721), a professor at Leipzig
University in Germany (Baskett 2001). Naboth
called these cysts “ovula,” i.e., eggs, as he imag-
ined that they represent a repository for ova.
The inner border of the transformation zone is
the original gland epithelium. This is considered
the “new” squamocolumnar junction. The outer
border is the transition between the newly formed
metaplastic squamous epithelium and the origi-
nal squamous epithelium (the “old” squamoco-
lumnar junction). Whereas the inner border can
be seen clearly unless it is located within the cer-
vical canal, the outer border of the transformation
Fig. 2.7 Three different positions of the newly formed
zone may be less well defined, especially when squamocolumnar junction (T1, T2, T3) after metaplastic
the metaplastic squamous epithelium has reached transformation
2.2 Colposcopic Appearance of the Normal Cervix 9

the sharp contrast between the more or less matured squamous epithelium consists of several
mature metaplastic epithelium and the deep cell layers so that the light from the blood vessels
red original glandular epithelium. If this tran- located within the stroma is only partially reflected.
sition is fully visualized during the colposcopy Therefore, normal squamous epithelium will
examination—if necessary by spreading the appear light red or pink. The original glandular
cervical os—the entire transformation zone epithelium, on the other hand, will be deep red,
can be viewed, and thus the area critical for because the stromal capillaries shine through the
the development of squamous epithelial neo- single-layered cylindrical epithelium. The meta-
plasia can be assessed colposcopically. plastic tissue of the transformation zone will
For every colposcopic examination, this is an reflect the white light from the colposcope,
important aspect, which needs to be documented. depending on its state of maturity.
Previous colposcopic nomenclatures have called Nabothian cysts are a characteristic phenom-
a colposcopic exam “satisfactory” when the enon of the normal transformation zone. There
squamocolumnar junction is fully visible (Walker may be only a discrete elevation of the squamous
et al. 2003). epithelium with a yellowish hue or there may be
multiple cysts, sometimes with a diameter of sev-
eral centimeters, so that they can be seen well on
2.2 Colposcopic Appearance vaginal ultrasound examination (Figs. 2.9, 2.10,
of the Normal Cervix and 2.11).
Frequently, large-caliber vessels are seen on
The colposcopic appearance of the normal trans- the surface of larger Nabothian cysts. Here, the
formation zone is highly variable. Therefore, the stromal capillaries are pushed toward the epi-
basic prerequisite for colposcopic recognition of
various dysplastic findings is to familiarize one-
self with the different manifestations of normal
metaplastic transformation (Fig. 2.8).
In part, the different colors of the cervix can be
explained by the different amount of light reflected
by the cervical epithelium. The completely

Fig. 2.9 Nabothian cysts. The cysts are of varying size

with a yellowish color covered by prominent but regular
blood vessels. This particular patient was sent to the col-
Fig. 2.8 Two characteristic phenomena of metaplastic poscopy clinic after multiple cysts had been documented
transformation: gland openings and Nabothian cysts on ultrasound exam
10 2 Normal Anatomy of the Cervix

thelial surface by the accumulated secretion.

The differential diagnosis of atypical vessels is
not difficult, since there are no fluctuations in
the caliber of the vessels; rather, the vessel
diameter tapers toward the periphery.
When 3–5% acetic acid solution is applied to
the normal cervix, it has little effect on squamous
cell or glandular epithelium. However, metaplas-
tic tissue sometimes produces a white (“aceto-
white”) staining. This is due to the fact that the
nuclei within the metaplastic tissue are enlarged
in relation to the cytoplasm. Acetic acid leads to
dehydration of the cytoplasm. Thus, the meta-
plastic nuclei are more densely packed resulting
in increased light reflection. After a few seconds
or a few minutes, this effect decreases, as the cell
cytoplasm is slowly rehydrated (Figs. 2.12 and
2.13).
Fig. 2.10 Nabothian cysts. The 50-year-old patient was Through colposcopy, the positive acetic
referred for further diagnosis of a “large exophytic tumor” acid reaction of immature metaplasia—when
the nuclear/cytoplasmic (N/C) ratio is high—
cannot be reliably distinguished from low-
grade squamous intraepithelial dysplasia.
In addition, stromal capillaries may become
more prominent during the process of metaplasia.
On colposcopic examination, after the applica-
tion of acetic acid, a fine mosaic pattern and/or
punctation are seen. These vascular phenomena
of metaplasia may complicate the colposcopic
diagnosis of intraepithelial neoplasia.
As the specificity of colposcopy for so-called
minor changes is low, one should not hesitate to
frequently perform colposcopically directed cer-
vical biopsy. Occasionally, even high-grade dys-
plasia can be found whereas the colposcopic
aspect suggests only metaplasia or low-grade dis-
ease. In some cases, the cytopathological diagno-
sis, which frequently is the reason for colposcopic
triage, can be helpful.
If the Pap smear suggests HSIL/CIN3 and
only minor lesions are seen, biopsy should be
Fig. 2.11 Large Nabothian cyst from a 34-year-old performed.
patient
2.2 Colposcopic Appearance of the Normal Cervix 11

Fig. 2.12 Metaplasia before and after the application of acetic acid

Fig. 2.13 Dynamics of the acetic acid reaction. The large image on the left shows the cervix before application of
acetic acid. Thirty seconds later, metaplasia with acetowhite squamous epithelium is visible at the ventral aspect of the
cervix. After 60 seconds, the epithelium still remains acetowhite in the periphery
12 2 Normal Anatomy of the Cervix

References Walker P, Dexeus S, De Palo G, Barrasso R, Campion

M, Girardi F et al (2003) International terminology of
colposcopy: an updated report from the International
Baskett TF (2001) On the shoulders of giants: eponyms
Federation for Cervical Pathology and Colposcopy.
and names in obstetrics and gynaecology. RCOG
Obstet Gynecol 101:175–177
Press, London
 Abnormal Findings of the Cervix
3

Contents
3.1 Introduction 13
3.2 Intraepithelial Neoplasia 23
3.3 Carcinoma of the Cervix 41
3.4 Special Considerations on Cervical Adenocarcinoma
and Adenocarcinoma In Situ (AIS) 52
References 53

After applying 4–5% acetic acid solution, thelial neoplasia” or “CIN” is just as commonly
vascular phenomena of the cervix can be used as the term “dysplasia.” Mild, moderate, and
visualized, which, for example, are described severe dysplasia are called CIN1, CIN2, and
as punctation or mosaic. When learning col- CIN3, respectively.
poscopy, the criteria for low- and high-grade The following criteria are helpful in order to
changes and, most importantly, for invasive distinguish between normal and abnormal col-
disease need to be practiced. This chapter poscopic findings:
describes in detail the relationship between
– The color of the epithelium before and after
precancerous and invasive processes with
application of acetic acid (“acetowhite”)
human papillomavirus (HPV).
– The type of delineation of acetowhite areas:
indistinct versus sharp borders
– The presence and appearance of capillary
vessels
3.1 Introduction
– The tissue reaction to Schiller’s test (Lugol’s
iodine solution)
Essentially, the main scope of colposcopy is the
recognition of precancerous changes. These Before describing and illustrating the typical
changes are also referred to as dysplasia and are colposcopic phenomena, an important aid for
classified into three grades: mild, moderate, and colposcopic evaluation, i.e., Schiller’s iodine test,
severe dysplasia or “carcinoma in situ.” Moderate will be addressed in more detail.
and severe changes are often summarized as Schiller used an iodine solution originally
high-grade lesions. The term “cervical intraepi- developed by Jean Guillaume Auguste Lugol

© Springer Nature Switzerland AG 2023 13

R. J. Lellé, V. Küppers, Colposcopy, [Link]
14 3 Abnormal Findings of the Cervix

(1788–1851) for the treatment of skin Schiller’s test is carried out after acetic acid
tuberculosis, in order to identify the glycogen- application and is the final step of the colposcopic
containing epithelium of cervix and vagina. examination procedure. Whenever possible, an
Therefore, Schiller’s iodine is also called Lugol’s image of Schiller’s iodine test has been added to
iodine solution (Table 3.1). the illustrations below.
The presence of iodine-negative epithelium is
Table 3.1 Preparation of Lugol’s solution for Schiller’s not always to be regarded as pathological. Normal
test (Sankaranarayanan and Wesley 2003)
glandular epithelium, an immature transforma-
Ingredients Quantity tion zone, or atrophic/inflammatory squamous
1. Potassium iodide 10 g epithelium do not contain glycogen and thus are
2. Distilled water 100 mL
iodine-negative.
3. Iodine crystals 5g
If an area with a usually dysplasia-related phe-
Preparation
(a) Dissolve 10 g potassium iodide in 100 ml of
nomenon such as punctation or mosaic turns out
distilled water. to be iodine-positive, dysplasia can be reliably
(b) Slowly add 5 g iodine crystals, while shaking. ruled out. Thus, the patient can be spared unnec-
(c) Filter and store in a tightly stoppered brown bottle. essary biopsies (Fig. 3.1).

Fig. 3.1 Iodine-positive “pseudo-mosaic” in a 19-year-old patient with no further evidence of cervical dysplasia. A
slightly raised gyrated pattern is seen on the ventral cervix that may be misinterpreted as dysplasia-associated mosaic.
However, the entire lesion is Lugol-positive, thus ruling out dysplasia
3.1 Introduction 15

3.1.1 Leukoplakia chronic infection (Fig. 3.3). Leukoplakia is par-

ticularly pronounced when cervical condyloma is
Leukoplakia presents as a white area that is vis- present (Fig. 3.4).
ible before acetic acid is applied. Leukoplakia Although the underlying causes of leukopla-
may be located both inside and outside the kia are usually benign, both cervical intraepithe-
transformation zone. Histologically, thickened lial neoplasia as well as keratinizing squamous
keratinized squamous epithelium is seen. cell carcinoma need to be ruled out.
Keratinized tissue areas will reflect the light In many cases of leukoplakia, tissue biopsy is
completely and therefore appear white on col- necessary in order to distinguish between benign
poscopic inspection (Fig. 3.2). and neoplastic squamous cell epithelium.
Leukoplakia may be caused by local irritation
of the epithelium, for example, by trauma or

Fig. 3.2 Leukoplakia. A thick keratinizing layer covers Fig. 3.3 Leukoplakia without dysplasia. The Pap smear
the squamous epithelium. This layer is opaque and is normal, and the HPV test (Hybrid Capture 2) is
appears white when viewed from above negative

Fig. 3.4 Typical cervical condyloma before and after application of acetic acid
16 3 Abnormal Findings of the Cervix

3.1.2 Acetowhite Epithelium

The acetic acid test is the most important part

of the colposcopic examination. Assessment of
the acetic acid reaction without the colposcope
is more meaningful than colposcopic examina-
tion without the application of acetic acid, as
demonstrated by VIA-based screening
programs.
A 4–5% acetic acid solution is applied to the
cervix. As a result, the cervical mucus coagulates
and can be removed mechanically. The acetic acid
solution has no visible effect on normal mature
Fig. 3.5 Acetic acid effect on normal epithelium (left) in
squamous epithelium or on glandular tissue. contrast to dysplasia (right). Dysplastic epithelium has a
However, when the protein content of the epithe- higher protein content. Acid leads to dehydration of the
lium is high, as it is the case in squamous dysplasia, cytoplasm. Subsequently, crowding of the abnormal
acetic acid application leads to a more or less pro- nuclei makes the tissue opaque or white (“acetowhite”)
when viewed from above
nounced white color change. High-grade squa-
mous intraepithelial lesions, and particularly CIN3,
are characterized by large nuclei surrounded by a lesions (HSIL) than in CIN1 (LSIL). Thus, CIN1
small amount of cytoplasm. Thus, protein content tissue will lead to a delicate acetic acid reaction,
within the epithelium layer is high, as there is an which remains flat within the normal squamous
increase of the nuclear/cytoplasmic ratio. epithelium that surrounds the abnormal tissue,
The hyperosmolar acetic acid causes the whereas CIN2 and CIN3 lead to pronounced and
cells to shrink by dehydration of the cytoplasm coarse acetowhite discolorization with a slightly
and coagulates the nuclear proteins. The cell elevated tissue surface.
nuclei move closer together and render the Thus, acetowhite epithelium is not necessarily
epithelium opaque. As a result, it loses its pink dysplastic, especially when there is still only a
color and appears to be white, as it reflects the delicate hue of acetowhite. Metaplastically devel-
incoming light beams (Fig. 3.5). oped, not yet mature squamous epithelium also
The nuclear/cytoplasmic ratio is high in pre- consists of cells with a high nuclear/cytoplasmic
cancerous lesions, especially in high-grade squa- ratio and thus have a high protein content. It is
mous intraepithelial lesions (CIN2 and CIN3). not possible to distinguish immature metaplasia
However, immature metaplastic cells will also from mild dysplasia by acetic acid reaction alone.
have a higher nuclear/cytoplasmic ratio and thus, In addition, dysplastic epithelium and immature
to a certain extent, will also react positive to the metaplastic epithelium may share other features
acetic acid test. such as a sharp delineation.
If, however, the cells contain abundant glyco- In summary, the following findings may be
gen, the acetic acid solution can only penetrate associated with enlarged nuclei and/or a high
into the superficial cell layers. The epithelium nuclear/cytoplasmic ratio and thus lead to a posi-
will retain its normal color. Normal squamous tive acetic acid reaction:
cell epithelium will therefore not change its color.
The most pronounced acetowhite reaction will be – Immature metaplasia
triggered in the presence of severe dysplasia, as – Squamous intraepithelial neoplasia (CIN2 and
the dysplastic tissue is characterized by large CIN3 more pronounced than CIN1)
nuclei with abundant protein. – Invasive carcinoma
Histologically and cytologically, the nuclear/ – Cell trauma and repair after infection or
cytoplasmic ratio is higher in CIN2 and CIN3 mechanical stress (pessary)
3.1 Introduction 17

3.1.3 Punctation and Mosaic

Punctation and mosaic are vascular phenom-

ena, which can be visualized by the applica-
tion of acetic acid.
Occasionally, punctation and mosaic may be
visible without the acetic acid, when the green fil-
ter is used, as the vessels can be seen more clearly.
The effect of punctation is caused by vessels
(Fig. 3.6), which are located in the dermal papil-
lae reaching close to the epithelial surface.
Punctation may be described as “fine” versus
“coarse” and “regular” as opposed to “irregular” Fig. 3.7 Fine punctation on the anterior (ventral) cervix
(Figs. 3.7 and 3.8).
Fine regular punctation is a frequent sign
of mild dysplasia as well as immature meta-
plasia, as these two diagnostic entities cannot
be distinguished colposcopically. On the other
hand, coarse and irregular punctation is seen
in the presence of high-grade precancerous
lesions.
Just like punctation, mosaic—originally
described by Hinselmann as “Felderung”—is
also a vascular phenomenon. Proliferating capil-
lary structures surround these polygonal or round
areas of dysplastic epithelial cells, resulting in a Fig. 3.8 Coarse and irregular punctation
mosaic-like appearance (Fig. 3.9).

Fig. 3.9 Mosaic-like punctation is a vascular phenome-

Fig. 3.6 Punctation is caused by single-looped terminal non, i.e., capillaries in stromal papillae perpendicular to
capillaries within stromal papillae of squamous epithe- the epithelial surface forming a honeycomb or
lium. As these vessels are situated perpendicular to the cobblestone-like pattern when viewed through the
epithelial surface, and the atypical epithelium is attenu- colposcope
ated after the application of acetic acid, red dots are seen
colposcopically. These dots may be regular or irregular,
fine or coarse
18 3 Abnormal Findings of the Cervix

A delicate or fine mosaic (Fig. 3.10) is more

likely associated with a low-grade dysplastic
lesion (or immature dysplasia), while a coarse
mosaic (Fig. 3.11) most likely corresponds to
higher-grade dysplasia.

Fig. 3.12 Coarse and irregular mosaic in a patient with

high-grade dysplasia. Every colposcope is equipped with
a green filter. The filter absorbs red light so that blood ves-
sels appear black and can be seen more clearly against the
green background

Since the red color of the blood vessels,

which causes the appearance of mosaic and
punctation, is more pronounced under green
light, every colposcope is equipped with a green
filter (Fig. 3.12).

3.1.4 Abnormal Vessels

The mature transformation zone can have numer-

ous prominent blood vessels (Fig. 3.13), espe-
cially when a Nabothian cyst bulges the squamous
Fig. 3.10 Fine and coarse mosaic on the anterior cervix epithelium. However, these do not meet the crite-
before and after the application of acetic acid ria for abnormal vessels as they gradually taper
toward the terminal end.
On the other hand, an abundance of vessels
within an acetowhite background, especially in
connection with ulcerative epithelial defects,
must be considered abnormal.
In the presence of cervical carcinoma, there
may be an irregular pattern of fragile vessels
with massive caliber fluctuations and bizarre
branching.
After introduction of the speculum, or upon
contact with a cotton swab, abnormal vessels will
bleed easily (Figs. 3.14 and 3.15).
Although abnormal vessels are typical for
invasive lesions, they may occasionally occur in
Fig. 3.11 Coarse and irregular mosaic on the posterior benign changes such as granulation tissue, radia-
cervix tion changes, inflammation, or exophytic condy-
lomata acuminata.
3.1 Introduction 19

Fig. 3.13 Normal vessels within the transformation

zone. The vessels are of regular caliber that gradually con-
stricts toward the periphery

Fig. 3.14 Abnormal vessels in a 29-year-old patient with

carcinoma of the cervix

Fig. 3.15 Abnormal vessels in a 30-year-old patient with squamous cell carcinoma of the cervix FIGO stage IB
20 3 Abnormal Findings of the Cervix

3.1.5 True Erosion Versus Ulceration 3.1.6 Atrophy

True erosion (Figs. 3.16 and 3.17) is caused by If there is prominent atrophy of the squamous
trauma, for example, when a tampon or speculum epithelium, characteristic petechial bleeding
is inserted into the vagina. If high-grade dyspla- occurs when the speculum is inserted, since the
sia is present, the abnormal epithelium is particu- atrophic epithelium is quite vulnerable
larly friable, since the number of desmosomes (Figs. 3.18, 3.19, 3.20, 3.21, and 3.22).
responsible for cell cohesion is reduced (“peeling
edges”).
True erosion must be distinguished from
ulceration. Ulceration leads to distinct epithe-
lial defects and is always suspicious of invasive
carcinoma.

Fig. 3.18 Atrophy. The epithelium is flat and vulnerable

Fig. 3.16 Erosion means sloughing of the squamous epi-

thelium due to a local trauma. Sometimes, the expression
“true erosion” is used to distinguish erosion from ulcer-
ation or ectopia

Fig. 3.17 True erosion. The epithelium on the posterior Fig. 3.19 Severe atrophy of the squamous epithelium
cervix is partially sloughed after inserting the speculum with petechial hemorrhage
3.1 Introduction 21

Due to atrophic epithelial changes, both cyto-

logical as well as colposcopic assessment can be
severely impaired so that satisfactory evaluation
is impossible. Usually, atrophy is due to estrogen
deficiency and should be treated by local estro-
gen application for 2–3 weeks. Then the col-
poscopic examination is repeated.

Fig. 3.20 Atrophy of the cervix in a 69-year-old patient

Fig. 3.21 A 59-year-old patient with atrophy. After acetic acid application, blood vessels are visible through the
thinned-out epithelium. As no glycogen is present, the ectocervix reacts Lugol-negative

Fig. 3.22 A 70-year-old patient with atrophic cervix and petechial bleeding. There is almost no difference after appli-
cation of acetic acid (center). The entire cervix is Lugol-negative
22 3 Abnormal Findings of the Cervix

3.1.7 Inflammation During inflammation, the capillary pattern is

more pronounced (Fig. 3.23). Colposcopically, a
Acute inflammatory changes of the vaginal and characteristic red discoloration may be seen,
cervical epithelium are another factor that may which differs from neoplastic punctation
severely impair colposcopic evaluation. Topical (Fig. 3.24). The glycogen storage capacity of the
anti-inflammatory treatment and/or estrogen will squamous epithelium is also impaired, resulting
be helpful to improve both cytological as well as in a partially negative Schiller’s test.
colposcopic assessment.

Fig. 3.23 Inflammation. The capillary pattern is more

pronounced, giving a typical spotty red color on col-
poscopic exam

Fig. 3.24 A 34-year-old patient with recurrent fungal infections. The cervix has a patchy red appearance with nonspe-
cific acetowhite staining (center) due to inflammation. At 9 o’clock, there is punctation that is fine and regular, and at
4–5 o’clock, discrete erosion. Only part of the tissue is able to store glycogen and therefore reacts Lugol-positive
3.2 Intraepithelial Neoplasia 23

3.2 Intraepithelial Neoplasia

The presence of human papillomaviruses

(HPV) is a necessary prerequisite for the for-
mation of intraepithelial neoplasia of the cer-
vix as well as various other anogenital lesions.
In 2008, Harald zur Hausen from the German
Cancer Research Center in Heidelberg received
the Nobel Prize for Medicine for the discovery of
the relationship between human papillomavirus
infection and cervical carcinoma (zur Hausen
2009). Also, cervical cancer precursors (cervical
intraepithelial neoplasia) are also triggered
through HPV infection. HP virus DNA alone is
sufficient for the development of dysplasia,
without the presence of particles capable of
replication. Fig. 3.25 Schematic representation of the HPV 16
It has been accepted as common knowledge genome. Presumably, the gene products of DNA regions
that in the vast majority of cases, human papillo- E6 and E7 interfere with apoptosis control mechanisms
mavirus infection is transmitted through sexual
intercourse. However, there is a certain percent- immunosuppression, for example, after organ
age of children and adolescents who may be HPV transplantation or in the case of HIV infection,
positive prior to sexual activity (Chap. 15). preneoplasia will develop more frequently.
Only certain HPV types, which are called Consequently, the cancer risk is increased.
“high-risk viruses,” are involved in the develop- Furthermore, a connection between smoking and
ment of dysplasia and ultimately cervical carci- dysplasia has been known for a long time.
noma, particularly HPV types 16, 18, 31, and Benzopyrenes in the cigarette smoke can be iden-
45. Presumably, the gene products of DNA seg- tified in the cervical mucus. These contaminants
ments E6 and E7 interfere with apoptotic con- are able to raise the titers of HPV high risk types
trol mechanisms (Fig. 3.25). E6 blocks the 16, 18, and 31 (Alam et al. 2007).
tumor suppressor protein p53, E7 the retinoblas- Although many aspects of cervical cancer
toma protein (pRB). Under normal circum- development through HPV infection are still
stances, damaged cells regularly occur, which unclear, several conclusions can be drawn from
are destroyed by apoptosis. Because of the pres- epidemiological studies: In sexually active women,
ence of viral E6 and E7, those damaged cells are HPV high-risk DNA is frequently detected within
no longer eliminated so that mutations will the cervical region. According to Ho et al. (1998),
accumulate, which may lead to malignant 43% of college students at one point were HPV
transformation. positive, when tested at 6 months intervals.
The disruption of cell replication caused by Therefore, women with a positive HPV high-
human papillomaviruses of the high risk type risk test result cannot be regarded as “ill” solely
occurs within the transformation zone, as this based on the HPV test. HPV positivity, especially
area is characterized by increased prolifera- in young women and/or after HPV-related dis-
tive activity, that makes it particularly suscep- ease has been ruled out, is a frequent source of
tible to HPV. misunderstandings between patients and clini-
Obviously, other cofactors are required for cians that needs to be clarified.
preneoplasia or neoplasia development from HPV high-risk positivity is not a disease by
HPV high-risk infection. The immune system itself but, depending on the individual circum-
certainly plays an important role. In drug-induced stances, may indicate a necessity of further
24 3 Abnormal Findings of the Cervix

observation or cytopathologic/colposcopic highly inappropriate. However, in 1993, Östör

triage. (1993) published an extensive literature review on
Only a very small percentage of patients with the natural history of precancer that encompasses
positive HPV high-risk test result will ever all pertinent articles published since 1950. Östör
develop cervical dysplasia. Furthermore, it takes concluded that in the vast majority of cases, CIN1
at least 3–4 years until preneoplasia of the cervix will either regress or persist and that only 10% of
may develop following HPV infection. Based on CIN1 lesions eventually progress to CIN3 with
the increasing knowledge about the human papil- only a 1% cancer risk. Even in CIN3 disease, one-
lomavirus and its influence on cervical disease, third of cases may regress. However, both in CIN2
highly effective prevention programs have been and CIN3, cancer risk is significantly higher (5%
established with well-defined screening intervals and >12%, respectively).
either in combination with Pap testing (Petry In the case of low-grade dysplasia, a wait-and-
et al. 2003) or as primary HPV screening see strategy, i.e., expectant management and
(Koliopoulos et al. 2007). observation, is acceptable. Based on a longitudi-
In the following section, the two most impor- nal study on 342 women, Duggan et al. (1998)
tant clinical manifestations of HPV-related dis- estimated a likelihood of progression (CIN1 to
ease are discussed: mild or low-grade dysplasia CIN2/3) of 18.6%. Another 18.6% of patients
(cervical intraepithelial neoplasia grade 1, CIN1) had persistent disease, and about two-thirds of
and high-grade dysplasia (cervical intraepithelial patients regressed (62.7%).
neoplasia grades 2 and 3, CIN2, and CIN3). The As shown by a statistical analysis of data
distinction of CIN1 versus CIN2 and CIN3 is of obtained from the British National Health
particular importance. Service, 80% of all high-grade lesions (CIN2 and
Low-grade cervical intraepithelial neopla- CIN3) will not progress to cancer (Raffle et al.
sia (CIN1) is an entity that is morphologically, 2003). A meta-analysis by Cantor et al. (2005)
clinically, as well as etiologically distinct from came to the conclusion that the average annual
higher-grade precancer (CIN2 and CIN3). transition probability for CIN1 to CIN2 or CIN3
Clinical management is also quite different. is only 7.20%. On the other hand, regression of
Thus, any cyto- or histopathology report has CIN1 is much more likely: 14.80% per year.
to make a clear distinction between low- and A British study shows what happens to women
high-grade disease. In Germany, where tradition- who are suspected to have high-grade intracervical
ally a five-class Pap system is used, a basic prob- disease on cytopathological screening: Those who
lem has only been fixed recently. Previously, the had to wait for more than 6 months until col-
“Pap IIID” designation had been used for the poscopic triage was performed were compared to
cytopathological diagnosis of both mild and patients who were examined in a more timely man-
moderate dysplasia. With the introduction of the ner. Surprisingly, the extended waiting period did
Munich III nomenclature (see below), this has not lead to an increase in the development of can-
eventually been corrected (“Pap IIID1” versus cer. Rather, significantly fewer operations were
“Pap IIID2”). necessary in this group (Fakokunde and Selo-
The basis for the clinical management of Ojeme 2008).
intraepithelial disease is its respective proba- Moderate and severe dysplasia (CIN2 and
bility for progression and regression. CIN3) are best summarized under the term of
In principle, all three grades of dysplasia (CIN1 high-grade dysplasia. In the American Bethesda
to CIN3) have the potential to regress spontane- classification, the term “high-grade squamous
ously. Understandably, data on the natural history intraepithelial lesion” or HSIL is used for both
of dysplasia are sparse as observation of untreated cytological and histopathological nomenclature
cervical intraepithelial neoplasia, possibly until as is the term “low-grade squamous intraepithe-
the development of invasive disease, would be lial lesion” (LSIL).
3.2 Intraepithelial Neoplasia 25

Probability of cancer development in the presence of high-risk HPV:

– Data on the natural history of cervical intraepithelial neoplasia are sparse despite the fact
that these data are the basis of clinical decision-making.
– Low-grade dysplasia (CIN1) is much more likely to regress than to progress to CIN2/3 or
cancer.
– Even high-grade dysplasia (CIN3) has only a limited potential of malignant progression
and may regress spontaneously.
Thus, most cases of higher-grade dysplasia (CIN2/3) are treated surgically (either ablation or
destruction), whereas for low-grade disease (CIN1), clinical follow-up exams are preferred,
as cell changes are more likely to regress spontaneously.

3.2.1 Low-Grade Dysplasia reported noncoital behavior that might have con-
tributed to HPV infection.
[Link] Pathogenesis The HP virus is thought to penetrate the cervi-
Low-grade squamous intraepithelial lesions cal epithelium during sexual intercourse. The
(LSIL) or cervical intraepithelial neoplasia immature metaplastic epithelium of the transfor-
grade 1 (CIN1) are the morphological expres- mation zone is more susceptible to HPV infec-
sion of HPV infection. The American ALTS tion than the original multilayered squamous
study (“ASC-US/LSIL Triage Study”) has epithelium. The virus is eventually integrated
shown that in 58.9% of cases, multiple HPV into the DNA of the basal cells.
types are present, with 86.1% being of the HPV However, HPV infection alone does not lead
high-risk variety (ALTS 2000). However, until to the morphologically detectable changes that
to date, it has not been shown conclusively that define intraepithelial neoplasia. Most HPV infec-
the identification of low- and high-risk virus tions do not result in cervical dysplasia. To date,
DNA allows identification of mild dysplasia it is not entirely clear which cofactors are required
with a high progression probability. to trigger the activation of viral DNA. Definitely,
It is not possible to reliably predict which the immune system plays an important role,
cases of low-grade dysplasia will regress spon- which is shown by the frequent occurrence of
taneously and when the diagnosis of CIN1 cervical dysplasia with an increased risk for pro-
must be considered as a first step to the devel- gression in immunosuppressed patients.
opment of high-grade dysplasia or even carci- Immunosuppression may be due to medication
noma. Thus, the group of low-grade dysplasia following organ transplantation or by HIV infec-
consists of two biologically different entities, tion. The negative influence of toxins in cigarette
which cannot be reliably distinguished by smoke has also been documented (Alam et al.
today’s laboratory tests. However, it is obvious 2007).
that the risk for progression to invasive cancer If the HP virus is activated under the influence
is very low (<1%). of these cofactors, replication of the viruses
Most likely, sexual intercourse is the most fre- occurs in the intermediate and superficial cells of
quent mode of HPV infection. However, Doerfler the squamous epithelium. The so-called koilocy-
et al. (2009) found that HPV high-risk DNA tosis constitutes the morphological expression of
could be detected in approximately 14% of girls HPV infection. The name “koilocyte” is derived
who not yet had had sexual intercourse after sex- from Greek “koilos,” which means “hollow” or
ual abuse had been ruled out. Shew et al. (2013) “hollowed out.” The term “koilocyte” or “koilo-
identified HPV DNA in 10 out of 22 adolescents cytotic atypia” was coined in 1956 by Koss and
(14–17 years old), who denied previous vaginal Durfee (1956), who described this cell type as
sex. However, 7 of the 10 HPV-positive girls “large cells with relatively small but irregular and
26 3 Abnormal Findings of the Cervix

significant risk factor for the subsequent develop-

ment of CIN3 (Castle et al. 2011).

[Link] Colposcopic Appearance

The most important prerequisite for colposcopic
assessment is the acetic test. Application of a
4–5% acetic acid solution to the cervix leads to a
whitish stain of the dysplastic epithelium
(Fig. 3.27). The acetic acid effect is only tempo-
rary. The white color will fade gradually as cells
reabsorb fluid from their environment, i.e., they
Fig. 3.26 Koilocyte surrounded by normal intermediate
squamous cells. Both nuclei are enlarged, slightly irregu-
are rehydrated. In the case of low-grade dysplasia,
lar, and surrounded by bright cytoplasm the intensity of the acetowhite reaction can be
quite variable. Usually, there is only a faint acetic
reaction. The white epithelium remains slightly
hyperchromatic nuclei surrounded by clear and
transparent. The acetowhite tissue stays level with
transparent cytoplasm” (Fig. 3.26). As early as
the surrounding squamous epithelium. Compared
1960, Ayre expressed the opinion that koilocytes
to high-grade lesions, it usually takes longer for
are caused by viral infections. According to
the staining to manifest itself. Furthermore, the
Naylor’s historical research (Naylor 2000), it was
acetic acid effect disappears faster in low-grade
Meisels and Fortin who identified human papil-
than in higher-grade dysplasia.
lomaviruses as the cause of koilocytic atypia.
The capillary loops in the epidermis are visi-
In koilocytosis, there is an irregularly shaped
ble through the epithelium. The capillaries are
nucleus with a distinct perinuclear transparency
compressed by the acetic acid effect and will
of the cytoplasm (“halo”) and an increased
extend toward the epithelial surface. This leads to
nuclear/cytoplasmic ratio. The cells often have
the appearance of fine punctation and/or mosaic.
more than one nucleus. Sometimes, the cyto-
In contrast to high-grade dysplasia, punctation or
plasm shows signs of disintegration with a patchy
mosaic form a regular pattern (Fig. 3.28).
eosinophilic pattern (“measle cells”).
Mild dysplasia usually has well-defined bor-
Low-grade dysplastic changes typically occur
ders toward the surrounding normal squamous
in younger women, and the rate of spontaneous
epithelium (Fig. 3.29). However, in contrast to
regression is high. According to a study on a col-
high-grade dysplasia, borders may also be less
lege population (Ho et al. 1998), 70% of new
well delineated.
HPV infections spontaneously regress within
In all dysplastic changes including mild dys-
8 months. As already mentioned, the progression
plasia, Schiller’s iodine test will be negative.
probability (low-grade to higher-grade dysplasia)
However, iodine negativity may be less pro-
is only 18.6%. A similar percentage of lesions
nounced in low-grade dysplasia. In the case of
persisted, and 62.7% regressed spontaneously
condylomatous changes, irregular iodine staining
(Duggan et al. 1998).
may be observed, sometimes called “tiger skin
Only in the case of persistent infection is
pattern” (Fig. 3.30).
there a risk for the development of clinically
Hellberg and Nilsson (1990) described the fol-
relevant intraepithelial neoplasia. However,
lowing colposcopic features in 165 women with
HPV persistence, i.e., continuing infection
histologically proven CIN1:
with the same HPV high-risk type, cannot be
conclusively diagnosed in individual patients. – Acetowhite staining 52.1%
According to the present management con- – Punctation 22.4%
cept, mild dysplasia (CIN1) is usually not to be – Mosaic 18.2%
treated immediately, as CIN1 lesions are not a – Atypical vessels 12.4%
3.2 Intraepithelial Neoplasia 27

Fig. 3.27 CIN1 in a 20-year-old patient. At 11 and 12 o’clock, there is a thin acetowhite lesion, which is not raised in
relation to the surrounding tissue. The abnormal tissue is Lugol-negative

As mentioned earlier, a reliable diagnosis of

mild dysplasia based on colposcopy alone is not
feasible. According to a literature review by
Hopman et al. (1998), only 42.8% of colposcopi-
cally diagnosed CIN1 lesions could be confirmed
by histopathologic evaluation. When the same
investigator was asked to reevaluate the col-
poscopic images after an interval of 3 months,
there was only a 51% intraobserver
concordance.
Flat condylomata do not have the characteris-
tic and easily recognizable appearance of condy-
lomata acuminata and may be confused with
mild dysplasia. The raised margin and the intense
acetowhite staining with sharp borders may even
suggest high-grade dysplasia. As shown in
Fig. 3.31, flat condylomata are frequently located
in the periphery of the cervix rather than near the
Fig. 3.28 Histologically confirmed CIN1 with fine and cervical canal or the squamocolumnar junction.
regular mosaic
28 3 Abnormal Findings of the Cervix

Fig. 3.29 CIN1 in a 25-year-old patient confirmed by biopsy at 6 o’clock. Characteristically, the acetowhite Lugol-
negative lesion exhibits sharp borders

Fig. 3.30 Condyloma as well as CIN2. A large condylomatous slightly raised lesion on the anterior cervix that is ace-
towhite and partially Lugol-positive (“tiger skin pattern”)
3.2 Intraepithelial Neoplasia 29

Fig. 3.31 Flat condyloma of the cervix. The 18-year-old Fig. 3.32 Fine transparent acetowhite areas within the
patient has already been surgically treated three times for transformation zone. On the anterior (ventral) cervix,
vulvar condylomata acuminata. Now similar condylomata there is also a regular mosaic. In a patient like this, it is
are found on the cervix: at 12 o’clock close to the cervical impossible to distinguish between immature metaplasia
canal and more peripherally at 4 and 7 o’clock and low-grade dysplasia

However, overinterpretation of metaplastic

changes is more likely than confusion of low- and
high-grade dysplasia. In particular, immature
metaplasia cells have a high protein content and
lead to a pale acetowhite reaction. The glycogen
content is not yet sufficient for a positive iodine
reaction. However, the borders are rather irregular
and less well defined than in dysplasia. Sometimes,
a fine and regular punctation or mosaic pattern is
visible (Figs. 3.32 and 3.33). Thus, colposcopic
differentiation between immature metaplasia and
low-grade dysplasia is not possible.
In postmenopausal women, the colposcopic
diagnosis of low-grade dysplasia should be made
with care as in these patients, colposcopic crite-
ria of high-grade dysplasia may be less promi-
nent and resemble those of CIN1 (Figs. 3.34,
3.35, 3.36, 3.37, 3.38, 3.39, 3.40, and 3.41).
Fig. 3.33 Immature metaplasia with fine mosaic. In this
22-year-old patient, the Pap smear is normal, and the
Hybrid Capture 2 test is negative for HPV DNA
30 3 Abnormal Findings of the Cervix

Fig. 3.34 A 41-year-old patient with HSIL of the vulva and a concomitant minor change of the cervix. Biopsy of the
Lugol-negative area reveals LSIL

Fig. 3.35 A 32-year-old patient with biopsy-proven LSIL (minor change). The squamocolumnar junction can be visu-
alized with the Kogan speculum

Fig. 3.36 Biopsy-proven LSIL in a 32-year-old nulligravid patient (minor change)

3.2 Intraepithelial Neoplasia 31

Fig. 3.39 Minor change with regular punctation. The

27-year-old patient, nulligravida, has no cervical
dysplasia
Fig. 3.37 Typical minor change in a 17-year-old patient
without cervical dysplasia

Fig. 3.38 A 27-year-old nulligravid patient with LSIL

Pap smear. The biopsy obtained at 12 o’clock close to the
squamocolumnar junction is negative
32 3 Abnormal Findings of the Cervix

Fig. 3.40 Minor change with transparent regular mosaic

Fig. 3.41 Ectopy with transparent acetowhite epithelium close to the anterior (ventral) squamocolumnar junction

3.2.2 High-Grade Dysplasia between LSIL and HSIL. Also, the cancer risk
for CIN2 and CIN3, which are based on infection
[Link] Pathogenesis with the same HPV high-risk types, is similar.
As pointed out earlier, the expression “high- HSIL assumingly has a significant risk of pro-
grade squamous intraepithelial neoplasia” or gression to invasive carcinoma. However, some
HSIL is used for both CIN2 and CIN3 lesions. evidence suggests that the diagnosis of CIN2/
The dramatic decline of the cervical cancer CIN3, based solely on morphological criteria,
incidence is primarily due to the correct diag- does not adequately reflect the biological progres-
nosis and treatment of high-grade cervical sion potential of the cells. Due to genetic varia-
dysplasia. The previous practice of surgical tions, the individual risk of developing cancer
treatment of only cytopathologically diag- from a high-grade precancer may be quite low.
nosed lesions led to significant overtreatment, The same applies to the presence of high-risk
which may be particularly harmful to younger HPV types in women without abnormal findings
women of childbearing age. who overall have a low cancer risk. If precancer
From a clinical point of view, it is important to and cancer does occur, this may be due to a yet
clearly differentiate low-grade (LSIL) from high- unidentified genetic variation. In the following
grade changes (HSIL). Furthermore, the cytolog- discussion about the association of HPV infection
ical and histological differences between CIN2 and high-grade cervical dysplasia, it should be
and CIN3 are less distinctive than the differences kept in mind that in a large percentage of sexually
3.2 Intraepithelial Neoplasia 33

active women, DNA of human papillomaviruses

can be detected without any evidence of disease.
This has been known for a long time. Moscicki
et al. (1990) demonstrated that more than 60% of
sexually active adolescents are diagnosed with at
least one of the following HPV high risk types:
16, 18, 31, 33, and/or 35.
Especially young women are able to elimi-
nate the HP virus, whereas older women are
more likely to develop persistent infection.
This is of crucial importance as HPV persis-
tence and not the transient HPV infection is
responsible for the subsequent development of
high-grade dysplasia and eventually invasive
disease.
The most common HPV type 16 plays a spe-
cial role as it may be harder to eliminate than
other HPV high risk types (Moscicki et al. 1990).
It can be assumed that high-grade dysplasia
originates from a small focus within an area of Fig. 3.42 A colposcopically guided biopsy from 12
low-grade dysplasia within the cervix. This focus o’clock showed high-grade dysplasia (CIN2 and CIN3).
expands gradually and finally replaces the low- After loop resection of the abnormal transformation zone,
grade dysplastic epithelium. This so-called pro- all three grades of dysplasia (CIN1 to CIN3) were identi-
fied on histopathological exam
gression theory would explain the age distribution
of patients with different degrees of dysplasia.
– Sexual intercourse with multiple partners (>4
Herrero et al. (2000) found that patients with
sex partners: relative risk 1.4 for CIN2 and 2.3
CIN1 are younger than patients with CIN2 and
for CIN3)
CIN3, respectively.
– Immunosuppression (due to medication, e.g.
Cervical lesions can be composed of both LSIL
following transplant surgery, or disease
and HSIL (Fig. 3.42). This could be due to one of
related, e.g. HIV infection)
the two of these mechanisms (Park et al. 1998):
[Link] Colposcopic Appearance
– Progression of CIN1 into CIN2/CIN3, in par-
The accuracy of colposcopic diagnosis of high-
ticular if two lesions found concomitantly on
grade dysplasia is higher and more reliable com-
the cervix differ only by one grade of dyspla-
pared to the abovementioned problematic
sia (progression theory)
discrimination between immature metaplastic
– Infection with more than one HPV type, espe-
changes and low-grade dysplasia. For the diagno-
cially if two concomitant lesions differ by
sis of high-grade dysplasia, Hopman et al. (1995)
more than one grade of dysplasia (CIN1 and
found a reproducibility of 70% for the same
CIN3 concomitantly)
investigator reevaluating previous colposcopic
The following cofactors can promote the images and 76.9% agreement when different
development from low- to high-grade dysplasia investigator evaluated the same cases. Similarly,
(Brisson et al. 1994; Koutsky et al. 1992): the reliability and reproducibility of the histo-
pathological diagnosis is also higher for the diag-
– Infection with HPV 16 (relative risk 8.7) nosis of HSIL than for LSIL.
– Smoking (≥20 cigarettes per day) If colposcopic triage is used to further eval-
– Hormonal contraception (relative risk 1.9) uate HSIL diagnosis on cytopathology, all
34 3 Abnormal Findings of the Cervix

acetowhite lesions should be biopsied in order were confirmed by Vercellino et al. (2013) who
to increase the sensitivity of colposcopic diag- found a sensitivity and specificity of 20% and
nosis (Massad et al. 2009). 99%, respectively.
On the cervix, LSIL and HSIL can be present Another colposcopic phenomenon that can be
simultaneously. The aim of colposcopically considered pathognomonic for high-grade dys-
guided tissue sampling is to identify the lesion plasia is the “ridge sign” (Fig. 3.48). The ridge
with the highest degree of dysplasia in order to sign is an opaque protuberance within the area of
avoid inadequate treatment. acetowhite epithelium located within the trans-
As a rule of thumb, high-grade lesions are formation zone. According to Scheungraber et al.
frequently located close to the cervical canal (2009a, b), CIN2 or CIN3 were diagnosed in
and/or in the vicinity of the squamocolumnar 63.8% of patients when the biopsy was taken
junction. Therefore, when in doubt, biopsies from the area of the protuberant acetowhite
should be taken from an area close to the cer- epithelium. Sensitivity and specificity for HSIL
vical canal. diagnosis was 33.1% and 93.1%, respectively,
Assuming that a high-grade lesion develops compared to 52.5% and 96.4%, respectively
out of low-grade dysplasia, the phenomenon of (Vercellino et al. 2013).
the so-called “inner border sign” can be explained. Both the inner border sign and the ridge sign
The inner border sign (Figs. 3.43 and 3.52) is have been included in the 2011 colposcopic
defined as a dull, oyster white area, which is nomenclature of the International Federation for
located within a less opaque acetowhite area Cervical Pathology and Colposcopy (Bornstein
(Scheungraber et al. 2009a, b; Vercellino et al. et al. 2012).
2013). According to Scheungraber et al. (2009a, A third pathognomonic colposcopic phe-
b), the inner border sign was identified in 7.6% of nomenon of HSIL has been suggested by
patients with an atypical transformation zone. In Vercellino et al. (2013), the “rag sign.” The rag
70% of these patients, HSIL could be confirmed sign is defined as an iatrogenic erosion of the
histopathologically. The sensitivity of the inner epithelium caused by mechanical trauma to the
border sign for the presence of HSIL was 20% cervix, for example, when a Pap smear is
with a specificity of 97%. These observations obtained or acetic acid and iodine solution are
applied using a cotton swab. Sensitivity and
specificity for the prediction of high-grade dys-
plasia are 38.4% and 96.0%, respectively.
With colposcopic evaluation, a high degree
of subjectivity is involved. However, inner bor-
der sign, ridge sign, and rag sign are three
pathognomonic criteria strongly associated
with the presence of high-grade dysplasia. In
order to improve the sensitivity and specificity
of colposcopically guided biopsy, pathognomic
lesions should always be sampled.
The dynamics of acetic acid staining is differ-
ent in high- compared to low-grade dysplasia:
Acetowhite staining occurs more quickly and
fades more slowly. The white color of high-grade
dysplastic epithelium is very intense, and the
abnormal epithelium becomes opaque, since the
light rays are reflected completely due to the high
Fig. 3.43 Inner border sign. Biopsy reveals high-grade
density of dysplastic nuclei and the osmotic with-
dysplasia (both CIN2 and CIN3) drawal of liquid caused by acetic acid (Fig. 3.44).
3.2 Intraepithelial Neoplasia 35

Compared to the epithelium of low-grade dys-

plasia, the epithelium of CIN2/CIN3 lesions is
thicker, and the acetowhite area will be slightly
raised (Fig. 3.45). The margins of the abnormal
epithelium are usually sharply demarcated.
Furthermore, atypical vascular phenomena
may be seen after the application of acetic acid.
As in low-grade dysplasia, the stroma capillaries
will be more pronounced and may be visible as
punctation and/or mosaic (Figs. 3.46, 3.47, 3.48,
3.49, 3.50, and 3.51). Both the pattern of puncta-
tion and of mosaic will be more irregular com-
pared to low-grade lesions or immature
metaplasia.
The atypical vascular phenomena described
do not always occur. Elevated, coarse acetowhite
areas without a recognizable vascular pattern
such as punctation or mosaic are also indicative
of high-grade dysplasia.
Sometimes, the raised and opaque epithe-
lium surrounds the glandular openings within
Fig. 3.44 A 32-year-old patient with biopsy-confirmed an atypical transformation zone (Figs. 3.52,
HSIL/CIN3. The characteristic features of a major change 3.53 and 3.54). This is called “periglandular
are seen with irregular mosaic, irregular punctation, and
cuffing” and may also be an indication of high-
inner border lesion
grade dysplasia (Fig. 3.55).

Fig. 3.45 CIN3 in a 40-year-old patient. There is opaque acetowhite staining with prominent blood vessels
36 3 Abnormal Findings of the Cervix

Finally, it is important to note that the col-

poscopic characteristics of high-grade dysplasia
as described above may be less prominent in
older postmenopausal women and may therefore
be overlooked or misinterpreted.
It should always be kept in mind that
there are limits to the accuracy of colposcopic
interpretation. According to a study by
Bekkers et al. (2008), experience did not
improve colposcopic accuracy. Rather, the
overall sensitivity and the positive predictive
value of colposcopy in identifying high-grade
lesions were low. Therefore, Bekkers et al.
came to the conclusion “that the role of col-
poscopy in the detection and treatment of
cervical abnormalities is to assess size, site,
and extent of an abnormality, rather than to
assess the severity of this abnormality.
Histology must remain the gold standard for
Fig. 3.46 CIN2 with intense acetowhite staining with treatment.”
sharp borders. The squamous epithelium is opaque and
slightly raised

Fig. 3.47 CIN3 with coarse and irregular mosaic

Fig. 3.48 Histologically confirmed HSIL/CIN3 of a 37-year-old patient: major change and ridge sign of the anterior
(ventral) cervix
3.2 Intraepithelial Neoplasia 37

Fig. 3.49 A 28-year-old patient with biopsy-proven HSIL/CIN3. There is a major change of all four quadrants. When
the acetic acid reaction slowly subsides, a characteristic irregular mosaic is visible
38 3 Abnormal Findings of the Cervix

Fig. 3.50 Irregular mosaic and irregular punctation in a 29-year-old with HSIL/CIN3
3.2 Intraepithelial Neoplasia 39

Fig. 3.51 Major change with discrete inner border lesion at 2 o’clock. Biopsy from the left ventral quadrant confirmed
HSIL/CIN3 in this 26-year-old patient
40 3 Abnormal Findings of the Cervix

Fig. 3.52 CIN3 with raised opaque epithelium surrounding the glandular openings. This phenomenon is called “peri-
glandular cuffing” and frequently associated with high-grade dysplasia

Fig. 3.53 Major change with periglandular cuffing

Fig. 3.54 Major change with irregular mosaic and inner border lesion. The mosaic pattern is more prominent after the
acetic acid reaction has partially subsided
3.3 Carcinoma of the Cervix 41

Fig. 3.55 Major change with prominent irregular mosaic

3.3 Carcinoma of the Cervix cancer of the cervix ranks fourth after cancers of
the breast (25.2%), colorectum (9.2%), and lung
3.3.1 Epidemiology and Clinical (8.7%).
Presentation These four sites also represent the most com-
mon causes of cancer death in women: 14.7%
The World Cancer Report published by the WHO (breast), 3.8% (lung), 9.0% (colorectum), and
in 2014 (Stewart and Wild 2014) shows a world- 7.5% (cervix).
wide cervical carcinoma incidence of 7.9% of all Unlike other cancer types, incidence and mor-
women who develop cancer (Fig. 3.56). Thus, tality of cervical cancer is unevenly distributed
42 3 Abnormal Findings of the Cervix

Fig. 3.56 Global cancer

incidence proportions in
women by cancer site. Breast: 1676633
Other: 1924710 (25.2%)
Cervical cancer ranks (28.9%)
fourth (Stewart and Wild
2014)
Liver: 228082
(3.4%) Colorectum: 614304
(9.2%)
Thyroid: 229923
(3.5%)
Ovary: 238719 Lung: 583100
(3.6%) (8.7%)
Corpus uteri: 319905 Cervix uteri: 527624
(4.8%) (7.9%)
Stomach: 320301
(4.8%)

between low- and high-resource countries due to

Czech Republic
the fact that cervical cancer and its precancerous
Denmark
lesions constitute a preventable disease as well as
a type of cancer that benefits strongly from early Poland
detection. According to the WHO world cancer Sweden
report (Stewart and Wild 2014), almost 70% of the Belgium
global cervical cancer burden occurs in areas with
Germany
low or medium levels of development. Although
England
from a global perspective cancer of the breast, the
colorectum, and the lung occurs more frequently, Netherlands
cervical cancer is the most common female cancer France
in 39 of 184 countries worldwide. In 45 countries, Austria
it is the leading cause of cancer deaths in women.
In recent decades, many countries in Europe USA
Incidence Switzerland
and elsewhere have developed a sophisticated
Mortality Finland
secondary prevention program based on cytologi-
cal screening. A further decline of cervical cancer 30 25 20 15 10 5 0
rates is to be expected when the positive effects
of HPV vaccination will become more and more Fig. 3.57 Cervical cancer. Age-standardized rates of new
diseases per 100,000 women in European countries and
apparent. the United States (GEKID 2019)
Looking at the age-standardized rate of new
cervical cancer cases in European countries and cancer (Fig. 3.58), cervical cancer ranks 13th
the United States, both incidence and mortality (1.9%) with breast cancer being by far the most
are low overall, although there are still differ- common malignant disease (29.5%). Other gyne-
ences between these countries (Fig. 3.57). cological cancers such as endometrial and ovarian
Data from Germany shall serve as an exam- occupy rank 5 (4.7%) and 8 (3.1%), respectively.
ple for a typical industrialized nation that has While the incidence is already low, there has
introduced cytological screening as early as been a further decrease in recent years (Fig. 3.59).
1971, when health insurance providers began to Mortality has also decreased. For 2020, 4400 new
cover screening expenses. The following data cases are projected. Thus, cervical cancer may even
have been made available through regular become less frequent than cancer of the vulva.
reports published by the Robert Koch Institute The mean age of cervical cancer patients in
(GEKID 2019). Germany is 55 years. The age-specific incidence
With a population of roughly 83 million, 4380 is highest in the group of 40 to 44-year-olds
patients have been diagnosed with cervical cancer (Fig. 3.60). Five- and 10-year survival rates are
in 2016. With regard to the percentage of female 67% and 63%, respectively.
3.3 Carcinoma of the Cervix 43

Breast 29,5% Forty-four percent of patients have FIGO

Bowel 11,1%
Lung 9,2% stage I disease (Fig. 3.61), where surgical treat-
Melanoma 4,8% ment is still feasible.
Endometrium 4,7%
Pancreas 3,9% Colposcopy should be used for triage of cytologi-
Non-Hodgkin lymphoma 3,7% cal abnormalities and not for screening. However,
Ovary 3,1%
Leukemia 2,6% historically, colposcopy has played a role for pri-
Stomach 2,5%
Kidney 2,3% mary screening especially in the eastern states of the
Thyroid 2,3% Federal Republic of Germany. A Health Technology
Cervix 1,9%
Bladder 1,8% Assessment Report of the German Institute for
Oral cavity and throat 1,8% Medical Documentation and Information (DIMDI,
Central nervous system 1,5%
Vulva 1,4% Deutsches Institut für Medizinische Dokumentation
Multiple myeloma 1,3%
Liver 1,2% und Information) has demonstrated unequivocally
Gallbladder 1,2% the superiority of cytological screening by a system-
Soft tissue without mesothelioma 0,8%
Esophagus 0,7% atic literature review (Nocon et al. 2007). Four stud-
Anus 0,6%
Small bowel 0,5% ies were identified that have compared sensitivity
Hodgkin’s disease 0,5% and specificity of colposcopic and cytological
Larynx 0,2%
Mesothelioma 0,1% screening. In all of these studies, sensitivity of col-
36 30 24 18 12 6 0 poscopy screening was lower than that of cytopa-
thology. The HTA report states:
Fig. 3.58 Percentage of new cancer cases among women The gold standard for the diagnosis of cervical
in Germany. Cancer of the cervix ranks 13th with 1.9% carcinoma is colposcopy with biopsy.
(GEKID 2019)

• The most important goals of colposcopy are:

– To rule out invasive disease (most importantly during pregnancy)
– Histological confirmation by targeted biopsies
– Gentle and targeted removal of microinvasive disease under colposcopic guidance
– Early detection of recurrent disease during follow-up

20
18
16
14
12 Incidence

10
Mortality
8
6 Prognosis of
incidence
4
2

1999 2001 2003 2005 2007 2009 2011 2013 2015 2017 2019

Fig. 3.59 Cervical cancer: age-standardized incidence and mortality per 100,000 women in Germany 1999 to 2017
including the incidence prognosis until 2020 (GEKID 2019)
44 3 Abnormal Findings of the Cervix

20
18
16
14
12
10
8
6
4
2

0–4 5–9 10–14 15–19 20–24 25–29 30–34 35–39 40–44 45–49 50–54 55–59 60–64 65–69 70–74 75–79 80–84 85+
age groups

Fig. 3.60 Cervical cancer: age-specific incidence in Germany 2015 and 2016 (new cases per 100,000 women of the
respective age group) (GEKID 2019)

Stage follow-up of 29 months in 108 patients who

50% underwent trachelectomy for invasive tumors
with a diameter of up to 2 cm and without lymph
44%
node involvement.

25%
3.3.2 Colposcopic Appearance
23%
20%
13% Histologically, microinvasive carcinoma has a
similar appearance as high-grade dysplasia.
0%
However, the cells already begin to penetrate the
I II III IV
basal lamina and invade the underlying stroma
Fig. 3.61 Cervical cancer: distribution of UICC stages I (Fig. 3.62).
to IV in Germany for 2015 and 2016 (UICC stages include The expression “carcinoma in situ,” which
local tumor spread as well as local and distant metastases is sometimes used to describe an undifferenti-
at the time of initial diagnosis) (GEKID 2019)
ated type of severe dysplasia, can be mislead-
ing to both patients as well as doctors. By
The early diagnosis of invasive cervical carci- definition, “carcinoma in situ” does not yet
noma is of paramount importance. The sooner constitute cancer as the cells are still “in situ,”
the cancer is detected, the higher the cure rate. i.e., “in their place” within the epithelium, and
The chances of preserving the uterus, especially there is no stromal invasion.
in young patients, are also better. In FIGO stage The majority of malignant cervical tumors
IA1 disease, conization may be sufficient. In the that present as FIGO stage IB or higher can be
case of more advanced carcinoma, radical cervi- diagnosed with the naked eye. The macroscopic
cal removal of the cervix (trachelectomy) can be appearances can be quite variable (Figs. 3.63,
carried out in selected cases (Halaska et al. 2015). 3.64, 3.65, 3.66, 3.67, 3.68, 3.69, 3.70, 3.71,
In trachelectomy, the uterine corpus is preserved 3.72, 3.73, 3.74, 3.75, 3.76, 3.77, 3.78, and 3.79).
and thus fertility. Also, the parametrial and pelvic In any case, it is advisable to take a tissue sample
lymph nodes are removed to rule out metastatic at the initial gynecological examination when
involvement. invasive disease is suspected macroscopically.
In 2006, Hertel et al. (2006) demonstrated a The biopsy of tumor tissue is not painful.
90.8% recurrence-free survival with a median Occasionally, there is increased bleeding from
3.3 Carcinoma of the Cervix 45

Fig. 3.62 Carcinoma of the cervix. There is invasion

through the basal lamina into the stroma. Also, there may
be hyperkeratosis indicative of keratinizing squamous cell
carcinoma

Fig. 3.64 Squamous cell carcinoma of the cervix. Instead

of a cervix, only fragile tumor tissue is seen that disinte-
grates and bleeds on contact with the speculum

Fig. 3.63 A 60-year-old patient with moderately differ-

entiated keratinizing squamous cell carcinoma FIGO
stage IB1. A significant tissue defect is seen, which is due
to ulceration

Fig. 3.65 Squamous cell carcinoma of the cervix with a

papillary-like appearance
46 3 Abnormal Findings of the Cervix

Fig. 3.68 A 26-year-old patient with a 6.1 cm adenocarci-

noma of the cervix (FIGO stage IB3) with ulceration and
Fig. 3.66 Advanced adenocarcinoma of the cervix. The
atypical vessels. The appearance of the ectocervix is dis-
24-year-old patient suffered from a heavy vaginal bleed-
torted. The arrow indicates the opening of the cervical os
ing 10 weeks after cesarean section and had been diag-
nosed with a 5 cm cervical mass
the biopsy site as well as from the tumor itself.
However, this can be easily controlled by vaginal
tampon.
Histological confirmation of invasive disease
should always be the first step before any further
diagnostic and therapeutic measures are taken. In
Chap. 17, clinical diagnosis of advanced cervical
carcinoma within a low-resource environment is
discussed in detail.
There are limits to the macroscopic and/or
colposcopic diagnosis of invasive cervical car-
cinoma as one of the early manifestations and
sometimes even more advanced tumors are not
accessible to visual diagnosis when they are
located within the cervical canal. This is par-
ticularly true for adenocarcinoma of the
cervix.
Furthermore, microcarcinoma of the cervix is
frequently impossible to diagnose based on col-
poscopic criteria alone. Usually, only high-grade
dysplasia is suspected. According to a literature
review by Hopman et al. (1998), approximately
Fig. 3.67 Adenocarcinoma located on the posterior cer-
vix in a 32-year-old patient who presented with heavy half of microinvasive carcinomas of the cervix
postcoital hemorrhage are not recognized based on colposcopic criteria
3.3 Carcinoma of the Cervix 47

Fig. 3.69 Carcinoma of the cervix in a 49-year-old patient before and after acetic acid

alone. Even an experienced colposcopist will be

faced with these problems because of the non-
specificity of the colposcopic features of
microcarcinoma.
Table 3.2 lists colposcopic features that may
indicate invasive cervical disease. However, speci-
ficity is low, and no single criterion can be used to
conclusively diagnose cervical carcinoma.
Microinvasive tumors with a depth of invasion
of less than 3 mm do not necessarily induce neo-
vascularization. Rather, the tumor cells are still
supplied by diffusion from the surrounding tissue.
Therefore, atypical vessels or ulceration/necrosis
as a manifestation of insufficient blood supply due
to rapid tumor growth are not seen. Instead, col-
Fig. 3.70 Squamous cell carcinoma of the cervix in a poscopic findings are consistent with intraepithe-
38-year-old patient arising from CIN3 (ventral lial neoplasia: acetowhite and iodine-negative
quadrants) epithelium, as well as punctation and mosaic.
48 3 Abnormal Findings of the Cervix

Fig. 3.71 Atypical vessels in a 32-year-old patient with adenocarcinoma of the cervix before and after acetic acid

Fig. 3.72 A 29-year-old patient with adenocarcinoma of the cervix. Although in advanced cervical carcinoma there are
no colposcopically distinguishable features between squamous and glandular carcinoma, the papillary appearance of
the tumor suggests adenocarcinoma
3.3 Carcinoma of the Cervix 49

Fig. 3.73 A 30-year-old patient with advanced squamous Fig. 3.75 A 32-year-old patient with a FIGO IB1 squa-
cell carcinoma. The cervix is replaced by a large exo- mous cell carcinoma with clearly recognizable tissue
phytic tumor defect

Fig. 3.76 A 52-year-old patient with locally advance

squamous cell carcinoma of the cervix
Fig. 3.74 A 52-year-old patient with a 7 × 7 cm squa-
mous cell carcinoma of the cervix (FIGO stage IB3)
50 3 Abnormal Findings of the Cervix

Fig. 3.77 A 31-year-old patient with FIGO stage IB1 squamous cell carcinoma. Atypical vessels are seen with the
green filter

Fig. 3.78 Locally advanced adenosquamous carcinoma of the cervix in a 60-year-old patient
3.3 Carcinoma of the Cervix 51

Table 3.3 FIGO classification of early stage cervical car-

cinoma (Bhatla et al. 2018) and therapeutic options
Stage Definition Therapy
I The carcinoma is Surgery (or primary
strictly confined to chemoradiation)
the cervix
(extension to the
corpus should be
disregarded).
IA Invasive carcinoma (Nonradical)
that can be surgery
diagnosed only by
microscopy, with
maximum depth of
invasion <5 mm
IA1 Measured stromal Simple
Fig. 3.79 A 49-year-old patient with a cervical metasta- invasion of <3 mm hysterectomy (no
sis from carcinoma of the sigmoid colon with widespread in depth vascular or
metastatic disease (ovaries, liver). The biopsy reveals lymphatic system
adenocarcinoma. The colposcopic image would be highly involvement)
unusual for primary carcinoma of the cervix and is or
explained by the fact that it is a metastatic lesion trachelectomy (if
fertility preservation
is desired)
Table 3.2 Differential diagnosis of colposcopic features
or
that may be associated with invasive disease
therapeutic
Colposcopic conization
observation Differential diagnosis IA2 Measured stromal Modified radical
Abnormal vessels Prominent vessels overlying invasion of ≥3 mm hysterectomy
Nabothian cysts: gradual and <5 mm in
tapering toward the periphery, depth
regular branching IB Invasive carcinoma Radical
Exophytic growth Cervical condylomata acuminata with measured hysterectomy
Hyperkeratosis Hyperkeratosis due to deepest invasion (Wertheim-Meigs)
condyloma or inflammation ≥5 mm (greater or
Friable tissue Inflammation, atrophy than stage IA), primary
Tissue defects True erosion: superficial lesion limited to chemoradiation
(ulceration, sloughing of the squamous the cervix uteri
necrosis) epithelial layer IB1 Invasive carcinoma Radical
Obstetrical trauma: cervical ≥5 mm depth of hysterectomy
defect without hemorrhage or stroma invasion (Wertheim-Meigs)
other abnormal findings and <2 cm in or
greatest dimension primary
chemoradiation
The colposcopic evaluation of an atypical or
cervical lesion alone is not sufficient to rule out trachelectomy (if
fertility preservation
invasive disease. A targeted biopsy is always is desired and tumor
required. If invasive disease is suspected, multi- ≤2 cm, no lymph
ple biopsies are appropriate. Surgical methods node involvement)
that do not allow comprehensive histopatho- IB2 Invasive carcinoma Radical
logic evaluation, such as CO2 laser vaporiza- ≥2 cm and <4 cm hysterectomy
in greatest (Wertheim-Meigs)
tion or cryotherapy, are contraindicated. dimension or
Carcinoma of the cervix is staged clini- IB3 Invasive carcinoma primary
cally using the FIGO system (Bhatla ≥4 cm in greatest chemoradiation
et al. 2018) (Table 3.3). Treatment decisions dimension
52 3 Abnormal Findings of the Cervix

(surgery versus radiation therapy) are based types in a synchronous squamous cell and adeno-
on clinical examination as well as imaging carcinoma: the squamous component expressed
techniques. HPV 33 while the adenocarcinoma was associ-
ated with HPV 18.
Both the cytological and the colposcopic
3.4 Special Considerations diagnosis of adenocarcinoma in situ is a diag-
on Cervical Adenocarcinoma nostic challenge. Frequently, squamous dys-
and Adenocarcinoma In Situ plasia is diagnosed first, as CIN and AIS may
(AIS) occur concurrently, whereas the glandular
component is much more difficult to detect.
The majority of cervical carcinomas originates Colposcopically, the acetic acid test has only a
from squamous epithelium. However, an increasing limited diagnostic significance, whereas
number of adenocarcinomas are diagnosed. Schiller’s test is of no value at all as both normal
According to the FIGO report (Benedet et al. 2003), and abnormal glandular epithelium are consis-
about 16% of cervical cancers are adenocarcino- tently Lugol-negative. In addition, adenocarci-
mas. Bray et al. (2005) reviewed epidemiological noma in situ and early invasive adenocarcinoma
data from 13 European countries. The age-adjusted without significant neoplastic growth and/or
incidence rates of adenocarcinoma had increased ulceration will not change the texture of the sur-
within all European countries studied. The rate of face epithelium, as neoplastic changes may be
increase was ranging from 0.5% per year in embedded within the glandular crypts or may be
Denmark, Sweden, and Switzerland to more than or located altogether within the cervical canal.
equal to 3% in Finland, Slovakia, and Slovenia. Punctation or mosaic are no features of intraepi-
The observed increase of adenocarcinoma thelial glandular neoplasia.
may be due to shifting risk factors. On the other Costa et al. (2007) analyzed cytological and
hand, cytology-based screening programs are colposcopic findings from 42 patients with
less effective in detecting glandular precursors. AIS. In less than half of patients (42.9%), atypi-
According to Bray et al. (2005), cytological cal cells were identified. Results from the col-
screening may have had at least some impact in poscopic evaluation were also disappointing: In
reducing cervical adenocarcinoma incidence in 54.8% of patients, the squamocolumnar junction
some of the countries studied. was not visible. In 16.7% of all colposcopic
HPV-based screening is more effective pre- exams, no abnormality was seen.
venting adenocarcinoma as shown by a review of Despite these difficulties, V. Cecil Wright
four European randomized controlled trials com- (2008) has attempted to define the colposcopic
paring cytology-based screening and HPV-based features of glandular changes. Wright distin-
screening (Ronco et al. 2014). guishes three appearances (reproduced in
With HPV vaccines and especially the nona- descending order):
valent type (Joura et al. 2015), prevention of pre-
1. Resemblance to immature metaplasia
malignant and invasive glandular neoplasia
2. Patchy red and white areas, again resembling
should be as effective as the prevention of the
an immature transformation zone
squamous variety.
3. A single isolated densely acetowhite and ele-
Analogous to squamous cell carcinoma, cervi-
vated lesion. This area may not be in contact
cal adenocarcinoma develops from a precursor,
with the squamocolumnar junction.
adenocarcinoma in situ (AIS). Adenocarcinoma
in situ is also related to HPV infection, mostly the Thus, the first two phenomena of AIS resemble
HPV high risk types 18 and 45. It is assumed that an immature transformation zone. As in cytologi-
gland cells are particularly susceptible to the cal diagnosis, concomitant squamous dysplasia,
transformational effect of these two HPV types which is present in about half of the cases, may
(Iwasawa et al. 1996). Jakob et al. (2000) distract from the more subtle features of glandular
observed expression of two different HPV sub- dysplasia.
References 53

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 International Nomenclature
of Colposcopy 4

Contents
4.1 Cervix 55
4.2 Vagina 57
4.3 Vulva 58
References 59

The colposcopic nomenclature was last equate” are used. This should be the first state-
updated in 2011/2012 by the International ment of any written colposcopic evaluation. If
Federation of Cervical Pathology and colposcopy is considered inadequate, a reason
Colposcopy (IFCPC). This system is used is given. For example, due to extreme obesity,
to describe and classify findings of the cer- the cervix might not be visible at all. Also, when
vix as well as of the vagina and vulva. inflammation or atrophy are present, no adequate
colposcopic evaluation is possible.
When colposcopy is considered inadequate,
a follow-up examination should be carried out,
for example, after local treatment of inflam-
4.1 Cervix mation and atrophy or after suitable instru-
ments have been procured (obesity).
At the 14th IFCPC World Congress in Rio Next, the visibility of the squamocolumnar
de Janeiro, the colposcopic terminology was junction is described. Formerly, the descriptors
updated by the nomenclature committee under “satisfactory” and “unsatisfactory” have been
the direction of Jacob Bornstein (Bornstein et al. used. A colposcopic exam was deemed unsat-
2012a; Quaas et al. 2013). The Rio classification isfactory, if the transformation zone was not
system of the cervix is divided into five sections: completely visible. However, the definitions of
general assessment, normal colposcopic findings, adequate/inadequate and satisfactory/unsatisfac-
abnormal colposcopic findings, suspicious for tory are not identical. According to the Rio 2011
invasion, and miscellaneous findings (Table 4.1). nomenclature, adequacy and the type of transfor-
The first step of colposcopic assessment mation zone observed are separate statements.
should be a statement regarding the quality of In fact, the IFCPC has abandoned the descrip-
this assessment. The terms “adequate” and “inad- tion of a colposcopic exam as “unsatisfactory”

© Springer Nature Switzerland AG 2023 55

R. J. Lellé, V. Küppers, Colposcopy, [Link]
56 4 International Nomenclature of Colposcopy

Table 4.1 2011 International Federation of Cervical Pathology and Colposcopy terminology of the cervix (Bornstein
et al. 2012a)
Section
General assessment • Adequate or inadequate for the reason (e.g., cervix obscured by inflammation, bleeding,
or scar)
• Squamocolumnar junction visibility: completely visible (type 1), partially visible
(type 2), not visible (type 3)
Normal colposcopic • Original squamous epithelium: mature, atrophic
findings • Columnar epithelium: ectopy/ectropion
• Metaplastic squamous epithelium: Nabothian cysts, crypt (gland) openings
• Deciduosis in pregnancy

Abnormal • Location of the lesion: by clock position, inside or outside the transformation zone
colposcopic findings • Size of the lesion: number of cervical quadrants the lesion covers and/or as percentage of
cervix
• Grade 1 (minor change): fine mosaic, fine punctation, thin acetowhite epithelium,
irregular, geographic border
• Grade 2 (major change): sharp border, inner border sign, ridge sign, dense acetowhite
epithelium, coarse mosaic, coarse punctation, rapid appearance of acetowhitening, cuffed
crypt (gland) openings
• Nonspecific: leukoplakia (keratosis, hyperkeratosis), erosion Lugol’s staining (Schiller’s
test): stained or nonstained
Suspicious for • Atypical vessels
invasion • Additional signs: fragile vessels, irregular surface, exophytic lesion, necrosis, ulceration
(necrotic), tumor or gross neoplasm
Miscellaneous • Congenital transformation zone, condyloma, polyp (ectocervical or endocervical),
findings inflammation, stenosis, congenital anomaly, posttreatment changes, endometriosis

altogether as it has the connotation of an insuf- mature transformation zone, the original squa-
ficient exam that needs to be repeated. mocolumnar junction may be difficult to identify.
In section two, normal colposcopic findings are The size of a lesion, which is relevant both
described. This includes both original squamous for follow-up exams as well as treatment plan-
and columnar/glandular epithelium as well as the ning, can be given as the number of quad-
phenomena of metaplasia such as gland openings rants involved, the percentage of the cervix
or Nabothian cysts. Deciduosis, when it occurs in affected, or both. Quadrants are usually called
pregnancy, is also considered a normal finding. right/left and anterior/posterior although the
In the third section, abnormal colposcopic correct anatomical term would be “ventral”
findings are listed starting with the location and and “dorsal.”
size of the abnormal lesion. By projecting a clock When abnormal colposcopic findings are
face onto the cervix, the approximate position described, the distinction between low-grade
and extent of a distinct area can be indicated. (CIN1) and high-grade (CIN2 and CIN3) pre-
Although the dysplastic epithelium will usually cancer are most important as this will be decisive
be located within the transformation zone, any for further clinical management. In colposcopic
abnormality should be described in relation to nomenclature, the terms “minor” and “major”
its location regarding the transformation zone. lesion are used, sometimes also called “grade 1”
“Inside the transformation zone” means that a and “grade 2” lesions, respectively. Whenever
lesion is located medial to the original squamo- appropriate, expressions such as “fine puncta-
columnar junction. However, in patients with a tion/mosaic” versus “coarse punctation/mosaic”
4.2 Vagina 57

and “regular” versus “irregular pattern” should transformation zone T1, T2, and T3. Whereas a
be used. In addition to the Rio nomenclature, the type 1 excision will resect a completely visible
distinction between “transparent” and “opaque” transformation zone, a type 2 excision includes
acetowhite areas is helpful. a small area of endocervical tissue that can be
Inner border sign and ridge sign—possibly visualized colposcopically. With a type 3 exci-
also rag sign—are considered to be pathogno- sion, a larger amount of endocervical tissue
monic for high-grade disease and will support the is resected. The type 3 excision is also used
colposcopic diagnosis of a major/grade 2 lesion to effectively treat adenocarcinoma in situ or
(Scheungraber et al. 2009; Vercellino et al. 2013). microinvasive disease.
For the colposcopic identification of a The type of excision that depends on the
major lesion, not only the high intensity of length of the surgical specimen, its thickness,
acetowhite staining is important but also the and its circumference should be reported by the
rapid development of the acetic acid reaction. pathologist.
However, even a slow reaction to the acetic
acid, which in some cases may take up to 3 min,
may be associated with major changes, especially 4.2 Vagina
if other criteria for a grade 2 lesion are met.
Periglandular cuffing, i.e., raised and opaque The IFCPC nomenclature also includes a classifi-
epithelium surrounding the glandular openings cation system for the vagina, which is analogous
within an atypical transformation zone, may also to that of the cervix (Bornstein et al. 2012a). The
be an indication of high-grade dysplasia. aim of this nomenclature is to create an evidence-
The classification of Lugol’s test (Schiller’s based terminology with relevance to management
test) as an unspecific reaction underlines the and especially to treatment of colposcopically
necessity of prior acetic acid application, espe- diagnosed changes of the vagina.
cially preoperatively in order to determine the The colposcopic nomenclature of the vagina is
extent of the excision, as the Lugol reaction similar to that of the cervix (Table 4.2). Initially,
alone is not a suitable criterium. Leukoplakia the adequacy of the examination is evaluated.
(keratosis, hyperkeratosis) is also considered If it is considered to be “inadequate,” an appro-
nonspecific. priate reason is given such as inflammation or
The fourth section describes all phenomena hemorrhage.
that are suspicious for invasion, especially atypi- Normal findings are classified either as
cal and fragile vessels, an irregular surface, and “mature” or “atrophic.” If an abnormality is iden-
ulceration and exophytic growth. tified, its location is given using descriptors such
According to the Rio nomenclature, the as “lower third of the vagina/upper two-thirds,
presence of atypical vessels is considered to be anterior/posterior, and left/right vaginal wall.”
pathognomonic for invasive disease, and his- As with the uterine cervix, the abnormal find-
topathological clarification by colposcopically ings are divided into “minor or grade 1 changes”
guided biopsy is mandatory. and “major or grade 2 changes.” As for the cer-
Finally, in the fifth section, several colposcopic vix, leukoplakia and the results of Lugol’s test
findings are summarized including condylomata (Schiller’s test) are classified as “nonspecific.”
and cervical polyps. Nonspecific findings also include the pres-
The IFCPC has added an addendum to its ence of columnar epithelium in the vagina, so-
nomenclature regarding surgical treatment. called vaginal adenosis.
The aim is to avoid poorly defined terms such The diagnosis “suspicious for invasion” is part
as cone biopsy, conization, or small and large of the “abnormal colposcopic findings” section.
loop excision. Instead, three excision types are Diagnosis is based on the same criteria as for sus-
defined, which correspond to the three types of pected cervical cancer.
58 4 International Nomenclature of Colposcopy

Table 4.2 2011 International Federation of Cervical Pathology and Colposcopy terminology of the vagina (Bornstein
et al. 2012a)
Section
General • Adequate or inadequate because of (e.g., inflammation, bleeding, or scar)
assessment • Transformation zone
Normal • Normal squamous epithelium: mature, atrophic
colposcopic
findings
Abnormal • Location of the lesion: Upper third or lower two-thirds, anterior, posterior, or lateral (right or
colposcopic left)
findings • Grade 1 (minor change): fine mosaic, fine punctation, thin acetowhite epithelium
• Grade 2 (major change): coarse mosaic, coarse punctation, dense acetowhite epithelium
• Suspicious for invasion: fragile vessels, irregular surface, exophytic lesion, necrosis,
ulceration (necrotic), tumor or gross neoplasm
• Nonspecific: columnar epithelium (adenosis)
• Lugol’s staining (Schiller’s test): stained or nonstained, leukoplakia
Miscellaneous • Erosion (traumatic), condyloma, polyp, cyst, endometriosis, inflammation, vaginal stenosis,
findings congenital transformation zone

4.3 Vulva • The vulvar nomenclature is also applicable

to the anus.
The terminoloy system for the vulva as pub-
lished by the International Federation of Cervical • The acetic acid test plays only a limited role
Pathology and Colposcopy (IFCPC) has been compared to its value in the diagnosis of the
established in collaboration with the International cervix, and Lugol’s test is not used at all.
Society for the Study of Vulvovaginal Diseases
(ISSVD). However, since the IFCPC publication Acetowhitening of the vulva is not as specific
by Bornstein et al. in 2012, the ISSVD has pub- as it is for the cervix, where the acetic acid test
lished a modification of its terminology for vulvar is mandatory for any colposcopic evaluation. In
intraepithelial neoplasia (VIN) (Bornstein et al. some circumstances, when intraepithelial neo-
2016). In the following section, the nomenclature plasia (VIN) is suspected, acetic acid may help
of the IFCPC terminology is discussed in detail. to better delineate the abnormal area and choose
Compared to colposcopy of the cervix and an appropriate biopsy site. For all other vulvar
vagina, colposcopic evaluation and nomenclature diseases, acetic acid does not contribute to diag-
of the vulva differ significantly: nosis. As Lugol’s test is based on the presence or
absence of glycogen with the intermediate cells
• Evaluation of the vulva is more complex. of nonkeratinizing squamous epithelium, it is not
applicable to the vulva.
There is a vast range of different dermatologic As Bornstein et al. (2016) point out, the vulvar
diseases that may affect the vulva. Due to the epithelium is more varied compared to the cervix
complexity of vulvar diagnosis, the reporting of and vagina as it encompasses both skin and mucosa.
vulvar lesions is frequently inconsistent. The skin is further divided into “hairy” such as the
labia majora and “nonhairy” such as the clitoris.
• The colposcope is not an indispensable tool Some diseases may be limited to the skin or the
for diagnosis of the vulva. mucosa alone, whereas others affect both types of
epithelium. The description of the structures con-
Many experts on vulvar diseases will use only stitutes the first section of vulvar nomenclature.
a magnifying glass or no magnification at all. The Hirsuties papillaris (micropapillomatosis),
IFCPC has taken this into account calling its ter- which is sometimes confused with condylo-
minology “colposcopic” as well as “clinical”. mata acuminata, sebaceous glands, and a mild
References 59

vestibular redness, are classified as normal Bornstein J, Bogliatto F, Haefner HK, Stockdale CK, Preti
findings and therefore should not be treated. M, Bohl TG, Reutter J (2016) The 2015 International
Society for the Study of Vulvovaginal Disease
In order to appropriately describe abnor- (ISSVD) terminology of vulvar squamous intraepithe-
mal findings, the dermatologic terminology is lial lesions. Obstet Gynecol 127(2):264–268. https://
applied. Besides lesion size and location, the [Link]/10.1097/AOG.0000000000001285
lesion type (macule, patch, papule, etc.) as well Quaas J, Reich O, Frey Tirri B, Küppers V (2013)
Erläuterung und Anwendung der kolposkopischen
as the secondary morphology such as eczema or Nomenklatur der IFCPC (International Federation for
lichenification is given. Cervical Pathology and Colposcopy) Rio 2011 Zu den
A weakness of present ISSVD nomenclature grundsätzlichen Hinweisen für die Kolposkopie der
is the fact that no distinction is made between Cervix uteri – adäquat/inadäquat. Plattenepithel-Zy
73:1–4
symptomatic and asymptomatic vulvar lesions. Scheungraber C, Koenig U, Fechtel B, Kuehne-Heid R,
In the differential diagnosis of diseases of Duerst M, Schneider A (2009) The colposcopic feature
the vulva, the consideration of symptoms is ridge sign is associated with the presence of cervical
decisive. intraepithelial neoplasia 2/3 and human papillomavirus
16 in young women. J Low Genit Tract Dis 13(1):13–
16. [Link]
Vercellino GF, Erdemoglu E, Chiantera V, Vasiljeva K,
References Drechsler I, Cichon G et al (2013) Validity of the col-
poscopic criteria inner border sign, ridge sign, and rag
Bornstein J, Bentley J, Bosze P, Girardi F, Haefner H, sign for detection of high-grade cervical intraepithelial
Menton M et al (2012a) 2011 colposcopic terminol- neoplasia. Obstet Gynecol 121(3):624–631. https://
ogy of the international federation for cervical pathol- [Link]/10.1097/AOG.0b013e3182835831
ogy and colposcopy. Obstet Gynecol 120(1):166–172.
[Link]
 Indications for Colposcopy
5

Contents
5.1 General Objectives 61
5.2 Triage of Abnormal Cytological Findings 62
5.3 Colposcopy as Triage of a Positive HPV Test Result 62
5.4 Other Indications 63
5.5 Contraindications 63
5.6 Summary of Colposcopy Indications of the Cervix 64
References 66

Colposcopy is most frequently used for tri- 5.1 General Objectives

age of patients with an abnormal cytologi-
cal cervical smear and/or a positive HPV The indication for colposcopy should be broad.
test result. In addition, there are special The colposcope as a magnification device is a
indications independent from cytological valuable tool for the inspection of the lower
or HPV testing. Besides diagnostic col- genital tract. In addition to the early detection
poscopy, all surgical procedures of the of gynecological malignancies, the diagno-
cervix and vagina are performed under col- sis of dermatologic and infectious diseases is
poscopic guidance. improved.

Colposcopy has three basic objectives:

1. Assessment of the vulva, anus, vagina, and cervix uteri in the context of cancer screening as
well as follow-up examination of patients who did or did not undergo surgical treatment of
dysplasia
2. Performance of operations on lower genital tract diseases under colposcopic guidance
3. Differential diagnosis of dermatologic and infectious diseases of the lower genital tract including
colposcopically guided biopsies

© Springer Nature Switzerland AG 2023 61

R. J. Lellé, V. Küppers, Colposcopy, [Link]
62 5 Indications for Colposcopy

Colposcopy is mostly used to evaluate the The sensitivity of any single parameter of
cervix. If intraepithelial neoplasia is suspected, colposcopic evaluation is far from perfect.
the most important task of colposcopy is grad- Sensitivity of colposcopic diagnosis is estimated
ing of a lesion, i.e., the distinction between minor to be 30–70% according to Huh et al. (2014).
change/grade 1 and major change/grade2 accord- Therefore, when all the test results from an
ing to the Rio 2011 colposcopic terminology (see individual patient have been gathered, a decision
Chap. 4). Usually, one or more colposcopically on further management is made based on the
guided biopsies are obtained. overall picture rather than any single param-
If no lesions are found despite cytological eter such as colposcopic grading or biopsy.
suspicion of HSIL and/or HPV16/18 infection,
random biopsies of the transformation zone are
helpful (Massad 2006; Massad et al. 2009) as 5.2 Triage of Abnormal
well as endocervical curettage (Pretorius et al. Cytological Findings
2012). Huh et al. (2014) found that a single
random biopsy could increase the sensitivity of The most common use of colposcopic triage is an
colposcopy to detect HSIL by about 20%, when abnormal cytologic screening result. Colposcopy
colposcopy was considered to be adequate and no is indicated if there is a 10% or higher probability
obvious lesion was identified. This was particu- of CIN3 and/or AIS. In addition, colposcopy may
larly true for patients positive for HPV16/18 on be useful in individual cases for reassurance of a
the cobas® HPV test. patient with a lower likelihood of high-grade dis-
By determining the type of transformation ease, e.g., when a low-grade squamous intraepi-
zone and by delineating the exact location and thelial lesion (LSIL) is suspected for the first time.
extent of the cervical lesion, appropriate treat- If the Pap smear result is repeatedly classified as
ment planning is possible. unsatisfactory, colposcopy is also required.

Colposcopy indications based on abnormal Pap test results using the Bethesda classification
(Nayar and Wilbur 2015) are as follows:
– Recurring atypical squamous cells of undetermined significance (ASC-US)
– Recurring low-grade squamous intraepithelial lesion (LSIL)
– Atypical squamous cells favor high grade (ASC-H)
– High-grade squamous intraepithelial lesion (HSIL)
– Atypical glandular cells (AGC)
– Adenocarcinoma in situ (AIS)
– Cytology suspicious for cancer

Decision-making for surgical procedures of an indication for colposcopy in women who are
the cervix such as a cone biopsy should not be older than 30 years.
based on the result of a cytological screening When primary HPV screening is performed
diagnosis alone but rather on thorough col- (≥30 years), cytology can also be used as triage
poscopic evaluation including targeted biopsy. of a positive HPV test result. If the HPV test is
able to identify HPV types 16 and 18 such as the
cobas® test, the screening algorithm as suggested
5.3 Colposcopy as Triage by Huh et al. (Huh et al. 2015) could be as follows
of a Positive HPV Test Result (Fig. 5.1): If the test is HPV16/18 positive, the like-
lihood of a patient already having or developing a
When a cytological ASC-US result is obtained high-grade lesion or cancer is high. According to
for the first time, HPV testing can be used for the trial data regarding the cobas® test (Wright
triage. ASC-US/HPV positivity is considered et al. 2015), the 3-year cumulative incidence rate
5.5 Contraindications 63

HPV16/18 positive Colposcopy

≥ ASC-US

Primary HPV
12 other Cytology
screening:
high risk HPV types
cobas© HPV test

NILM

Follow up in 12 months

negative Routine screening

Fig. 5.1 Primary HPV screening algorithm based on the cobas® HPV test (Huh et al. 2015)

is as high as 21.16% (95% confidence interval – Young patients with HSIL that have not
18.39–24.01%). Therefore, colposcopy without been treated initially and instead are fol-
the need of preceding cytology is indicated. If the lowed at regular intervals for spontaneous
test is positive for other HPV high risk types (12 regression
different HPV types are included in the cobas® test
system), cytologic triage is required.

5.5 Contraindications
5.4 Other Indications
It should always be kept in mind that col-
Besides triage of screening tests such as cytology, poscopic triage, although not an invasive pro-
HPV test, or co-testing, colposcopy is indicated cedure such as surgery for actual or suspected
if any abnormality is identified without magni- cervical dysplasia, is still uncomfortable for
fication with or without the use of acetic acid. most patients, both physically and psychologi-
Recurrent postcoital bleeding is also a frequent cally (O’Connor et al. 2017). However, in many
reason for colposcopic inspection of the cervix. instances, colposcopy will be helpful to avoid
Patients with conditions involving immuno- unnecessary or even harmful surgical proce-
suppression due to HIV/AIDS or following trans- dures. Therefore, no absolute contraindications
plant surgery are at higher risk for developing for colposcopy exist.
lower genital tract neoplasia. Therefore, colpos- Relative contraindications include atro-
copy in combination with Pap smear and HPV phy and inflammation, as hormonal or anti-
testing at regular intervals is advisable. inflammatory pretreatment may be appropriate.
Other indications for colposcopy include: Menstruation may also be a relative contraindi-
cation depending on the particular clinical cir-
– Follow-up after surgical treatment of intraepi- cumstances. For example, bleeding might be due
thelial cervical disease to malignant disease. Also, if there is only minor
– Follow-up after HSIL cytology without identi- menstrual bleeding, colposcopic evaluation may
fication of a lesion on initial colposcopic triage still be possible.
64 5 Indications for Colposcopy

5.6 Summary of Colposcopy

Indications of the Cervix

• Colposcopy indications due to cytological findings

– Recurring atypical squamous cells of undetermined significance (ASC-US)
– Recurring low-grade squamous intraepithelial lesion (LSIL)
– Atypical squamous cells favor high grade (ASC-H)
– High-grade squamous intraepithelial lesion (HSIL)
– Atypical glandular cells (AGC)
– Adenocarcinoma in situ (AIS)
– Cytology suspicious for cancer
• Colposcopy indications related to HPV test results
– HPV high-risk positivity in combination with equivocal cytology (ASC-US)
– HPV16/18 positivity in women ≥30 years of age
• Colposcopy due to other findings
– Postcoital bleeding
– Cervical abnormality when viewed without magnification
– Immunocompromised patients (e.g., HIV/AIDS or following transplant surgery)
– After surgical treatment of CIN
– After HSIL cytology without a visible lesion
– Follow-up for possible spontaneous regression of CIN
• Relative contraindications
– Atrophy
– Inflammation
– Menstrual bleeding

As an example for a practical approach to

colposcopic triage, the algorithms for cervi-
cal screening in Germany are given (Figs. 5.2
and 5.3).
5.6 Summary of Colposcopy Indications of the Cervix 65

Cytology

HSIL (severe dysplasia)

ASC-H
Normal

up to the age of 29 years from the age of 30 years AIS

AGC favor neoplastic
ASC-US, AGC, LSIL ASC-US, AGC, LSIL Squamous cell carcinoma
HSIL (moderate dysplasia)
Endocervical adenocarcinoma

6–12 months

cytology
HPV
6–12 months interval

after 24 months
Normal

HPV HPV
HSIL ASC-US, AGC
negative positive
LSIL

within 3 months within 3 months within 3 months within 3 months immediately

Colposcopy

Fig. 5.2 Algorithm for cervical screening in German. Women 20–34 years are screened by annual Pap tests

Co-Testing (HPV + cytology)

HPV pos/neg HPV pos/neg

HPV negative HPV positive ASC-H, AGC HSIL
HPV positive HPV negative
Normal ASC-US, AGC favor neoplastic, (severe dysplasia)
Normal LSIL
ASC-US, AGC LSIL HSIL AIS
(moderate dysplasia) Carcinoma

12 months 12 months

Co-Testing (HPV + cytology)

HPV positive
HPV negative
ASC-US, AGC
Normal
LSIL

within 3 months within 3 months within 3 months immediately

Colposcopy

Fig. 5.3 Algorithm for cervical screening in German. Women from the age of 35 years are screened by co-testing
66 5 Indications for Colposcopy

References Nayar R, Wilbur DC (2015) The Bethesda system for

reporting cervical cytology: definitions, criteria, and
explanatory notes. Springer
Huh WK, Sideri M, Stoler M, Zhang G, Feldman R,
O’Connor M, O’Brien K, Waller J, Gallagher P, D’Arcy
Behrens CM (2014) Relevance of random biopsy at
T, Flannelly G et al (2017) Physical after-effects
the transformation zone when colposcopy is nega-
of colposcopy and related procedures, and their
tive. Obstet Gynecol 124(4):670–678. [Link]
inter-relationship with psychological distress: a
org/10.1097/AOG.0000000000000458
longitudinal survey. BJOG: an international. J
Huh WK, Ault KA, Chelmow D, Davey DD, Goulart RA,
Obstetr Gynaecol 124(9):1402–1410. [Link]
Garcia FA et al (2015) Use of primary high-risk human
org/10.1111/1471-0528.14671
papillomavirus testing for cervical cancer screening:
Pretorius RG, Belinson JL, Azizi F, Peterson PC,
interim clinical guidance. Gynecol Oncol 136(2):178–
Belinson S (2012) Utility of random cervical biopsy
182. [Link]
and endocervical curettage in a low-risk population.
Massad LS (2006) More is more: improving the sensitivity
J Low Genit Tract Dis 16(4):333–338. [Link]
of colposcopy. Obstet Gynecol 108(2):246–247. https://
org/10.1097/LGT.0b013e3182480c18
[Link]/10.1097/[Link].0000229430.04828.83
Wright TC, Stoler MH, Behrens CM, Sharma A, Zhang G,
Massad LS, Jeronimo J, Katki HA, Schiffman M (2009)
Wright TL (2015) Primary cervical cancer screening
The accuracy of colposcopic grading for detec-
with human papillomavirus: end of study results from
tion of high-grade cervical intraepithelial neoplasia.
the ATHENA study using HPV as the first-line screen-
J Low Genit Tract Dis 13(3):137–144. [Link]
ing test. Gynecol Oncol 136(2):189–197. [Link]
org/10.1097/LGT.0b013e31819308d4
org/10.1016/[Link].2014.11.076
 The Significance of Cytology,
Biopsy, and HPV Testing 6

Contents
6.1 Cytology 67
6.2 Biopsy 87
6.3 HPV Test 95
References 101

a former co-worker of George N. Papanicolaou,

Cytology is recognized as the gold stan- wrote about the impact of cytologic diagnosis
dard of cervical screening that has led to (Naylor 2000) and concluded: “These and other
a significant reduction of cervical cancer events and achievements in cytopathology, from
cases in numerous countries. However, its meager beginnings in the early twentieth cen-
Pap smear results alone do not provide a tury to its worldwide use and acceptance today,
reliable diagnosis. Therefore, colposcopic mark this century as the ‘century for cytopathol-
triage is required, which includes target ogy’.” Although during the twenty-first century,
biopsy. HPV testing plays an important the role of cytology, especially as the sole screen-
role as a screening tool either alone or in ing tool, will change significantly, cytology will
combination with cytology. For the colpos- continue to play an important role in cervical
copist, knowledge of the strengths and lim- cancer prevention in the foreseeable future.
itations of these methods are indispensable. Colposcopy training courses require a basic
knowledge in the field of morphology and addi-
tional methods based on molecular biology.
Therefore, the following text will present the
6.1 Cytology knowledge that is indispensable in the context of
colposcopy.
Cytology as a screening tool has been at the core The following explanations refer to the cervix.
of screening for cervical cancer and its precursors. The statements made here only partially apply to
It is responsible for a significant reduction in cer- other regions of the genital tract as well as the anus.
vical cancer cases and thus has saved numerous Currently, the Pap smear is the best docu-
lives. In the year 2000, Bernard Naylor, himself mented screening procedure for early diag-

© Springer Nature Switzerland AG 2023 67

R. J. Lellé, V. Küppers, Colposcopy, [Link]
68 6 The Significance of Cytology, Biopsy, and HPV Testing

nosis of cervical carcinoma and especially its sisting of multiple layers of squamous cells, the
precursors. Colposcopy including targeted smear shows mainly superficial and intermediate
tissue removal, however, is used for triage, i.e. cells, while in the case of atrophy only parabasal
for further clarification of abnormal cytologi- cells may be present.
cal findings and treatment planning. In normal squamous epithelium, the cells
In Germany, the country of origin of colpos- become larger and the nuclei smaller the further
copy, there has been a discussion whether cytol- they are from the basal layer. Therefore, super-
ogy or rather colposcopy is the most effective ficial and large intermediate cells have a large
screening tool. This dispute has long been settled, amount of cytoplasm with a small round and
and cytology has become internationally accepted pyknotic nucleus. In the Papanicolaou stain, the
as the screening method of choice. As summa- superficial cells are eosinophilic (red), while
rized in a Health Technology Assessment report other cells are usually basophilic (blue) (Fig. 6.1).
(Nocon et al. 2007), colposcopy has a lower sen- In addition to the squamous epithelial cell
sitivity compared to cytology for detecting cervi- component, endocervical glandular cells are
cal dysplasia. found in the cervical smear (Fig. 6.2). The Pap
In everyday clinical practice, Pap smears smear report has to include a statement, whether
are frequently taken from the vagina and vulva. endocervical or metaplastic cells are identified.
While the Pap test after hysterectomy for benign The presence of these cell types signifies that the
disease is not always required (Fox et al. 1999), smear was taken from the transformation zone.
vaginal intraepithelial neoplasia (VAIN) may Such a smear is more likely to be representative
occur after a previous diagnosis of cervical and therefore more sensitive/reliable as a smear
intraepithelial neoplasia. Therefore, Pap smear containing squamous epithelial cells only.
screening is indicated in the latter situation. Cervical intraepithelial neoplasia (CIN) leads
Conversely, the usefulness of vulvar cytol- to changes of both the cell nucleus as well as the
ogy is less well documented, as the keratinizing cytoplasm. In higher grade CIN, the cell nucleus
squamous epithelium of the external genitalia is is larger and has a more abnormal chromatin
much more difficult to evaluate cytologically as structure (uneven distribution, coarseness, clump-
are cells from the non-keratinizing epithelium ing), while at the same time the cytoplasmic vol-
including glandular tissue. In vulva smears, the ume decreases. This results in an increase in the
cell yield is usually much lower, and the mor- nuclear-to-cytoplasmic ratio (N/C ratio).
phological assessment of keratocytes may be
inconclusive.
If an abnormal or suspicious vulvar lesion
is seen, a punch biopsy should be performed to
rule out vulvar intraepithelial neoplasia (VIN)
or invasive carcinoma, as vulvar cytology may
yield a false negative result.

6.1.1 Cytological Morphology

The normal squamous epithelium of the cervix

consists of basal cells, parabasal cells, small and
large intermediate cells, and superficial cells.
Keratinization does not usually occur on the cer- Fig. 6.1 Superficial (eosinophilic = red) and large inter-
vical epithelium. mediate cells (cyanophilic = blue) of normal squamous
epithelium in a conventional cervical smear. The nuclei
In the cytological smear, with the exception of
are small with large cytoplasm. Erythrocytes have a diam-
basal cells, all the above-mentioned cell groups eter of approximately 7 μm and can be used to estimate
can be found. In non-atrophic epithelium con- cell size
6.1 Cytology 69

Table 6.1 summarizes the features of mild

a (CIN1), moderate (CIN2), and severe dysplasia
(CIN3) (Flenker 2003; Lellé et al. 2007a, b).
Figures 6.3, 6.4, 6.5, and 6.6 show examples
of the different degrees of dysplasia. It is impor-
tant to note that grading of dysplastic changes
always involves a certain amount of subjectivity
on the part of the cytopathologist. Also, it is not
uncommon for cells of different grades of dyspla-
sia to coexist within the same smear. Of course,
the final cytologic diagnosis is always based on
the highest grade of dysplasia observed.
b An unequivocal cytological diagnosis of
malignant disease is not possible. The morpho-
logical proof of cancer is the presence of inva-
sion, i.e. the presence of atypical cells within
the stroma after these cells have broken through
the basal membrane. Such a diagnosis is possi-
ble only on tissue sections and not on cytologi-
cal smears. Therefore, cytological diagnosis, or
rather suspicion of malignancy is primarily based
on the presence of severely abnormal cells, par-
ticularly those with prominent nuclear changes.
However, similar cells may be present in high
grade squamous dysplasia (CIN3).
Fig. 6.2 Endocervical cells in a conventional Pap smear. Therefore, cytological suspicion of malig-
When viewed from above, (a) endocervical cells form a nancy always includes indirect signs of poten-
honeycomb pattern. When viewed from the side, cells tial invasive disease such as fresh or hemolyzed
have a palisade-like appearance (b)
blood cells and cellular debris (Figs. 6.7 and

Table 6.1 Cytological features of the three grades of dysplasia (CIN1–3) (Flenker 2003; Lellé et al. 2007a, b)
CIN1 (LSIL) CIN2 (HSIL) CIN3 (HSIL)
Cytoplasm Similar to superficial cells Similar to intermediate cells Similar to parabasal cells
or large intermediate cell,
koilocytosis
Nucleus 2–3 times larger than in 2–3 times larger than in large Slightly smaller than in mild
large intermediate cells, intermediate cells, marked increase (CIN1) or moderate (CIN2)
irregular and of N/C ratio, irregular nuclei, dysplasia, further increase of
hyperchromatic; chromatin indentations of the nuclear N/C ratio, severe deformation
evenly distributed membrane, hyperchromasia with of the nucleus with indentations
slightly coarsened and granular and invaginations of the nuclear
chromatin membrane, severe invagination
of the nuclear membrane,
increased hyperchromasia with
coarse and granular chromatin
70 6 The Significance of Cytology, Biopsy, and HPV Testing

Fig. 6.6 Cells from severe squamous dysplasia (CIN3).

Fig. 6.3 Cells of low grade squamous dysplasia (CIN1). Compared to the superficial cell at the top of the image,
Superficial cells with enlarged nuclei with anisokaryosis the dysplasia cells are much smaller and have a basal- or
and pleomorphism. The cytoplasm has turned orange in parabasal-cell like appearance. The nuclei almost fill out
the Papanicolaou stain due to atypical keratinization the cells with only a narrow cytoplasmic margin remain-
ing. The dysplastic cells are arranged in a “cobblestone
road manner”

6.8). This so-called malignant or dirty back-

ground is due to tumor necrosis and called
tumor diathesis.1 Unfortunately, tumor diathe-
sis is not a very reliable indicator of malignancy.
As Rushing and Cibas (1997) point out, tumor
diathesis is only present in about half of cytologi-
cal smears obtained from squamous cell carcino-
mas. Also, there is a positive correlation between
the presence of tumor diathesis and the depth of
invasion, i.e. tumor diathesis is absent when the
Fig. 6.4 Typical koilocytes. There is bright cytoplasm depth of invasion is less than 5 mm.
around the nuclei. One of the koilocytes has two nuclei. In summary, the accuracy of cytological diag-
Koilocytes are the morphological sign of HPV infection and nosis of malignancy is far from that of histo-
are frequently associated with low grade dysplasia (CIN1)
logical assessment. This should always be kept
in mind when management decisions are made
based on cytologic findings.
Examples of dysplasia and carcinoma
depicted here all refer to squamous epithe-
lium. The cytological diagnosis of precan-
cerous lesions of the endocervical columnar
epithelium (AIS, adenocarcinoma in situ) as
well as invasive tumors (adenocarcinoma) is
much more difficult and is less accurate than
the cytological diagnosis of squamous lesions.
The morphology of abnormal cells from the
keratinizing epithelium is far more difficult to
Fig. 6.5 Cells from moderate squamous dysplasia (CIN2).
interpret compared to the non-keratinizing cervi-
Next to a red superficial cell with a slightly enlarged nucleus
are two smaller intermediate cells with enlarged nuclei and
nuclear pleomorphism. The N/C ratio is increased 1
“Diathesis” from Greek means “arrangement”.
6.1 Cytology 71

Fig. 6.9 Squamous cell of the vulva (ThinPrep® cytopa-

thology). The tumor cells, which are called “dyskerato-
cytes,” show prominent pleomorphism, both of the
nucleus and the cytoplasm, the latter sometimes taking on
bizarre cell forms. The N/C ratio shifted in favor of the
cytoplasm, and not the other way around as with squa-
mous epithelial dysplasia or carcinoma of non-keratinizing
epithelium, where the nucleus pushes the cytoplasm
towards the cell periphery

cal epithelium (Fig. 6.9). In Nauth’s textbook and

atlas “Gynecological Cytology” (Nauth 2007),
vulvar cytology is discussed in detail. Otherwise,
Fig. 6.7 Squamous cell carcinoma of the cervix. In this
conventional smear, the background is bloody and
the literature is sparse, and the discussion about
inflammatory (tumor diathesis). A group of tumor cells its relevance continues (Bae-Jump et al. 2007;
clings to the erythrocytes. The N/C ratio of these cell is Dennerstein 2009; Jimenez-Ayala and Jimenez-
increased Ayala 2002; van den Einden et al. 2012).
In summary, there is no cytological screening
of the vulva for the detection of vulvar intraepi-
thelial neoplasia or early vulvar cancer. If a sus-
picious lesion of the vulva is seen, a punch biopsy
should be obtained rather than a Pap smear.

6.1.2 Cytological Nomenclature

In the USA as well as in many other countries

worldwide, the original cytological classification
system using five Pap groups has been replaced
by the Bethesda classification first established in
1988 (Table 6.2), last updated in 2014 (Nayar and
Fig. 6.8 Squamous cell carcinoma of the cervix. Tumor Wilbur 2015). Instead of Pap groups, emphasis is
cell nuclei are enlarged with prominent macronucleoli. placed on a descriptive morphological diagnosis.
Characteristics of isolated cells may be identical to those
Instead of the designations CIN1 to CIN3, the
of severe dysplasia (CIN3) (Table 6.1). The cytological
suspicion of malignancy is based on a subjective impres- term “squamous intraepithelial lesion” (SIL) is
sion of an experienced cytopathologist used, which applies not only to squamous precan-
72 6 The Significance of Cytology, Biopsy, and HPV Testing

Table 6.2 The 2014 Bethesda system for reporting cervical cytology (Nayar and Wilbur 2015)
Specimen type
Indicate conventional smear (Pap smear) vs. liquid-bused preparation vs. other
Specimen adequacy
• Satisfactory for evaluation (describe presence or absence of endocervical/transformation zone component and
any other quality indicators, e.g., partially obscuring blood, inflammation, etc.)
• Unsatisfactory for evaluation … (specify reason)
– Specimen rejected/not processed (specify reason)
– Specimen processed and examined, but unsatisfactory for evaluation of epithelial abnormality because of
(specify reason)
General categorization (optional)
• Negative for Intraepithelial Lesion or Malignancy
• Other: See Interpretation/Result (e.g., endometrial cells in a woman ≥45 years of age)
• Epithelial Cell Abnormality: See Interpretation/Result (specify ‘squamous’ or ‘glandular’ as appropriate)
Interpretation/result
Negative for intraepithelial lesion or malignancy
(When there is no cellular evidence of neoplasia, state this in the General Categorization above and/or in the
Interpretation/Result section of the report--whether or not there are organisms or other non-neoplastic findings)
Non-neoplastic findings (optional to report optional to report: List not inclusive)
• Non-neoplastic cellular variations
– Squamous metaplasia
– Keratotic changes
– Tubal metaplasia
– Atrophy
– Pregnancy-associated changes
• Reactive cellular changes associated with:
– Inflammation (includes typical repair)
Lymphocytic (follicular) cervicitis
– Radiation
– Intrauterine contraceptive device (IUD)
• Glandular cells status post hysterectomy
Organisms
• Trichomonas vaginalis
• Fungal organisms morphologically consistent with Candida spp.
• Shift in flora suggestive of bacterial vaginosis
• Bacteria morphologically consistent with Actinomyces spp.
• Cellular changes consistent with herpes simplex virus
• Cellular changes consistent with cytomegalovirus
Other
• Endometrial cells (in a woman ≥ 45 years of age)
(Specify if “negative for squamous intraepithelial lesion”)
Epithelial cell abnormalities
Squamous cell
• Atypical squamous cells
– of undetermined significance (ASC-US)
– cannot exclude HSIL (ASC-H)
• Low-grade squamous intraepithelial lesion (LSIL)
(encompassing: HPV/mild dysplasia/CIN 1)
6.1 Cytology 73

Table 6.2 (continued)

• High-grade squamous intraepithelial lesion (HSIL)
(encompassing: moderate and severe dysplasia, CIS; CIN 2 and CIN 3)
– with features suspicious for invasion (if invasion is suspected)
• Squamous cell carcinoma
Glandular cell
• Atypical
– endocervical cells (NOS or specify in comments)
– endometrial cells (NOS or specify in comments)
– glandular cells (NOS or specify in comments)
• Atypical
– endocervical cells, favor neoplastic
– glandular cells, favor neoplastic
• Reactive cellular changes associated with:
– Inflammation (includes typical repair)
• Lymphocytic (follicular) cervicitis
– Radiation
– Intrauterine contraceptive device (IUD)
• Glandular cells status post hysterectomy
Organisms
• Trichomonas vaginalis
• Fungal organisms morphologically consistent with Candida spp.
• Shift in flora suggestive of bacterial vaginosis
• Bacteria morphologically consistent with Actinomyces spp.
• Cellular changes consistent with herpes simplex virus
• Cellular changes consistent with cytomegalovirus
Other
• Endometrial cells (in a woman ≥45 years of age)
(Specify if “negative for squamous intraepithelial lesion”)
Epithelial cell abnormalities
Squamous cell
• Atypical squamous cells
– of undetermined significance (ASC-US)
– cannot exclude HSIL (ASC-H)
• Low-grade squamous intraepithelial lesion (LSIL)
(encompassing: HPV/mild dysplasia/CIN 1)
• High-grade squamous intraepithelial lesion (HSIL)
(encompassing: moderate and severe dysplasia, CIS; CIN 2 and CIN 3)
– with features suspicious for invasion (if invasion is suspected)
• Squamous cell carcinoma
Glandular cell
• Atypical
– endocervical cells (NOS or specify in comments)
– endometrial cells (NOS or specify in comments)
– glandular cells (NOS or specify in comments)
• Atypical
– endocervical cells, favor neoplastic
– glandular cells, favor neoplastic
(continued)
74 6 The Significance of Cytology, Biopsy, and HPV Testing

Table 6.2 (continued)

• Endocervical adenocarcinoma in situ
• Adenocarcinoma
– endocervical
– endometrial
– extrauterine
– not otherwise specified (NOS)
Other malignant neoplasms: (specify)
Adjunctive testing
Provide a brief description of the test method(s) and report the result so that it is easily understood by the clinician
Computer-assisted interpretation of cervical cytology
If case examined by an automated device, specify device and result
Educational notes and comments appended to cytology reports (optional)
Suggestions should be concise and consistent with clinical follow-up guidelines published by professional
organizations (references to relevant publications may be included)

cer of the cervix but also to preinvasive lesions of Table 6.3 German system for reporting cervical cytol-
the vagina, vulva, and anus. ogy using the traditional Pap groups in relation to the
Bethesda system
Furthermore, squamous intraepithelial lesions are
subdivided into LSIL (low grade squamous intraepi- Pap group Bethesda
0 Unsatisfactory for evaluation
thelial lesion) and HSIL (high grade squamous
I NILM
intraepithelial lesion). This dichotomy satisfies the
II-a NILM
clinical requirements better than the classification II-p ASC-US
of CIN1 to CIN3, which has been in use since the II-g AGC endocervical NOS
1970s. LSIL therefore stands for minor cell abnor- II-e Endometrial cells
malities that need to be followed rather than treated III-p ASC-H
surgically, whereas HSIL may require treatment. III-g AGC endocervical favor neoplastic
Another advantage of the Bethesda classification III-e AGC endometrial
is the fact that the same nomenclature can be applied III-x AGC favor neoplastic
to both cytological and histological findings. IIID1 LSIL
IIID2 HSIL
Occasionally, HSIL and CIN3 are called “car-
IVa-p HSIL
cinoma in situ,” especially when the mitotic activ-
IVa-g AIS
ity is high, and the basal-type dysplastic cells are IVb-p HSIL with features suspicious for invasion
not stratified anymore. In carcinoma in situ, the IVb-g AIS with features suspicious for invasion
basal membrane is still intact. Consequently, it is V-p Squamous cell carcinoma
not a “carcinoma.” Moreover, due to its potential V-g Endocervical adenocarcinoma
for spontaneous regression, albeit rarely, the term V-e Endometrial adenocarcinoma
“carcinoma in situ” is not justified. Unfortunately, V-x Other malignant neoplasms
“squamous cell carcinoma in situ” is still men- p squamous, g glandular (endocervical), e endometrial, x
tioned as “acceptable terminology” for “HSIL unknown. II-a patient with a history of previous abnormal
cytological/histological/colposcopic findings (Griesser
(CIN3)” in the 2020 WHO classification update et al. 2013)
(WHO Classification of Tumours Editorial Board
2020). Also, TNM and FIGO staging systems rec- Designations such as p (“plattenepithelial” = squa-
ognize “in situ cancer” as Tis (TNM) and stage 0 mous), g (glandular/endocervical), e (endometrial),
(FIGO), respectively (Compton et al. 2012). and x (unknown) are used for the presumed origin
Some countries such as Germany have not of cells. This may potentially overstretch the accu-
adopted the Bethesda classification. Based on the racy of cytological diagnosis. In contrast to the
traditional Papanicolaou Pap groups I to V, an update Bethesda classification, the Pap group system tries
has been published in 2013 called the Munich III to make a clear distinction between CIN2 and CIN3
classification (Griesser et al. 2013) (Table 6.3). (Pap IIID2 and Pap IVa-p).
6.1 Cytology 75

If a cytological sample is classified as Pap This is also demonstrated by Marquardt et al.

IVa-p, there is indeed a very high likelihood that (2007, 2011). The previous Pap smear history
CIN3 is present. According to Hilal et al. (2015), from 617 patients, who were eventually diag-
in 22 out of 25 patients CIN3 was confirmed nosed with invasive cervical cancer, was docu-
histologically (85%). Marquardt and Ziemke mented. Most of these cancer patients (60%) had
(2018), who analyzed 4162 Pap smears classi- not had a Pap smear taken within the previous
fied as abnormal according to Munich III from 5 years. In 31% of the patients, gynecological
3396 women, found similar results: Pap IVa-p examinations had been done at irregular inter-
had a high specificity of 87.47% for CIN3+ and vals. However, 9% of patients with a cervical
98.07% für CIN2+. Compared to Pap IVa-p, the cancer diagnosis participated regularly in the
respective percentages for Pap IIID2 (suspect- annual screening program.
ing CIN2) were 45.80% and 62.44% and for Another retrospective analysis of cytology
Pap IIID1 (suspecting CIN1/LSIL) 17.07% and examinations prior to the histopathological diag-
9.32%. The CIN2+ group of Pap IVa-p consisted nosis of “carcinoma in situ” (CIN3) was done by
of 10.60% histologically confirmed CIN2 lesions, Bergeron et al. (1997). Whereas the smear imme-
84.82% CIN3, 0.24% AIS, and 2.4% squamous diately preceding the biopsy-proven diagnosis of
cell carcinomas. CIS had been positive in 518 out of 585 women
One should be aware that a clear distinction (88.5%), 20% of previous smears were false neg-
between CIN2 and CIN3 is difficult, both cyto- ative as they were found to be positive for HSIL
logically and histologically. As no evidence exists on review.
that CIN2 can be considered a distinct intermedi- Doctors, patients, and cytopathologists
ate state between CIN1 and CIN3, it may as well tend to overestimate the sensitivity of exfolia-
be a mix of true CIN1 and CIN3 (Kurman et al. tive cytology.
2014; WHO Classification of Tumours Editorial Despite its proven effectiveness in secondary
Board 2020). Despite these limitations, an effort cervical cancer prevention, cytological screening
should be made to identify CIN2 as a subgroup has a surprisingly low sensitivity. A meta-analysis
with a certain potential for spontaneous regres- by Cuzick et al. (2006) found a sensitivity of only
sion, thus limiting overtreatment especially in 53%, whereas specificity was 96%. The authors
younger patients with the intention to avoid conclude that purely cytology-based screening
pregnancy-related complications. can only be effective if two or three consecutive
annual examinations are performed before the
screening interval may be extended. Statistically,
6.1.3 Cytology as a Screening Tool sensitivity (based on the detection of CIN2+
lesions) will improve from 53% to 76%, when
The effectiveness of cervical cytology, i.e. its two consecutive Pap smears are obtained and
sensitivity and specificity, is far from ideal. improve up to 88% after three Pap tests.
Screening error is estimated to be between 10% Due to the low sensitivity of the cytological
and 67%, and sampling error occurs at a rate of examination, every effort must be made to mini-
20% to 39% (quoted from Bergeron et al. 1997). mize potential sources of error.

• The following factors can be the cause of false negative cytological results:
– Sampling from the wrong area.
– No intracervical collection.
– Insufficient transferral of cells to the slide.
– Blood.
– Inflammation.
– Poor fixation.
– Interpretation error.
76 6 The Significance of Cytology, Biopsy, and HPV Testing

The choice of the sampling device plays an – The cervical broom.

important role in the quality and representative- – A spatula (wooden or plastic) in combination
ness of the cell sample. In cervical screening, with an endocervical brush.
both the ectocervix and the endocervix need to – The extended tip spatula.
be sampled, in order to cover the entire trans-
formation zone. The European Guidelines for There are certain national and certain indi-
Quality Assurance in Cervical Cancer are giv- vidual preferences regarding the use of sampling
ing recommendations on sampling methods devices. For example, the extended tip spatula is
(Arbyn et al. 2007). The use of a cotton tip the preferred device in the UK.
applicator is considered inappropriate. Instead, Another device used is the PapCone®, a soft
three devices or combination of devices are rec- polyurethane foam swab which is compressible
ommended (Fig. 6.10): and can be used for both ectocervical and endo-
cervical sampling (Petruzziello et al. 2011). When
there is any doubt that a representative intracervical
a b c d sample has been obtained, the additional use of the
cervical brush is recommended. Conversely, the
endocervical brush should not be used by itself.
The presence of a cellular component from
the transformation zone (endocervical and/or
metaplastic cells) is a valuable quality assur-
ance measure. However, it is not required for
the adequacy of each individual sample.
Several studies (Elias et al. 1983; Luzzatto and
Boon 1996) showed that the presence or absence
of cellular transformation zone components cor-
relates with the reliability of the detection of
dysplasia, since dysplastic cells from squamous
epithelium always occur within the transfor-
mation zone, which may be located partially or
completely within the cervical canal. However,
women with Pap smears lacking the endocervi-
cal/metaplastic component do not develop cervi-
cal dysplasia more frequently (Bos et al. 2001;
Kivlahan and Ingram 1986). The Bethesda cyto-
logical terminology takes this into account (Nayar
and Wilbur 2015). The presence or absence of
the transformation zone components should still
be reported as a quality indicator. However, its
absence should not lead to early repeat Pap smear
testing as can be concluded from a Canadian
meta-analysis (Elumir-Tanner and Doraty 2011).
Fig. 6.10 Three of the cytological sampling devices that If there already is a cytological suspicion of
are recommended by the European Guidelines for Quality high grade dysplasia (HSIL) or even carcinoma,
Assurance in Cervical Cancer Screening as well as the so- the presence or absence of a transformation
called PapCone®. (a) extended tip (Szalay) spatula (top)
and Ayre spatula (bottom). (b) endocervical brush. (c) cer-
zone component is irrelevant. Also, HPV testing
vical broom (Cervex® brush). (d) PapCone® (Arbyn et al. in women 30 years and older is independent of
2007; Kaur and Kushtagi 2013; Petruzziello et al. 2011) transformation zone sampling.
6.1 Cytology 77

6.1.4 Liquid-Based Cytology Not all LBC procedures that are available
have been adequately tested in clinical tri-
Liquid-based cytology (LBC) has become als. Although approval by the Food and Drug
increasingly popular over the last 20 years. Many Administration (FDA) is not required outside
cytology labs are using LBC as an alternative to of the USA, the use of FDA-approved methods
conventional cytology. Most studies show that its only should be considered.
performance is at least as good as the standard In Europe no institution comparable to the
methodology. The major advantage of LBC is FDA exists, and there are no binding approval
the fact that several cell samples can be obtained criteria for medical devices to assure that only
from one vial and then used for additional stud- scientifically tested methods are used. Bollmann
ies such as HPV testing or immunocytochemical et al. (2006) and Bollmann and Jordan (2005)
analyses. have tried to establish criteria for the use of LBC
methods in cytological screening. These criteria
[Link] Principle of LBC are fulfilled by both ThinPrep® and SurePath®.
Several methods of cell preparations for LBC Both cell preparation systems have been stud-
are available. They all use the same principle, ied extensively and are the only two procedures
i.e. transfer of the cell sample into a vial with which obtained FDA approval (in 1996 and 1999,
a preservative medium by rinsing the collection respectively). The FDA accepted that these tech-
device instead of spreading cells directly onto a niques were significantly more effective com-
glass slide. Thus, cell fixation is optimized and pared with conventional Pap smear (ThinPrep®)
there are no drying artifacts. or at least of similar efficiency (SurePath®) (Gibb
For Pap testing, only an aliquot from the vial and Martens 2011). Furthermore, both tests were
is needed. A representative cell sample is pre- recognized by the FDA to improve specimen ade-
pared and transferred onto a glass slide. There are quacy and HSIL detection.
several automated or semi-automated methods Several alternative LBC methods have been
for cell preparation, some of which are described developed using similar slide preparation meth-
below. The two major principles of cell prepara- ods as ThinPrep® or SurePath®. However, none of
tion are centrifugation and filtration. these methods has been studied on large cohorts
LBC has the potential to significantly improve of patients and therefore are not FDA approved.
the quality of the Pap test. Almost the entire cell Although these methods are legal in many coun-
specimen is transferred into the vial by rinsing tries, they should not be used.
the collection device. Blood cells, inflamma- Presently, LBC (ThinPrep® or SurePath®) is
tory cells, or mucus, which may all interfere the preferred methodology for cytological test-
with cytological evaluation, are dissolved in the ing in the USA. Also, the British National Health
transport medium or removed by filtration. LBC System has been officially using ThinPrep® and
also standardizes the distribution of cells onto the SurePath® testing since 2004.2
glass slide. Ideally, a cell sample resembling a The two FDA-approved LBC methods,
monolayer is achieved. ThinPrep® and SurePath®, are described in more
detail below.
[Link] LBC Procedures
Several LBC procedures have been developed. [Link].1 ThinPrep® (Hologic)
These are all based on one or more of the follow- ThinPrep® specimen preparation is based on
ing principles: filtration. The cells are collected as described

– filtration 2
[Link]
– density gradient centrifugation uploads/system/uploads/attachment_data/file/436647/
– cell sedimentation [Link].
78 6 The Significance of Cytology, Biopsy, and HPV Testing

Fig. 6.12 ThinPrep® filter after cell preparation. After

stamping the filter onto the slide, some of the cells still
remain on the filter membrane. The filter pores are gener-
ated by shooting neutrons through a membrane. Thus they
all have the same diameter

Fig. 6.11 ThinPrep® 2000 processor

above. The FDA certified collection device for

ThinPrep® is the Cervex Brush®, which is rinsed
in the so-called PreservCyt® medium, which con-
tains methanol. However, its precise composition
has not been published by Hologic.
In the cytologic laboratory, the cell sample is
processed by the ThinPrep® processor (Fig. 6.11),
a semi-automated slide preparation unit. A plas-
tic cylinder, which is closed at the bottom with a
filter membrane, is used for each sample. These
filters are manufactured by shooting neutrons
Fig. 6.13 ThinPrep® glass slide. The enlarged area shows
through a membrane, thus producing irregularly a typical cell distribution after stamping the cylinder onto
distributed pores, which all have the same diam- the glass slide with conglomerates of cells and cell-free
eter (Fig. 6.12). areas. Therefore, the term “monolayer cytology” is not
The filter is immersed into the vial and rotated really appropriate
to produce a homogenized cell solution. Larger
cell conglomerates are dissolved. A vacuum is a sufficient amount of cells adheres to the filter.
then applied, and cells are aspirated onto the The filter is then pressed against the glass slide
filter surface. Smaller particles such as erythro- in order to transfer the cell sample into a 20 mm
cytes, leukocytes, and part of the cell detritus are circle (Fig. 6.13). Finally, the slide is ejected into
sucked through the filter pores and do not reach the fixative and stained in the usual manner.
the slide. Some of the cells adhere to the filter
membrane and close the pores. The ThinPrep® [Link].2 SurePath® (Becton Dickinson)
unit measures the relaxation time of the vacuum. In contrast to rinsing the collection devices in the
These steps—aspiration and measurement of transport medium as with the ThinPrep® tech-
relaxation times—are repeated several times until nique, SurePath® requires to leave the tip of the
6.1 Cytology 79

collection devices in the vial in order to increase Table 6.4 Comparison of the two validated and FDA-
approved LBC methods
cell yield.
The storage medium contains ethanol. Its ThinPrep® SurePath®
exact composition has not been published by Manufacturer Hologic BD (Becton,
Dickinson and
Becton Dickinson. The cell preparation process Company)
is quite different from that of the ThinPrep® Website [Link] [Link]
method. SurePath® uses repeated density gradi- Technical Filtering via a Density gradient
ent centrifugation with subsequent sedimenta- principle computer- centrifugation and
tion instead of filtration (Figs. 6.14 and 6.15). controlled sedimentation
vacuum
During the first centrifuge step, mucus, erythro-
Cell Sampling device Sampling device is
collection is rinsed in the left in the vial
vial and then
removed
Computer- Location guided Ranking of slides
ized screen- screening (FocalPoint
ing option (ThinPrep® imaging)
imager)

cytes, and leucocytes are removed. Slide prepa-

ration is automated and carried out by a slide
processor.
Table 6.4 compares the properties of the two
liquid-based cytological procedures ThinPrep®
and SurePath®.

[Link] Morphological Features

of Liquid-Based Cytology
Fig. 6.14 SurePath® slide. The circular cell area is The staff performing cytological assessment on
smaller than that of the ThinPrep® procedure. The cell conventional Pap smears must be trained when
sample is obtained by sedimentation. Cell aggregates are
still present LBC is introduced in the cytology laboratory.
Furthermore, when changing the LBC method,
formal training is required again, as there are
important differences in the morphology of cells
and cell elements. A lack of appropriate knowl-
edge may result in screening errors. Some of
these morphological peculiarities are explained
below for ThinPrep®.
A purified but still representative sample of the
cell material is transferred to the slide. Although
LBC is frequently called “monolayer cytology,” a
preparation of individual cells lying side by side
without any overlap is rarely achieved. Instead,
small and large cell conglomerates are seen,
which are usually not as thick as in conventional
smear. The expression “thin layer” is therefore
Fig. 6.15 Normal squamous epithelium using the
more appropriate than “monolayer.”
SurePath® method. In this example, a cellular monolayer
is almost achieved. However, the terms “liquid-based Compared to conventional smears, cell num-
cytology” or “thin layer cytology” are more appropriate bers and cell density are lower with ThinPrep®.
80 6 The Significance of Cytology, Biopsy, and HPV Testing

Therefore, the diagnosis of dysplasia may be fixation of the cell sample. This is especially
based on a rather small number of diagnostic true for preservation of the chromatin structure
cells. ThinPrep® preparations have to be screened (Figs. 6.18 and 6.19).
extra careful and more slowly than conventional
slides (Fig. 6.16). The potential disadvantage of
a longer screening time per slide is offset by the
fact that a smaller area has to be assessed.
Although LBC procedures remove part of the
background material, detection of specific inflam-
matory changes such as mycosis (Fig. 6.17) or
trichomonas is as good as in conventional cytol-
ogy. Sometimes these microbiological elements
may even be more prominent against the “clean”
background compared to conventional cytology
(Renshaw et al. 2004).
The recognition of HPV-related as well as
dysplastic cell changes is facilitated by optimal

Fig. 6.17 Candida infection ThinPrep®. Hyphae and

spores are easily recognized

Fig. 6.16 HSIL/CIN3 ThinPrep®. The background is

clean. Most of the cells are from the intermediate and
some from the superficial layer of normal squamous epi- Fig. 6.18 HSIL/CIN3 ThinPrep®. The pleomorphic
thelium. In between are a few small conglomerates of nuclei have a well-defined chromatin structure. Many
basal-type dysplastic cells, which might be overlooked nuclei are hyperchromatic
6.1 Cytology 81

Fig. 6.19 Adenocarcinoma in situ (AIS) ThinPrep®. The

chromatin structure of the abnormal endocervical cells is
well preserved. Macronucleoli and chromocenters are
easily visible

Cells tend to be slightly smaller and rounder

than in conventional Pap smears, due to round-
ing of the cells when transferred into the liquid
medium. Therefore, cells of squamous epithelial
dysplasia may sometimes be misinterpreted as
glandular (Renshaw et al. 2006). However, the
N/C ratio remains unchanged.
Also, the cytoplasmic details are better visible
in LBC, which is helpful for diagnosis of HPV-
related changes such as koilocytosis. Some diag- Fig. 6.20 Squamous cell carcinoma ThinPrep®.
nostic patterns that result from manual spreading “Clinging tumor diathesis” is seen consisting of a “dirty”
of cells onto the glass slide as, for example, the background of hemolyzed blood and cellular debris that
are attached to the tumor cells
arrangement of CIN3 cells in a cobblestone road
manner (Fig. 6.6) are not seen in LBC.
Most importantly, tumor diathesis is preserved from squamous cell carcinomas and hypoth-
in liquid-based slide preparations, although it has a esize that cellular particles of tumor diathesis
slightly different morphological appearance com- and inflammation may decrease cellularity by
pared to conventional cytology. Tumor diathesis, blocking filter coverage by epithelial cells. They
i.e. a “dirty” cell background due to hemolyzed suggest that low cellularity in combination with
blood and cellular debris, is helpful to establish tumor diathesis is a useful clue for the diagnosis
the cytological diagnosis of cancer when highly of squamous cell carcinoma.
abnormal and possibly malignant cells are present. As stated above, the presence of endocervical
Squamous cell carcinoma typically presents with cells is considered to be a marker for adequate
“clinging tumor diathesis” as blood and cellular cervical cell sampling and is correlated with the
debris are attached to epithelial cells (Fig. 6.20). detection of dysplasia (Elias et al. 1983; Luzzatto
A study by the College of American Pathologists and Boon 1996). Similarly, the presence of meta-
comparing conventional and ThinPrep® educa- plastic cells also indicates adequate cell collec-
tional slides (Renshaw et al. 2004) also suggested tion from the transformation zone. Compared
some difficulty of participants to identify squa- to conventional slides, ThinPrep® slides more
mous cell carcinoma on ThinPrep® slides. frequently do not contain endocervical or meta-
Clark and Dawson (2002) observed a pattern plastic cells (Lellé et al. 2007a, b): 11.6% versus
of decreased cell coverage in ThinPrep® slides 2.3%, respectively. This may partly be due to the
82 6 The Significance of Cytology, Biopsy, and HPV Testing

so-called split-sample bias, as in these studies for ThinPrep® cytology compared to 38% for the
conventional smears are obtained first, and sub- conventional Pap test.
sequently, the cell collection device is rinsed in However, on the basis of several meta-
the liquid medium. analyses, these advantages of liquid-based cytol-
However, ThinPrep® cytological samples ogy over conventional cytology have been called
without endocervical or metaplastic cells are not into question (Arbyn et al. 2008; Davey et al.
necessarily less sensitive. According to Selvaggi 2006; Sawaya 2008; Sawaya and Sox 2007;
and Guidos (2002), ThinPrep® smears without an Siebert et al. 2003).
endocervical component are diagnosed with dys- Macharia et al. (2016) noted, that when using
plasia as frequently as smears containing endo- colposcopy as the gold standard for further triage,
cervical cells. This also applies to the detection of LBC also has a higher specificity than conven-
glandular lesions (Ashfaq et al. 1999). The FDA tional Pap tests. They therefore recommend that
has even attributed the ThinPrep® procedure an for repeat Pap tests liquid-based cytology is used.
increased detection rate of abnormal glandular Another study using the Dutch registry for
cells. histopathology and cytology analyzed data from
While the discussion on the importance of 3,118,685 conventional Pap smears, 1,313,731
endocervical cells in ThinPrep® slides is still SurePath® samples, and 1,584,587 ThinPrep®
ongoing, the routine use of the SurePath® pro- samples (Rozemeijer et al. 2015). Compared
cedure in the UK has revealed a statistically to conventional cytology, SurePath® led to an
significant correlation between the presence of increased probability to detect CIN2+. However,
cell components of the transformation zone and the number of CIN1 diagnoses also increased
the diagnosis of dysplastic changes (Narine and resulting in overdiagnosis.
Young 2007).
[Link] Computer-Aided Cytological
[Link] Comparison of Conventional Evaluation
Cytology, ThinPrep®, Computer-aided evaluation systems have been
and SurePath® developed to increase the accuracy of cytological
Numerous studies suggest that ThinPrep® and examinations as well as overall efficiency. Such
SurePath® identify high grade dysplasia signifi- systems exist for conventional cytology as well as
cantly more often than conventional cytology and for ThinPrep® and SurePath® (Ikenberg 2011). The
reduce the number of unsatisfactory samples. BD FocalPoint GS Imaging System is able to ana-
The so-called Rhine-Saar study (Klug et al. lyze both SurePath® and conventional Pap smears.
2012) is of particular relevance for Germany with A computer algorithm assigns the cytology
non-organized opportunistic screening and evalu- samples to specific groups according to the statis-
ation of samples by a large number of different tical probability that abnormal cells are present.
cytology laboratories operated by gynecologists 25% of slides that are assigned to the category
or pathologists without centralized quality con- with the lowest risk do not need to be reviewed.
trol. A total of 20 gynecologists in private prac- Thus, the laboratory staff can focus on the high-
tice participated in this study. Overall, 20,627 est risk groups and review those slides more
women were examined. At weekly intervals, thoroughly.
conventional smear preparation and ThinPrep® Hologic has developed the ThinPrep® Imaging
procedure were alternated. For positive cyto- System. It uses an automated microscope attached
logical findings, colposcopy was performed, to a computer (Fig. 6.21) that uses algorithms,
and the detection rates of the two methods were which results in location guided screening. After
compared with respect to CIN2+ lesions. The the imager has screened the entire slide, 22 areas
relative sensitivity of ThinPrep® cytology was of special interest are marked. These areas are
significantly higher by a factor of 2.74. The posi- reviewed manually. Only when abnormal cells
tive predictive value for CIN2+ lesions was 48% are detected, the entire slide is reviewed.
6.1 Cytology 83

Both the FocalPoint GS Imaging System® and

the ThinPrep® Imager are at least as sensitive as
manual screening and make the workflow more effi-
cient. Studies with both systems provide evidence
of a higher sensitivity for detection of high grade
cervical dysplasia without increasing the number
of ASC-US or LSIL (Wilbur et al. 2009; Davey
et al. 2007), whereas other authors found equal
HSIL detection rates for the ThinPrep® Imaging
System without increasing ASC-US or AGUS rates
(Quddus et al. 2009; Thrall et al. 2008).
The Rhine-Saar study cited above (Klug et al.
2012), which showed a higher detection rate of
CIN2+ lesions by ThinPrep®, also included an
imager study arm. No additional improvement
in the detection rate of HSIL lesions (CIN2+)
could be demonstrated under the conditions of
the German screening system.
Computerized optimization of cytology-based
screening will become increasingly important, as
the HPV vaccine effect will decrease the preva-
lence of cervical dysplasia. Statistically, this will
reduce the predictive value of the screening test.
Fig. 6.21 ThinPrep® Imaging System. A computer algo- In other words, dysplastic cell changes may be
rithm marks 22 visual fields, which may contain abnormal
cells and need to be reviewed manually
overlooked more easily.

Summary: Liquid-Based Cytology

The aim of liquid-based cytology is to improve the quality of cell preparations by

– increasing the number of cells transferred from the collection device to the transport medium
– optimizing cell distribution on the slide (thin layer)
– removal of mucus, blood, and cell debris
– optimal fixation without drying artifacts

Two LBC methods are available: ThinPrep® based on cell filtration and SurePath® based on
centrifugation and sedimentation. Both methods have been extensively studied and approved by the
American Food and Drug Administration (FDA).
With liquid-based cytology, several cell samples can be obtained so that additional tests can
be carried out, such as testing for human papillomavirus (“reflex testing”).
The superiority of LBC over conventional cytology by diagnosing more high grade lesions
is controversial in the literature. For the German screening system, the Rhine-Saar study
demonstrated a significantly higher detection rate for CIN2+ lesions using the ThinPrep®
method. However, a computer-assisted evaluation using the ThinPrep® imager did not result in
further improvement of sensitivity.
Computer-assisted evaluation methods, which are available for both LBC methods and conven-
tional cytology, will become increasingly important in the future as the prevalence of high grade
dysplasia decreases due to the anticipated effects of HPV vaccination.
84 6 The Significance of Cytology, Biopsy, and HPV Testing

6.1.5 Adjunctive Cytological Tests to establish the correct morphological diagnosis.

Conversely, aneuploidy may be used as a progres-
According to Pinto et al. (2012), it is likely that a sion marker. According to Bocking and Nguyen
better understanding of HPV effects on cell cycle (2004) and Bocking et al. (2004), DNA euploidy
control will one day lead to the “ideal biomolecu- has a high negative predictive value of around
lar Papanicolaou test.” Numerous adjunctive tests 95% regarding progression of LSIL. Conversely,
are already available which complement morpho- DNA aneuploidy has a positive predictive value
logical evaluation of a cytological sample with of up to 100% with respect to the development of
Papanicolaou staining. However, none of these CIN3 within a follow-up period of 3 years.
tests is yet incorporated in screening algorithms. Nghiem et al. (2015) calculated that DNA
From the many adjunctive cytological mark- ploidy analysis may be more cost-effective for
ers that are available, three tests will be discussed cervical screening than liquid-based cytology. As
in more detail: DNA ploidy analysis, HPV L1 measurements of ploidy can be automated and do
capsid protein detection, and p16/Ki67 dual not need the highly skilled staff required for cyto-
stain (CINtecPLUS®). The most important of logical screening, the authors suggest that it may
all biomarkers, HPV testing, will be discussed be a promising alternative, feasible also in a low
separately. resource setting.
All three methods can be carried out on con-
ventional cytological preparations after destain- [Link] HPV L1 Capsid Protein
ing of the slide. With liquid-based cytology Detection
additional slides can be obtained from the cell The L1 protein is a major part of the HPV virus
suspension, so that the original slides remain shell or capsid. The presence of the L1 capsid
available for morphological evaluation. protein can be determined immunocytochemi-
The biomarkers available should not yet cally on cytological samples (Cytoactiv test®,
be incorporated into routine clinical practice. Cytoimmun Diagnostics GmbH, Pirmasens,
Presently, there is no sufficient evidence to jus- Germany). If L1 is found to be positive in the
tify the use of these tests for primary screening case of HPV infection, the immune system is still
or triage. capable to produce antibodies against the virus.
As Pinto et al. (2012) point out, those If the L1 capsid is absent, cellular dysregulation
biomarkers need “to be evaluated in large may already be too advanced, and an adequate
prospective clinical trials with appropriate immune response is no longer possible. Thus, L1
colposcopic, histological, and clinical end- protein detection may discriminate between true
points as well as adequate follow-up.” cervical precancer and transient HPV infection.
Mehlhorn et al. (2013) studied women with a
[Link] DNA Ploidy Analysis positive HPV test and the cytological diagnosis
One of the first steps of carcinogenesis is genetic of LSIL. The patients were subsequently fol-
instability. Genetic instability results in cells lowed by colposcopic examinations and biopsies
with an aneuploid set of chromosomes. Several for up to 54 months. If L1 was positive, only 20%
methods are available for measuring ploidy of of low grade lesions progressed to CIN3 com-
a cell sample, such as Feulgen staining of the pared to 84%, if L1 was negative. With a cyto-
nucleic acids (Feulgen and Rossenbach 1924). logical HSIL/CIN2 diagnosis and L1 negativity,
Measurements are semi-automated using elec- only 5% of patients had a spontaneous remission
tronic image analysis. The area of the cell nucleus compared to 68% in the LSIL/L1 positive group.
is directly proportional to DNA content and L1 capsid protein detection on cytological
ploidy. Leukocyte nuclei, which have a diploid samples may be combined with a rapid human
set of chromosomes, are used for standardization. papillomavirus type 16 L1 specific antibody test
The degree of aneuploidy serves as an objec- to improve risk assessment and clinical man-
tive measure of dysplasia. This may be helpful agement of low risk dysplastic lesions, in order
6.1 Cytology 85

to avoid overtreatment, especially in women of For CINtecPlus® testing, Ki67 is visualized

reproductive age (Mehlhorn et al. 2014). by APAAP (alkaline phosphatase anti-alkaline
phosphatase) staining, resulting in a red nucleus.
[Link] p16/Ki67 Dual Stain For p16, which is located in the cytoplasm, DAB
(CINtecPlus®) (diaminobenzidine) immunostaining is used.
The CINtecPlus® test (Roche) or p16/Ki67 Positive cells have a brown colored cytoplasm.
dual stain may be the most interesting Pap test One single dual-stained cell in the cyto-
based assays which has been developed to date. logical sample already indicates cell cycle
CINtecPlus® is an immunocytochemical double dysregulation and oncogenic transformation
stain (p16 and Ki67), which is preferably done on (Figs. 6.22 and 6.23), and the CINtecPLUS®
thin layer cytological specimens. test is considered to be positive.
P16 (p16INK4a protein) is overexpressed in Nevertheless, the number of dual-stained cells
transforming HPV infections and cervical pre- is also significant as it correlates with the grade
cancer, whereas Ki67 is a marker of cell prolif- of dysplastic changes of the cervix (Yoshida
eration. Under physiological conditions, the p16 et al. 2011): The more p16/Ki67 positive cells are
protein serves as a cell cycle regulator that trig- present, the more likely is the diagnosis of high
gers a cascade of biochemical events which even- grade dysplasia.
tually lead to cell cycle arrest. In precancerous For the p16/Ki67 dual stain, intra- and interob-
lesions of the cervix, p16 is overexpressed. Ki67 server agreement is very good for both patholo-
is expressed in several phases of the cell cycle gists and cytotechnologists (Goh et al. 2017;
and indicates active proliferation. When cells McMenamin et al. 2017).
are found to express both p16 and Ki67 simul- Wentzensen et al. (2012) performed the
taneously, this is a clear indication of cell cycle CINtecPlus® test on patients referred for col-
dysregulation, as under normal circumstances, poscopy and found a statistically significant cor-
p16 positive cells are not able to proliferate. In relation between different grades of CIN and
normal cells, the expression of p16 and Ki67 CINtecPlus® positivity: When histopathology
is mutually exclusive. Simultaneous p16/Ki67 was found to be negative, 26.8% of cytological
positivity is associated with HPV-induced onco- preparations were positive compared to 46.5%
genic transformation. Therefore, a strong correla- for CIN1, 82.8% for CIN2, and 92.8% for CIN3.
tion exists between the presence of cells that are In addition, the CINtecPlus® test could
simultaneously positive for both biomarkers and improve the detectability of glandular lesions.
the diagnosis of high grade cervical disease. According to Ravarino et al. (2012), 92.5% of the

a b

Fig. 6.22 CINtecPlus® and HSIL/CIN3. Basal-type cells with increased N/C ratio indicate HSIL/CIN3 (a), and the
immunohistochemical double stain (b) is positive for both p16 (brown) and Ki67 (red)
86 6 The Significance of Cytology, Biopsy, and HPV Testing

a b

Fig. 6.23 CINtecPlus® and HSIL/CIN3. The atypical cells (a) are simultaneously positive for p16 and Ki67 (b)

Fig. 6.24 CINtecPlus® Women ≥30 years

Triage of patients HPV positive, NILM
screened HPV positive 100%
(HC2) and normal Pap
smear result (NILM) in
the Wolfsburg trial.
Almost all CIN2+
CINtecPlus® CINtecPlus®
diagnoses fall within the
negative positive
CINtecPlus® positive
75 % 25 %
group (Petry et al. 2011)

< CIN2 CIN2+ < CIN2 CIN2+

99 % 1% 68 % 32 %

cytologic preparations with glandular lesions are ity than for the HPV positive test (HC2): 78.7%
p16/Ki67 positive. versus 60.4% for ASC-US and 53.3% versus
Presently, three indications for the CINtecPlus® 15.6% for LSIL, respectively. Thus, the number
test are discussed: of colposcopy referrals for ASC-US and LSIL
triage could be greatly reduced by introducing a
– Triage of ASC-US and LSIL. CINtecPlus® based algorithm.
– Reduction of the number of colposcopy exams CINtecPlus® testing is also of value within a
within a system based on primary HPV primary HPV screening algorithm. Petry et al.
screening. (2011) investigated the CINtecPlus® test as part
– CINtecPlus® based primary screening. of the Wolfsburg project for the triage of patients
who had a positive HPV test with normal cyto-
Schmidt et al. (2011) have shown that the logical finding. About one quarter of these
CINtecPlus® test is able to identify ASC-US patients also had a positive CINtecPlus® result.
and LSIL patients with high grade dysplastic In one-third of these patients, high grade dyspla-
changes. These study results were updated in sia (CIN2+) was detected, compared to only 1%
2015 (Bergeron et al. 2015). Positive predictive if the CINtecPlus® test was negative (Fig. 6.24).
values for the presence of CIN2+ lesions were Similar results were found for the ATHENA
significantly higher for CINtecPlus® positiv- trial using the cobas® HPV test (Wright et al.
6.2 Biopsy 87

2017). Referral for colposcopy of all HPV

16/18 positive women combined with p16/
Ki67 dual stain triage of women positive for 12
other HPV genotypes detected by the cobas®
test provides the highest sensitivity for the
detection of CIN3+ lesions (86.8%; 95% CI:
81.9–90.8).
Wentzensen et al. (2017) also used HPV16 and
18 genotyping. Within a group of 7124 HPV positive
women, 315 CIN3 lesions were detected. 88.9% of
these patients were CINtecPlus® positive, whereas
only 55.9% were positive for HPV16 or 18.
Sun et al. (2018), who performed a meta-
analysis on p16/Ki67 dual immunostaining,
found a pooled sensitivity for the detection of
HSIL of 0.88 (95% CI 0.86–0.90) and a pooled
specificity of 0.58 (95% CI 0.56–0.60). The sen-
sitivity of the HPV test (0.94; 95% CI 0.93–0.96)
is comparable, whereas its specificity is lower
(0.32; 95% CI 0.29–0.34). Sun et al. therefore
suggest that CINtecPlus® should be evaluated as
an initial screening assay.

6.2 Biopsy

Colposcopy requires a basic understanding

of histopathological methods, particularly the
interpretation, validity, and reliability of cervi-
Fig. 6.25 “Der Krebs der Gebärmutter” (Cancer of the
cal biopsy, which plays a central role in col- Uterus) by Ruge and Veit in 1881 was the world’s first
poscopic triage. comprehensive monograph on cervical cancer (Ruge and
Early on, Rudolf Virchow (1821–1902), Veit 1881)
the father of anatomical histopathology, and
his contemporaries recognized the problem of ment diagnostics) was taken up by Carl Ruge
representativeness of tissue samples that are (1846–1926), a nephew of Virchow. Ruge took
only a few millimeters in size. It has been said care of numerous gynecological patients as
that early pathologists flatly refused to evaluate part of his general medicine practice in Berlin,
tissue fragments such as from uterine curettage. Germany (Becker 1979). In 1881, Ruge pub-
At the time it was argued that histopathologi- lished the world’s first comprehensive text-
cal diagnosis should only be made on the organ book on cervical cancer (Ruge and Veit 1881)
that has been removed completely. Eventually, (Fig. 6.25), which includes detailed histopatho-
this so-called “Stückchendiagnstostik” (frag- logical drawings (Fig. 6.26).
88 6 The Significance of Cytology, Biopsy, and HPV Testing

Cervical dysplasia is characterized as follows:

LSIL (CIN1)
– proliferation of the basal/parabasal cell layer
to no more than one-third of the epithelium
– mitotic figures confined to the lower one-third
of the epithelium, usually not abnormal
– differentiation/maturation towards the surface
of the epithelium with an increasing amount
of cytoplasm
– enlargement of nuclei with an increased N/C
ratio
– koilocytic atypia

HSIL (CIN2 and CIN3)

– less cytoplasmatic differentiation with exten-
sion of the proliferating cells up into the mid-
dle (CIN2) or the upper third (CIN3).
– mitotic figures more abundant, more fre-
quently abnormal, extending into the middle
or upper third of the epithelium (Fig. 6.27).
– increased nuclear size, irregular nuclear mem-
branes, and a further increase of the N/C ratio.

Unfortunately, the designation “carcinoma

Fig. 6.26 Some of the earliest drawings of cervical
in situ” (“Tis”) as a distinct form of high grade
biopsy specimens with invasive squamous cell carcinoma
(Ruge and Veit 1881) cervical dysplasia is still used according to the
official histopathological WHO classification
(Kurman et al. 2014; WHO Classification of
6.2.1 Histological Morphology Tumours Editorial Board 2020), not however by
FIGO (Pecorelli 2009).
Histopathological assessment of cervical lesions If cervical carcinoma is not yet visible mac-
is standardized and based on the WHO publica- roscopically, it is called microcarcinoma. Depth
tion “Female Genital Tumors” last published in of invasion should not exceed 5 mm, whereas the
2020 (WHO Classification of Tumours Editorial lateral extent of the tumor is no longer considered
Board 2020). Cervical dysplasia is subdivided (Bhatla et al. 2018). FIGO stage IA is subdivided
into three grades of severity, CIN1, CIN2, and into stage IA1 and IA2, depending on whether
CIN3, based on evaluation of hematoxylin-eosin the depth of invasion is <3 mm or ≥3 mm.
stained slides. As with all such grading systems, a For glandular preinvasive disease, only one
high degree of subjectivity is involved, especially grade of severity is defined: high grade cervical
since CIN constitutes a continuum of abnormal glandular intraepithelial neoplasia (HGCGIN) or
changes. According to the WHO classification, as it is more frequently called: adenocarcinoma
a system of LSIL and HSIL is both more bio- in situ (ACIS or AIS).
logically relevant and more reproducible than the Diagnosis of AIS on cytological slides can be
three-tier CIN1 to 3 terminology. challenging and is associated with a significant
6.2 Biopsy 89

Fig. 6.27 Severe squamous dysplasia (CIN3). The abnormal cells extend to the upper one-third with several mitotic
figures within the middle and upper third of the squamous epithelium (arrow)

Fig. 6.28 Adenocarcinoma in situ (AIS) of the cervix. The Pap smear shows a dense group of abnormal endocervical
cells with washed-out chromatin und fuzzy margins, which is adjacent to a normal superficial squamous cell.
Histopathology shows densely packed partially superimposed nuclei. Characteristically, areas of AIS are juxtaposed to
normal endocervical epithelium

degree of uncertainty. In comparison, AIS diag- surface or within endocervical glands. Nuclei
nosis on histopathological slides (Kurman et al. are enlarged with hyperchromatic and coarse
2014; WHO Classification of Tumours Editorial chromatin structure. Prominent nucleoli may
Board 2020) is less difficult (Fig. 6.28). be present. Usually, mitotic figures are found.
Manifestations of AIS are confined to areas AIS may be of the usual/endocervical type,
of pre-existing normal endocervical epithe- or it may show intestinal or endometrioid
lium. AIS may be located on the endocervical differentiation.
90 6 The Significance of Cytology, Biopsy, and HPV Testing

In contrast to carcinoma, in adenocarcinoma 6.31, and 6.32 show p16 staining examples in
in situ there is no desmoplastic stromal reaction, moderate and severe intraepithelial neoplasia
and no neoplastic glands are found beyond the and Fig. 6.33 in invasive squamous cell car-
deepest normal gland. cinoma. Moderate dysplasia (CIN2) may be
In invasive adenocarcinoma, a variety of sub- diagnosed, when the middle third is also p16
types are distinguished, including mucinous, positive. In severe dysplasia (CIN3), the entire
endometrial, clear cell, mesonephric, and villo- depth of the epithelium may be p16 positive
glandular adenocarcinoma. (Fig. 6.31).
According to the current FIGO staging rules Diagnosis and grading of cervical dysplasia
(Bhatla et al. 2018), the definition of microcarci- should by not be based exclusively on p16 stain-
noma as quoted above also applies to adenocarci- ing. According to a meta-analysis by Tsoumpou
nomas, even though the depth of invasion is more et al. (2009), biopsies with normal squamous
difficult to determine in glandular neoplasms as epithelium may be diffusely p16-positive in 2%
there is no basal membrane for reference. of cases (82% in CIN3 lesions). P16 positivity
in LSIL does not have any predictive value, i.e.
[Link] p16 (p16INK4a Protein) p16 is not a potential progression marker for low
Immunohistochemistry grade disease (Sagasta et al. 2016). Therefore, in
As has already been explained in connection LSIL/CIN1 lesions, p16 immunostaining should
with the p16/Ki67 dual stain, p16 is a cell-reg- be used in equivocal cases only.
ulating protein which is overexpressed in HPV Galgano et al. (2010) studied 1455 cervical
transformed cells under the influence of high risk biopsies and used the conventional slide diagno-
HPV-associated oncoproteins. The abnormal p16 sis as the gold standard for a consensus diagnosis
protein can no longer trigger cell cycle arrest. of three experienced pathologists. In comparison
Of the multitude of immunohistochemical to conventional slide evaluation, the immunohis-
markers, p16 has been found to be particu- tochemical diagnosis with p16 was more sensi-
larly useful in diagnosing and grading squa- tive, however less specific. Overall, staining for
mous intraepithelial dysplasia, especially when p16 was found to be a useful adjunct for identify-
only a limited amount of tissue is available as ing patients with CIN2+ lesions.
is the case with colposcopic biopsy material. Stoler et al. (2018) reported that through
Therefore, a number of pathology departments p16 immunostaining as an adjunct to hema-
are routinely using p16 immunohistochemistry toxylin and eosin staining, diagnostic accuracy
for tissue samples from the cervix. of cervical biopsies is higher. Both sensitivity
As in the CINtecPlus® test, immunoperoxidase (11.5%) and specificity (3.0%) are significantly
p16 staining is used, which results in a brown increased.
color of the cytoplasm, if p16 is overexpressed. Obviously, p16 immunohistochemistry can
Under normal conditions, only isolated squa- also be helpful in cervical screening of HIV
mous epithelial cells are p16 positive. In LSIL, infected women. As an adjunctive test, p16
only cells in the basal epithelium are stained. In immunostaining increases specificity and the
HSIL, also the middle (CIN2) and upper third positive predictive value of HPV and VIA for
(CIN3) are involved (Fig. 6.29). Figures 6.30, CIN2/CIN3 (McGrath et al. 2017).
6.2 Biopsy 91

normal CIN1 CIN2 CIN3

Fig. 6.29 p16 immunostaining of different grades of mous epithelium is p16 positive. In CIN2, positive cells
squamous dysplasia. In accordance with the WHO defini- are also present in the middle one-third, and in CIN3, all
tion of CIN1–3, in CIN1 the lower one-third of the squa- cell layers stain positive

Fig. 6.30 p16 immunohistochemistry in CIN2. The

lower and the middle one-third of the squamous epithe-
lium are p16 positive Fig. 6.31 p16 immunohistochemistry in CIN3. The epi-
thelium is homogeneously p16 positive
92 6 The Significance of Cytology, Biopsy, and HPV Testing

Fig. 6.32 p16 immunohistochemistry of CIN3. The dysplastic epithelium, which is homogeneously p16 positive,
extends into the endocervical glands. This may not be confused with true invasion

diagnostic conization, as this procedure usually

requires general anesthesia, has a significant
risk of bleeding, and may lead to complications
of future pregnancies. Therefore, in the context
of dysplasia diagnosis, colposcopy and targeted
biopsy is the best available triage at present (Versi
1992), although its sensitivity and specificity are
well below 100%.
Taking one or several biopsies from the cer-
vix does not have any adverse effects. Castle
et al. (2009) showed that multiple biopsies do
not increase the risk of subsequent HPV infec-
tion. Biopsies may even induce clearance of HPV
Fig. 6.33 p16 immunohistochemistry of invasive squa- infections and/or shorten the time to HPV clear-
mous cell carcinoma. Numerous nests of p16 positive
tumor cells infiltrate the stroma
ance (Petry et al. 2018).
The influence of the number of biopsies
on diagnostic sensitivity has been studied by
[Link] Reliability of Colposcopically Gage et al. (2006). Patients from the ASC-US/
Guided Biopsy Sampling LSIL triage study who developed CIN3 or car-
Histological diagnosis of dysplasia is subject cinoma within 2 years were identified. By tak-
to considerable intra- and interobserver vari- ing two instead of one biopsy, sensitivity can be
ability (Ceballos et al. 2008). These inaccura- increased significantly: 68% and 82%, respec-
cies can be improved to some extent by p16 tively. However, three or more biopsies do not
immunohistochemistry. lead to any further increase of sensitivity.
However, both cervical biopsies (Fig. 6.34) Interestingly, the overall accuracy of col-
and endocervical curettage (ECC) (Fig. 6.35) poscopic diagnosis was similar across people
are small volume samples and will not always with different types of medical training, such as
be representative. Nevertheless, colposcopically nurse practitioners, general gynecologists, and
guided cervical biopsy is considered to be the gynecologic oncologists.
gold standard of cervical diagnosis, a fact that According to these data, about one-third of
has been called into question by cytopathologists diagnoses based on a single targeted biopsy do
such as Mangold and Nauth (2008) and Nauth not accurately reflect the findings on subsequent
and Mangold (2009). However, in practice not LEEP resection or cone biopsy. Several other
all cytological abnormalities can be clarified by studies have led to similar results. Stoler et al.
6.2 Biopsy 93

Fig. 6.34 Cervical

biopsy specimen. The
tissue sample is only a
few millimeters in size
and has been cut into 12
sections

Fig. 6.35 Endocervical

curettage specimen.
Endocervical curettage
has been performed with
the Stiefel® curette.
Nevertheless, the
amount of tissue is
small, which is a
contributing factor of the
low sensitivity of ECC
94 6 The Significance of Cytology, Biopsy, and HPV Testing

(2011a, b) examined 594 patients who had one or According to data derived from the ALTS
more target biopsies taken on the day of coniza- study (ASC-US/LSIL triage), CIN2+ was diag-
tion. Dysplasia (CIN or AIS) in the cone specimen nosed in only 3.7% of patients by ECC com-
was frequently underdiagnosed in the cervical pared to 21.7% of patients who were diagnosed
biopsy or biopsies. The overall agreement was by biopsy (Solomon et al. 2007). Thus, sensi-
only 56% with underestimation of CIN2/CIN3/ tivity of ECC was only 12.2% compared to
AIS being 57%. In accordance with the data from 72.5% for the biopsy. The relative contribution
Gage et al. (2006), taking more than one biopsy of ECC to the diagnosis of CIN2+ lesions was
improved the accuracy of colposcopic triage. higher in older patients: 13.0% among women
Zuchna et al. (2010) found a sensitivity of 40 years and older versus 2.2% in women
52.0% for the detection of CIN2+ lesions when younger than 40.
taking a single biopsy, and 65.2% when two biop- Fan et al. (2017) tried to correlate accuracy
sies were obtained. A third biopsy did not lead to of colposcopically directed cervical biopsy with
a significant improvement in sensitivity, again in clinical factors. The agreement between histo-
accordance with the data from Gage et al. logical results of biopsy and cone was 74.1%
Byrom et al. (2006) found a sensitivity of (78.2% for high grade lesions). Univariate analy-
74% for the detection of CIN2+ by taking a sin- sis showed that underdiagnosis was significantly
gle biopsy immediately prior to conization. All more frequent in older patients (≥50 years of
patients had a type T1 or T2 transformation zone. age), postmenopause and transformation zone
Two out of three microinvasive carcinomas were type T3, and when fewer biopsies were taken. On
not detected by biopsy. multivariate analysis, cone width was the only
Data from Stubbe et al. (2011) are very simi- independent factor correlated with the accuracy
lar: In 240 correlations between biopsies and of cervical biopsy.
confirmational result, only 67% of the patients Based on the limited sensitivity of cervical
had an exact match. biopsies, Massad (2006) suggests to consider
According to Pretorius et al. (2004), the immediate loop excision in older women who
diagnosis was correct in 57% of colposcopi- have completed childbearing when cytology sus-
cally guided biopsies from 364 patients with pects high grade disease without prior confirma-
a CIN2+ lesion and a T2 or T3 transformation tion by targeted biopsy.
zone. Interestingly, a random biopsy would still As pointed out above, the German Munich
diagnose 37% of CIN2+ disease. In a subsequent III classification of Pap groups places particu-
publication, Pretorius et al. (2012) found that 61 lar emphasis on the distinction between CIN2
out of 295 CIN3+ cases (21%) were diagnosed and CIN3 cytology (Pap IIID2 and Pap IVa-p,
with CIN2+ after random biopsy and/or ECC. respectively). Specificity of a Pap IVa-p diag-
Endocervical tissue removal, i.e. endocervi- nosis is high with 85% to 87% for a CIN3 diag-
cal curettage, is by definition a blind biopsy. Its nosis and 98% for CIN2+ (Hilal et al. 2015;
accuracy is much lower than targeted cervical Marquardt and Ziemke 2018). In contrast, as
biopsy/biopsies (Driggers and Zahn 2008). In shown above, biopsies are only concordant with
Chap. 7, a Stiefel® curette is recommended for conization diagnosis in 52% to 74%. Therefore,
ECC. However, whether significantly more mate- biopsy—but not colposcopy—may be omit-
rial can be obtained compared to a conventional ted prior to therapeutic resection in selected
curette has not yet been confirmed by a study. patients.
6.3 HPV Test 95

Summary: Target Biopsy

• Colposcopically guided biopsy as the sole diagnostic criterion would be unreliable and devi-
ates from the definitive diagnosis in about one-quarter to one-third of patients.
• Taking two biopsies instead of one significantly improves accuracy.
• Technically, endocervical curettage constitutes a “blind biopsy” and has a low negative pre-
dictive value.
• Cervical biopsy and ECC are only two of the several decision criteria, and their interpretation
in the context of clinical decision-making must take into account the inherent error rate.
• Prior to the colposcopic triage decision, an overall assessment is required including.
– age
– reproductive desire
– previous and present cytological results
– colposcopic evaluation (T1-T3, minor versus major change)
– individual risk factors (immunosuppression)
– individual patients preferences

6.3 HPV Test responds with CIN2+ detection. The oversensi-

tive detection of an HPV infection per se is not
The HPV test plays an important role, both in screen- clinically useful (de Thurah et al. 2018). Rather,
ing as a primary test option or as a co-test in combi- this could lead to an unnecessary increase in the
nation with cytology. Also, the HPV test can be used number of colposcopic triage examinations, as
as triage of ASC-US and as a test of cure following Kinney et al. (2010) pointed out when the FDA-
treatment of preinvasive cervical disease. The fol- approved Cervista test (Hologic) was found to
lowing chapter focuses on basic requirements for be two to four times more often positive than the
HPV tests that are used in clinical decision-making. other FDA-approved tests.

“Misguided attempts to achieve the lofty

6.3.1 HPV Assays Suitable
goal of perfect clinical sensitivity (via
for Clinical Use
increasing analytic sensitivity) have the
potential to result in significant patient
Test sensitivity is one of the most important issues.
harm from unnecessary clinical follow-up,
Analytical sensitivity, i.e. the detection of a
unnecessary diagnostic procedures, and
very low number of viruses, is less important
unnecessary treatment of healthy women.”
than clinical sensitivity, i.e. the capability to
(Kinney et al. 2010)
detect CIN2+ lesions. Therefore, an HPV assay
is only useful for screening, if it has a certain
accuracy to predict the presence of HSIL. Results from different HPV assays do not
The Hybrid Capture 2 test (HC2), which is necessarily have to be concordant. However,
discussed in more detail below, can be considered in the presence of CIN2+ disease, the concor-
the benchmark assay, as most clinical studies on dance between different HPV test procedures
CIN2+ detection are based on HC2. Newer and is as high as 84%, compared to only 29% of
more advanced tests have to prove their efficacy the entire screening population. This has been
through comparison with HC2 data. Therefore, shown by Rebolj et al. (2016) who compared
these newer assays need to be calibrated, so that four different assays (HC2, CLART®, cobas®,
their thresholds of high risk HPV detection cor- APTIMA®).
96 6 The Significance of Cytology, Biopsy, and HPV Testing

• The following basic guidelines have been established for the requirements of HPV tests that
are applied clinically, especially when used for primary cervical screening:
• (Stoler et al. 2007; Meijer et al. 2009)
– The test should be capable of detecting the following 13 HPV high risk types: 16, 18, 31,
33, 35, 39, 45, 51, 52, 56, 58, 59, and 68.
– Sensitivity to detect CIN2+ should be at least 90% compared to an established and clini-
cally validated HPV test.
– Specificity to diagnose CIN2+ should be at least 98% compared to an established and
clinically validated HPV test.

hybrids. Subsequently, antibodies are added,

[Link] Hybrid Capture II Test (HC2) which are conjugated with alkaline phosphatase
The hybrid capture assay is a signal-amplifying and bind specifically to DNA–RNA hybrids.
hybridization assay for the qualitative detection of Finally, hybridization is detected by che-
DNA of human papillomaviruses. The assay can miluminescence. A special reagent serves as a
distinguish two HPV groups: the A probe identi- substrate of alkaline phosphatase. As a result of
fies the low risk HPV types 6, 11, 42, 43, and 44 this enzymatic reaction, light is emitted, which
and the B probe the high risk HPV types 16, 18, is measured in the luminometer and reported
31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. as “relative light units” (RLU). By comparison
Overall, 13 high risk HPV types are qualita- with a positive and a negative sample serving as
tively analyzed with the Hybrid Capture II test. a standard, it is determined whether the sample is
Identification of individual virus types (geno- positive for either the A or B probe or both.
typing) is not possible. Nor can the viral load Although the A probe, which identifies four
be quantified. The test is positive, when at least different HPV low risk viruses is still avail-
5000 viral copies/ml (1 pg DNA) are present. able, no clinical indication for its use does exist.
During gynecological examination, the speci- Rather, the diagnosis of HPV low risk types,
men is obtained from the cervix or from the pos- which are of no diagnostic or therapeutic conse-
terior vaginal vault using a brush-like sampling quence, may only be disconcerting to the patient
device. The brush is placed into the transport and should therefore be avoided.
medium and sent to the laboratory without the
need for refrigeration. The sample can be stored [Link] Other HPV Assays
for up to 2 weeks at room temperature. Although FDA approval is not required outside
In the laboratory, the DNA is released from the USA, FDA certification can still be consid-
the cells using sodium bicarbonate and then ered a quality criterion. FDA approval of labo-
hybridized with the specific HPV RNA probes ratory tests, that are used for clinical decision
A and B. In this step, DNA–RNA hybridization making, requires extensive documentation.
occurs if the sample contains the HPV DNA tar- Presently, the following four HPV tests are
get sequences. FDA approved:
The next phase involves the so-called cap-
ture step, as the DNA–RNA hybrids are bound – Digene Hybrid Capture 2 High Risk HPV DNA
to the surface of a microtiter plate. Each well Test (Qiagen, Gaithersburg, MD, USA). HPV
of the microtiter plate contains on its surface types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58,
anti-RNA–DNA antibodies which capture the 59, and 68 (antigen: DNA, whole genome).
6.3 HPV Test 97

– Cobas® HPV Test (Roche Diagnostics). HPV – APTIMA® HPV Assay (Hologic). HPV 16,
16/18 (genotyping), 31, 33, 35, 39, 45, 51, 52, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66,
56, 58, 59, 66, and 68 (antigen: DNA, L1). and 68 (antigen: RNA, E6/E7).
– Cervista® HPV HR and GenFind DNA
Extraction (Kit) (Hologic). HPV 16, 18, 31, A more comprehensive list of commercially
33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 available HPV tests is given in Table 6.5.
(antigen: DNA, E6/E7/L1).

Table 6.5 List of commercial HPV tests (Poljak et al. 2012, updated 2018)
hrHPV DNA tests Current status
Hybrid Capture® 2 (HC2) HPV DNA Test (QIAGEN Inc., Gaithersburg, MD; USA US FDA-approved (2003)
(previously Digene Corp.)) EIA kit HPV GP HR (Diassay, Rijswijk, The clinically validated
Netherlands) US FDA-approved (2009)
Cervista® HPV HR Test (Hologic, Madison, WI) clinically validated
CareHPVTM Test (QIAGEN Inc., Gaithersburg, MD; USA)
hrHPV DNA tests with concurrent or reflex partial genotyping for the main hrHPV US FDA-approved (2011)
types clinically validated
Tests with concurrent partial genotyping for the main hrHPV types US FDA-approved (2009)
cobas® 4800 HPV Test (Roche Molecular Systems Inc., Alameda, CA, USA)
RealTime High Risk HPV test (Abbott Molecular, Des Plaines, IL)
Tests with reflex partial genotyping for the main hrHPV types
Cervista HPV 16/18 Test (Hologic, Madison, WI)
digene® HPV Genotyping PS Test, RUO (Qiagen, Hilden, Germany)
HPV DNA full genotyping tests Widely used test widely
Strip, filter or microtiter-well hybridization based full genotyping tests used test widely used test
Linear Array® HPV Genotyping Test (Roche Molecular Systems Inc., Alameda, widely used test
CA, USA) INNO-LiPA HPV Genotyping Extra (Innogenetics NV, Gent, Belgium) Clinically validated widely
HPV SPF10 LiPA25version 1 (Labo Bio-Medical Products, Ev Rijswijk, The used test
Netherlands) digene HPV Genotyping RH Test (Qiagen, Hilden, Germany)
Medium or low density microarray-based full genotyping tests
PapilloCheck® HPV Screening Test/High risk Test (Greiner Bio-One,
Frickenhausen, Germany)
Clart® HPV 2 - Papillomavirus Clinical Arrays (Genomica, Coslada, Spain)
Microsphere beads based full genotyping tests
Gel electrophoresis based full genotyping tests
Capillary electrophoresis based full genotyping tests
Real-time PCR based full genotyping tests
PCR combined with matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry
HPV DNA type- or group-specific genotyping tests
Real-time PCR based tests
Gel electrophoresis based test
hrHPVE6/E7 mRNAtests US FDA-approved (2011)
APTIMA® HPV Test (Gen-Probe Inc., San Diego, CA) widely used test
PreTect HPV-Proofer (NorChip, Klokkarstua, Norway)/NucliSENS EasyQ® HPV
(Biomerieux, Marcy l’Etoile, France)
In situ based hybridization HPV tests
HPV human papillomavirus, hrHPV high risk human papillomavirus, PCR polymerase chain reaction
98 6 The Significance of Cytology, Biopsy, and HPV Testing

6.3.2 Clinical Application of HPV et al. 2001). This is particularly true for ASC-US
Testing cytology, whereas in women with LSIL its value
is less obvious (Arbyn et al. 2012). Figure 6.36
HPV tests play an important role in the present shows the recommendations of the Italian Group
concepts of secondary cervical cancer prevention for Cervical Cancer Screening for management
(Arbyn et al. 2012). In the USA and elsewhere, of women with HPV positive ASC-US (Carozzi
HPV testing as a primary screening strategy et al. 2015). For LSIL, the same triage algorithm
has been approved for women of ≥30 years. In is proposed with the HPV test result playing a
other countries, such as Germany, co-testing limited role only.
with cytology has been introduced for women of However, Kyrgiou et al. (2016) state that imme-
≥35 years. diate colposcopy for LSIL does not increase the
Besides primary screening, HPV testing is detection of CIN2+ lesions, compared to repeat
useful for the triage of equivocal (ASC-US) or cytology over 2 years. Unfortunately, compli-
LSIL Pap smear result. Also, in post-LEEP or ance decreases with the length of follow-up, so
conization follow-up, the HPV test can is consid- that immediate colposcopy might be considered
ered as a “test of cure.” for women with LSIL/HPV+ findings.
As a general rule, women under the age of For triage of glandular cytological abnormali-
30 years should not be tested for HPV, regard- ties, HPV testing plays only a very limited role.
less of the indication for the HPV test, as there According to a systematic review by Verdoodt
are too many incident HPV infections which et al. (2016), the risk for high grade squamous
are not clinically significant. and/or glandular dysplasia or invasive disease
(CIN2+/AIS+) in women with atypical glandular
[Link] HPV Triage of ASC-US and LSIL cells (AGC) was 19.8%, and as high as 55.7% in
The HPV test can successfully triage women women with concurrent squamous lesions (AGC/
with equivocal or low grade cytological abnor- ASC-US+). Therefore, a cytological AGC diag-
malities at screening. Thus, the number of refer- nosis warrants referral to colposcopy regardless
rals to colposcopy can be reduced (Solomon of HPV status. However, a negative HPV test in

ASCUS HPV+
LSIL HPV+ (or HPV-)

colposcopy CIN 2+ treatment and follow up

negative for CIN2+

HPV test at 12 months

HPV negative HPV positive

return to screening negative for CIN2+ colposcopy

HPV test at 12 months CIN 2+

HPV positive

colposcopy and pap test

Fig. 6.36 Recommendations of the Italian Group for Cervical Cancer Screening for management of women with
ASC-US using HPV triage and LSIL with or without HPV testing (Carozzi et al. 2015)
6.3 HPV Test 99

patients with atypical glandular cells is linked According to a meta-analysis by Arbyn et al.
with an 18% chance of extra-cervical neoplasia (2012), the summary accuracy estimates for the
such as endometrial, fallopian tube, or ovarian HPV test 6 months after treatment were higher
malignancy. than for cytology (Fig. 6.37).
However, it has to be kept in mind that a posi-
[Link] HPV Test for Post-Treatment tive HPV test on follow-up is not necessarily
Follow-Up (“Test of Cure”) associated with persistent CIN. According to the
HPV tests are useful in post-treatment follow-up study by Verguts et al. (2006), 23% of women
(Alonso et al. 2006; Bae et al. 2007; Prato et al. who did not relapse within a two-year follow-up
2007) since numerous studies have shown that continued to be positive for high risk HPV.
after successful surgical dysplasia treatment the In patients treated conservatively for cervical
HPV test frequently becomes negative. When the adenocarcinoma in situ, post-treatment moni-
HPV test is negative, most likely the precancer- toring by colposcopy and cytology is even less
ous lesion has been removed completely. effective than for squamous lesions, and an addi-
In fact, a negative HPV test result is more reli- tional parameter such as the HPV test is advan-
able than a negative margin on histopathological tageous. Costa et al. (2007) found a diagnostic
examination, or a normal Pap smear obtained sensitivity of 90% for a combination of cytology
during follow-up (Verguts et al. 2006). Alonso and HPV testing (HC2) with a specificity of 50%
et al. (2006) found that 11.8% of patients with for follow-up of adenocarcinoma in situ.
a negative resection margin still developed a Globally, an increasing number of algorithms
relapse, whereas 63.6% with a positive margin incorporate HPV testing as a “test of cure” during
had no recurrence. post-treatment follow-up.

Fig. 6.37 Sensitivity hrHPV testing

1

and specificity of HPV with HC2 or PCR

testing versus
cytological surveillance
cytology
following treatment of
HSIL to predict
.8

treatment failure. The

summary accuracy
estimates for the HPV
test (filled orange circle)
.6

and cytology (filled gray

Sensitivity

square) 6 months after

treatment are given
together with the 95%
confidence interval as an
.4

ellipse around the

summary estimates.
According to this
review, HPV testing is
superior to cytology in
.2

the post-treatment
follow-up (Arbyn et al.
2012)
0

1 .8 .6 .4 .2 0
Specificity
100 6 The Significance of Cytology, Biopsy, and HPV Testing

Fig. 6.38 Primary HPV HPV test

screening project in Cytology
Wolfsburg, Germany,
with cytology co-testing
for women ≥30 years HPV negative HPV negative HPV positive HPV positive
Cytology negative Cytology positive Cytology negative Cytology positive
(Luyten et al. 2009)

Repeat HPV + cytology

ASCUS/LSIL: Repeat cytology
after 5 years immediate
observation after 6 months;
Routine exam colposcopy
HSIL: colposcopy if positive: colposcopy
annually

[Link] Primary HPV Screening review.3 According to this proposal, co-testing

Although Pap smear based screening has been is no longer recommended. Instead, women
proven to be very effective in secondary preven- aged 30 to 65 years should receive primary
tion of cervical cancer, additional or alternative HPV testing every 5 years as an alternative to
testing is needed in order to increase test sen- cervical cytology every 3 years.
sitivity, especially in a population that has a low Similar to the USA, there is an increasing
prevalence of HPV-related cervical disease due to number of national cervical screening programs
previous screening effort as well as the effects of and guidelines that incorporate HPV testing into
HPV vaccination. Primary HPV screening, either primary screening. However, in some countries
alone or in combination with cytology, has been such as Germany that has a tendency to over-
shown to decrease false negative screening results diagnose cytological abnormalities by perform-
and improve test sensitivity (Mayrand et al. 2007; ing Pap tests every year, health care officials are
Naucler et al. 2007). As a negative HPV test result reluctant to change the current practice of exclu-
implies a very high probability that no dysplasia sive Pap smear screening. Eventually, starting in
and no carcinoma presently exists and will not 2020, co-testing every 3 years from the age of
occur in the next few years, screening intervals of 35 years has been approved as an alternative to
three to 5 years are discussed. Sherman et al. (2003) annual cytological screening.4
demonstrated that, over a 10-year period, HPV In Germany, Petry et al. (2003) initiated the
negative women have a minimal risk of developing “Wolfsburg model,” a long-term prospective clin-
severe dysplasia compared to women who tested ical study of an HPV and cytology-based screen-
positive for high risk HPV. These groups of women ing program (Fig. 6.38), which has been shown
developed high grade HPV-related disease in 4.3% to avoid unnecessary surgical procedures and to
within 3 years and in 7% within 10 years. be cost-effective due to longer screening intervals
The American College of Obstetrics and (Luyten et al. 2009). Also, acceptance for the
Gynecology (ACOG) Practice Bulletin Nr. “Wolfsburg model” is high for both patients and
168 as of October 2016 recommends either participating gynecologists.
Pap smears every 3 years or HPV/cytology Colposcopic triage for the detection of CIN3
co-testing every 5 years, with the latter being lesions and invasive disease appears to be safe in
the preferred screening method. Screening by participants of primary HPV screening programs
HPV testing alone is not recommended. Due to
data from the ATHENA study (Addressing the
Need for Advanced HPV Diagnostics) (Stoler
et al. 2011a, b; Wright et al. 2012), and after 3
[Link]
Practice-Advisories/Practice-Advisory-Cervical-Cancer-
interim suggestions for using the cobas® HPV
Screening.
test, the U.S. Preventive Services Task Force 4
[Link]
(USPSTF) in 2017 published new cervical 09- 15_Aenderung_Beauftragung-IQWiG_Einladung_
cancer screening recommendations for public Zervixkarzinom-Screening_vom-[Link].
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 Colposcopic Examination
7

Contents
7.1 History Taking 107
7.2 Colposcopes 109
7.3 Handling of the Colposcope and Speculum 110
7.4 Cytological Smear 114
7.5 Tissue Sampling 114
7.6 Documentation 118
7.7 Practical Colposcopic Examination Step by Step 119
References 120

The colposcopic examination starts with 7.1 History Taking

the collection of relevant patient data fol-
lowed by colposcopic inspection includ- Prior to colposcopic examination, a detailed his-
ing cytological smear collection and then tory is taken. Table 7.1 lists key points that are
colposcopically guided biopsy or biopsies. relevant to colposcopy. Usually, the patient her-
In the following section, practical aspects self will provide this information. However, it
of colposcopy are explained including the is advisable to obtain copies of prior medical
use of colposcopic instruments. reports, especially on previous Pap smears and
HPV test results (type of HPV test).
Cervical dysplasia does not cause any symp-
The main objective of colposcopic examina- toms. However, many patients are worried and
tion is the diagnosis of high-grade dysplastic anxious and may associate unspecific symp-
disease, i.e., the identification of major changes. toms with serious disease and cancer. Vaginal
Most importantly, invasive disease must not be bleeding during and after sexual intercourse
overlooked. Furthermore, the site or sites with does not necessarily indicate carcinoma and
the most abnormal changes are identified, and may occur because of a benign condition such
targeted biopsies are obtained. as a large ectopy or inflammation. In the case

© Springer Nature Switzerland AG 2023 107

R. J. Lellé, V. Küppers, Colposcopy, [Link]
108 7 Colposcopic Examination

Table 7.1 Key points for history taking in colposcopy

Reason for referral to • Abnormal Pap smear, presently and in the past
the colposcopy clinic/ • HPV test results
presenting complaint • Other relevant test results
• Abnormal findings of cervix, vagina, vulva, and anus
Complaints • Vaginal discharge (inflammatory, bloody)
• Vaginal bleeding/spotting (intermenstrual bleeding)
• Pain (dyspareunia)
• Itching
• Genital warts
• Abnormal skin areas (including oral mucosa, scalp)
Past medical history • Particularly any diseases, conditions, or medications that may impair the immune
(including system
medication)/previous – Inflammatory bowel diseases (medication)
surgery – Transplant surgery (medication)
– Rheumatic arthritis (medication)
– HIV infection, etc.
• Previous surgery
• Gynecologic operations (see below)
• Other operations (bowel, anus)
Previous (gynecologic) • Conization, LEEP, laser surgery
surgery • Sterilization of patient or partner
• Hysterectomy (indication)
Pregnancies • Gravidity and parity
• Previous deliveries (cesarian section, obstetrical trauma to the lower genital tract)
• Infertility treatment
• Future family planning
Sexual history • Age at first intercourse
• Regular sexual partner, number of partners (male/female)
• Types of sex (vaginal, oral, anal)
• Condom use
• Previous STDs
• Sexually transmitted diseases (including partner, including drug or surgical condyloma
removal)
Contraception/ • IUD with or without hormones
hormones • NuvaRing®
• Oral contraceptive
• Hormone replacement therapy
• Topical estrogen treatment
• Barrier contraception (condom)
Smoking • For how many years
• How many cigarettes a day
Allergies • Iodine

of intermenstrual bleeding (metrorrhagia), how- The presence of an intrauterine device

ever, invasive disease of the uterus needs to be should be noted before the colposcopic exami-
considered. nation, as well as vaginal contraceptive devices
In contrast to cervical precancer, HSIL of the such as a NuvaRing®. The NuvaRing® should
vulva may cause local discomfort such as itch- be removed before the examination and rein-
ing or pain. In cases of lichen planus of the vulva serted within 3 hours so as not to impair
or vagina, the oral mucosa and/or scalp may also contraception.
be affected, and the patient should be specifically The sexual history should be documented
asked if she has any symptoms in areas outside also. Considering anxiety and the negative asso-
the lower genital tract. ciation between dysplasia, HPV infection, and
7.2 Colposcopes 109

sexual activity, anything should be avoided that

may cause or intensify feelings of guilt.
Smoking habits should be documented too,
and the association between tobacco use and the
risk of intraepithelial neoplasia, especially of
the vulva, should be explained. Enrollment in a
smoking cessation program should be offered.
Furthermore, it is important to find out if the
patient wants to become pregnant in the near
future. If the patient does not use hormonal con-
traception, condoms should be recommended
until colposcopic diagnosis and possible surgical
treatment have been completed.
Further important aspects of the patient’s his-
tory are previous surgery, especially procedures
directly related to the colposcopic examination,
such as conization or hysterectomy. Also, com-
prehensive information should be gathered about
the patient’s past medical history. Any diseases
or medications that may compromise the immune
system are of particular interest.

7.2 Colposcopes

Since Hinselmann’s invention in 1925, the basic

Fig. 7.1 A classic Leisegang colposcope (Leisegang
design of the colposcope has not changed. With Feinmechanik Optik GmbH, Berlin, Germany) with mag-
an optical colposcope, a magnification of up nifications of 3.75, 7.5, and 15 times. The attached camera
to 15 or 30 times is possible. For example, the records three-dimensional images
Leisegang colposcope (Leisegang Feinmechanik
Optik GmbH, Berlin, Germany) has three steps of patient and doctor. Greimel et al. (1997) did a
magnification: 3.75, 7.5, and 15 times (Fig. 7.1). prospective randomized study on the effect of
Usually, the focal length of colposcopes is such video colposcopy. Patient satisfaction was sig-
that the working distance is about 30 cm. nificantly higher when using video colposcopy.
A more recent concept is the video colpo- However, video colposcopy does not signifi-
scope. This device does not need complex optical cantly reduce the anxiety levels of patients when
lenses like the classical colposcope. As with lapa- tested in a controlled trial setting (Hilal et al.
roscopy, the person doing the colposcopy looks 2017; Ketelaars et al. 2017).
at the monitor while performing the examination, In addition to his well-known colposcope,
including tissue removal. Although in the year Hinselmann described the “von Eicken’sche
2000 the majority of physicians still gave pref- Stirnlampe,” a magnification device with a light
erence to the classical optical colposcope (Ferris source mounted on a headset (Fig. 7.2). A mod-
et al. 2000), the present generation of gynecolo- ern version of this device using a 4× magnifi-
gists is well versed in laparoscopic techniques cation (“Muenster Colposcopic Magnification
and is already used to working via a video screen. Device”) can be used as a simple and economi-
Patients also react positively to video colpos- cal alternative to the conventional colposcope
copy, as it helps to explain the colposcopic find- (Lellé 2007), especially when performing sur-
ings and to improve communication between gery (Fig. 7.3).
110 7 Colposcopic Examination

7.3 Handling of the Colposcope

and Speculum

First, the colposcope needs to be adjusted to the

person doing the exam. For the binocular colpo-
scope, first the interpupillary distance is set. As
with a microscope, the distance between the ocu-
lars is indicated on a scale located between the eye-
pieces. The diopter settings are initially set to “0,”
if the colposcopist has 20/20 vision or uses cor-
rective glasses or contact lenses. Alternatively, the
diopter settings are adjusted accordingly. Now, the
colposcope is focused by moving the head of the
colposcope back and forth. Focus is only accom-
plished when the distance to the target is within
the range of the focal distance, which is usually
about 30 cm. Some colposcopes are equipped with
Fig. 7.2 “von Eicken’sche Stirnlampe” as described by a mechanism to achieve final focus by moving the
Hinselmann in 1925 as an alternative to the classical col- colposcope in small increments only.
poscope (Hinselmann 1925)
When an optical colposcope is equipped with a
video camera, both the optical and the digital image
are brought into focus simultaneously by first adjust-
ing to the image on the monitor. Subsequently, each
eyepiece is focused separately using the diopter set-
tings without moving the colposcope.
The colposcopic examination begins with the
inspection of the vulva and anus. Occasionally,
HSIL of the vulva is identified in an asymp-
tomatic patient, who presents with cervical
dysplasia.
Furthermore, the vagina should be carefully
inspected. The upper (proximal) portion of the
vagina can be evaluated together with the por-
tio. The middle and lower (distal) portion is best
examined by slowly retracting the speculum. The
partially closed speculum is rotated in order to
assess the anterior (ventral) and posterior (dor-
Fig. 7.3 “Muenster Colposcopic Magnification Device” sal) aspects of the vagina. If there is suspicion of
as a modern version of Hinselmann’s device (Fig. 7.2) vaginal intraepithelial neoplasia (VAIN), Lugol’s
with a 4× magnification. The headset design is particu-
larly useful when performing surgery under colposcopic
solution is applied.
guidance (Lellé 2007) Some colposcopists are using two separate
specula for spreading the vagina (e.g., combin-
ing the Seidl speculum with the Kristeller specu-
With the improvement of digital imaging, lum). However, “duckbill” specula such as the
a whole range of portable imaging devices has Cusco1 type are better suited for colposcopy, as
been developed based on smartphone technology
(Chap. 17). 1
Edward Gabriel Cusco, French physician 1819–1894.
7.3 Handling of the Colposcope and Speculum 111

both hands remain free. The Grave speculum is Sometimes, the vaginal walls may still not be
also commonly used. In addition to a two-bladed sufficiently unfolded, for example, in very obese
mechanism similar to the Cusco speculum, the patients or during a second or third trimester
valves can be moved further apart. pregnancy. In these situations, the use of a con-
The speculum chosen for colposcopy must dom, which is pulled over the speculum and cut
be sufficiently large and adequately shaped. distally, can be helpful (Fig. 7.5).
Usually, it is not the size of the speculum that Before inserting the speculum, the inside of
causes discomfort to the patient but an inser- the thigh is lightly touched with the back of the
tion technique that does not take into account hand as the immediate contact of the vulva with
the anatomy of the lower genital tract. If the the speculum may startle the patient. The small
speculum is too small and/or too narrow, labia are spread using the left hand. Then the
the lateral vaginal walls are not sufficiently closed speculum is inserted under pressure in
unfolded and exposed and obstruct the view the direction of the posterior fourchette. As the
of the ectocervix (Fig. 7.4). vulvar opening has a vertical axis, the speculum
is inserted accordingly. The closed speculum is
then rotated clockwise by 90° and fixed by turn-
ing the screw (also see Chap. 17).
After inserting the speculum, the position of
the examination chair is adjusted so that the col-
poscopist sits in a comfortable upright position.
The patient is typically in a slight head-down
position (Fig. 7.6).
First, the colposcope is set to its lowest mag-
nification, as this facilitates focusing. All subse-
quent steps are now performed under colposcopic
guidance.
Fig. 7.4 Two Cusco type specula. The speculum in the The cervix and upper vagina are inspected
upper part of the image may be too narrow so that the
before any manipulation of the cervix. However,
view of the ectocervix is partially obstructed as the lateral
vaginal walls are not sufficiently unfolded excessive mucus may be initially removed using

Fig. 7.5 A condom is pulled over the speculum to help to unfold the vaginal wall and improve visualization of the
ectocervix
112 7 Colposcopic Examination

Fig. 7.6 The patient is

positioned for
colposcopy with the
pelvis elevated

a cotton swab. If leukoplakia is identified, a

biopsy is obtained after colposcopic inspection
using acetic acid and then Lugol’s solution has
been completed.
Small abnormal vessels are best visualized prior
to the application of acetic acid. Acetic acid has a
mild vasoconstrictive effect on small blood vessels
through tissue edema, thus making these vessels
less visible. Some colposcopists use normal saline
to remove mucus and improve visualization.
A cytological smear is now obtained. A 3–5%
acetic acid solution is now abundantly applied
using a cotton swab (Fig. 7.7). Alternatively, a
spray bottle may be used.
Usually, application of acetic acid of up to 5%
does not cause discomfort. Occasionally, patients
will feel a temporary burning sensation.
Using a prospective study design with video doc-
umentation, Hilal et al. (2020) were able to deter-
mine optimal timing of colposcopic assessment
following the application of acetic acid. A 5% ace-
Fig. 7.7 Large and small cotton swabs for the removal of
tic acid solution was used. From this well-designed mucus and blood and for application of acetic acid and
study, the following conclusions can be drawn: Lugol’s solution
7.3 Handling of the Colposcope and Speculum 113

100 At this point of the examination, one should

determine whether the area where the squamous
and glandular epithelium meet (squamocolum-
nar junction) is completely visible (T1/T2). It
80 P = .016 may also be helpful to try to define the original
squamocolumnar junction, before the physiologi-
cal changes of metaplasia had occurred. This is
only possible if there is still immature metapla-
Proportion not faded (%)

60 sia or if gland openings and/or Nabothian cysts

are visible, because, in older women with mature
squamous metaplasia, identification of the origi-
nal squamocolumnar junction is not possible
anymore. The transformation zone, i.e., the area
40
between the current and the original squamo-
columnar junction, is the major area of interest,
where squamous intraepithelial neoplasia is most
likely to occur.
20 According to a survey from eight colposcopy
HSIL centers (Luyten et al. 2015), the squamocolum-
nar junction can be visualized in the majority of
LSIL
patients (81%). A T3 transformation zone was
0 found to be the most commonly reported type in
0 100 200 300 women older than 50 years (70%).
Time (seconds) Visualization of the entire squamocolumnar
junction is usually accomplished by spreading
Fig. 7.8 Time dynamics of acetowhite staining and fad- the outer cervical canal with the Kogan endocer-
ing. HSIL lesions (filled circles) fade significantly more
quickly than LSIL lesions (open circles). The optimal
vical speculum (Fig. 7.9) or by using two dry cot-
time for diagnosis is about 1 min after the application of ton swabs.
acetic acid (Hilal et al. 2020) Visualization of vascular structures can be
enhanced by using the green filter. The filter
– Acetowhite staining develops very quickly. should be used both before and after the applica-
HSIL lesions become visible after a median of tion of the acetic acid. Under the green filter, ves-
7 seconds, whereas LSIL is first recognized sels appear black and have a better contrast to the
after a median of 19 s. surrounding tissue. Also, punctation and mosaic
– Subsequently, staining of both LSIL and HSIL are seen more clearly, as these two phenomena
lesions intensifies and reaches its maximum are due to the peculiar morphology of subepithe-
after 50 s. lial capillaries.
– A plateau is reached between 1 and 3 min. Now, Lugol’s solution is applied to the cervix
After that, fading of the acetic acid effect is and upper vagina, if the patient is not allergic
observed that occurs significantly more to iodine, as an anaphylactic-like reaction with
quickly in HSIL as in LSIL (Fig. 7.8). vaginal and generalized pruritus, vaginal edema,
hypotension, tachycardia, and breathing difficul-
Thus, the optimal time to identify both HSIL ties has been documented by Indraccolo et al.
and LSIL lesions is 1 min after the application (2009).
of acetic acid, and colposcopic observation Finally, any abnormal findings of the ectocer-
should not be longer than approximately 3 min vix or upper vagina are biopsied including endo-
because of fading of the acetowhite staining. cervical curettage.
114 7 Colposcopic Examination

Fig. 7.9 Kogan speculum. A pedunculated Nabothian cyst is pushed aside to visualize the squamocolumnar
junction (T2)

Zardawi and Rode (2002) saw some benefit of

7.4 Cytological Smear the repeat Pap test, if the previous smear had
shown only mild dysplasia.
When colposcopic triage for an abnormal Pap Hall et al. (2011) concluded that repeat cytol-
smear is performed, repeat cytology is not ogy provides useful clinical information in only
required in the majority of cases. 3.6% of patients. However, when the patient is
The Pap test is intended for screening and not referred for evaluation of HSIL, the repeat Pap
for definitive diagnosis, as it does not come close test provided potentially useful information in
the specificity of histopathologic evaluation. Also, 41% of cases.
if the interval between two cytological smears is If the Pap test is repeated at the time of col-
short, false-negative results seem to be more com- poscopic triage, it should be obtained prior to
mon. This problem has been known for a long time the application of acetic acid.
(Meisels 1990). Drescher et al. (1983) obtained a After the acetic acid test, the cell yield is sig-
cytological smear immediately prior to colposcopic nificantly lower and the rate of unsatisfactory
biopsy in 540 patients. Pap smears were false nega- false-negative results is higher (Griffiths et al.
tive in 26.4% of patients. In a more recent study, 1989; Cronje et al. 1997).
Kolben et al. (2017) showed that the risk of a false-
negative result of a second smear taken immedi-
ately after the first is not statistically significant, 7.5 Tissue Sampling
although a tendency toward a more benign out-
come in the second smear was observed. Obtaining one or more colposcopically directed
However, only 1.2–2.0% of repeat cytological biopsies from the cervix or vagina and perform-
examinations obtained during colposcopy have ing endocervical curettage are integral parts of
an influence on management decisions (Young colposcopy, as colposcopic assessment alone has
et al. 1993; Panos et al. 2001). Panos et al. found a low specificity, even when done by an experi-
a high false-negative rate of 19%. However, enced colposcopist.
7.5 Tissue Sampling 115

The procedure of tissue sampling should be Kevorkian, or Burke. The tips of these instruments
explained to the patient in advance, and her con- are of different sizes and shapes. However, size is
sent should be obtained as for any surgical pro- not decisive, as it is not necessary to make use of
cedure. If the patient is relaxed and confidently the entire cutting surface. For histological evalu-
goes into the examination, she will find colpos- ation, a few millimeters of tissue are sufficient.
copy and tissue sampling less uncomfortable. It To prevent the biopsy forceps from slipping
is also crucial that the medical assistant is well off a smooth and firm tissue surface, a hook-
acquainted with the procedure and contributes to like instrument can be used to immobilize the
a anxiety-free atmosphere. cervix. Similar problems may be encountered
As biopsy results will only be available at a in the vagina, especially on the lateral vaginal
later date, these results have to be related to the walls. By slightly closing and retracting the
patient in a timely fashion together with possible speculum, tension is released and the biopsy
treatment or follow-up suggestions. forceps can be applied to the slightly folded
vaginal wall.
If the tissue sample is taken too superficially,
7.5.1 Biopsy only dysplastic epithelium that is detached
from the stroma may be seen on histopatho-
Colposcopic biopsy is usually done without local logical examination so that possible invasion
anesthesia as most patient will experience only cannot be assessed. If ulceration is present,
minor discomfort. However, a recent prospec- biopsies should not be limited to the ulcerat-
tive randomized trial suggests that injection of a ing area. The lesion’s margin also needs to be
local anesthetic makes the procedure less painful biopsied so that the pathologist is better able
(Kiviharju et al. 2017). Another controlled pro-
spective study has shown that forced coughing
can also provide significant pain relief (Akavia
et al. 2018).
Tissue samples are always “targeted,” i.e.,
taken under direct colposcopic guidance.
Biopsies are obtained from areas with the most
severe changes. As a rule, higher-grade lesions
are more likely located close to the squamoco-
lumnar junction. Also, colposcopic phenomena
that are considered to be pathognomonic for
high-grade disease such as inner border sign,
ridge sign, or rag sign should be biopsied.
Several biopsy forceps types are available such Fig. 7.11 Targeted biopsy under colposcopic guidance
as Tischler (Figs. 7.10 and 7.11), Eppendorfer, with a Tischler forceps

Fig. 7.10 Tischler biopsy forceps

116 7 Colposcopic Examination

to ascertain invasion of tumor cells through tampon can be applied, which the patient can
the basal membrane. remove herself after a couple of hours.
The cervix is well supplied with blood, and For better control of bleeding during the pro-
the amount of bleeding resulting from even a cedure, the biopsy forceps is held in one hand
small biopsy can be upsetting to a less expe- and a cotton swab in the other to immediately
rienced colposcopist. However, satisfactory apply pressure onto the biopsy site. A hemostatic
hemostasis can always be achieved if there are agent such as Monsel’s solution can then be used
no coagulation disorders. (Fig. 7.12). Monsel’s solution is ferric subsulfate
If one is uncomfortable with the amount of solution named after Léon Monsel, a military
bleeding, especially during pregnancy, a loose apothecary of Bordeaux, France, who in 1856
reported on its hemostatic effect (Monsel 1856).
There are different ways for prepar-
ing Monsel’s solution or Monsel’s paste.
The procedure that is recommended by the
World Health Organization (2014) is given in
Table 7.2.
If necessary, a large tampon is inserted, the tip
of which has been immersed in Monsel’s solu-
tion. The patient must be made aware that she
will have a greenish black discharge for some
time after the biopsy.
Hilal et al. (2016) have tested the efficacy of
Monsel’s solution in a randomized trial setting.
Fig. 7.12 Monsel’s solution for hemostasis after cervical
The control group did not receive any hemostatic
biopsy agent. Overall, the clinical benefit of Monsel’s

Table 7.2 Preparation of

Ingredients Quantity
Monsel’s solution (World
Health Organization 2014) 1. Ferric sulfate base 15 g
2. Ferrous sulfate powder a few grains
3. Sterlle water for mixing 10 ml
4. Glycerol starch (see 12 g
preparation on next page)

Preparation:
Take care, as the reaction is exothermic (emits heat).
1. Add a few grains of ferrous sulfate powder to10 ml of sterile water in a glass beaker.
Shake.
2. Dissolve the ferric sulfate base in the solution by stirring with a glass stick. The
solution should become crystal clear.
3. Weigh the glycerol starch (see preparation instructions below) in a glass mortar.
Mix well.
4. Slowly add the ferric sulfate solution to the glycerol starch constantly mixing to get a
homogeneous mixture.
5. Place in a 25-ml brown glass bottle.

Note: Most clinics prefer to leave the stopper of the bottle loose, to allow the mixture
to evaporate until it has a sticky paste-like consistency and looks like mustard. This
may take 2-3 weeks, depending on the environment. The top of the bottle can then
be secured for storage. If necessary, sterile water can be added to the paste to thin it.
Label: Monsel’s paste
Store in a cool place
For external use only
Use by: [day/month/year] (one year from date of preparation)
7.5 Tissue Sampling 117

Fig. 7.13 Small electrosurgical loop for targeted biopsy,

performed as in-office procedure (LEEP)
Fig. 7.14 Ring curette (Stiefel®) for endocervical curet-
tage in comparison to a conventional instrument

solution was low. Although bleeding was reduced

significantly for 6 h after biopsy, it did not reduce more times in order to sample all four quadrants
pain or increase overall satisfaction. before the tissue is transferred into the fixative.
Targeted colposcopic biopsies may also be Endocervical curettage is always done while
obtained by using a small electrosurgical loop looking through the colposcope.
(Fig. 7.13). In some cases, a small lesion can be The use of a ring curette (Stiefel® curette) is
removed completely as an in-office gynecologi- recommended (Fig. 7.14). Originally, this type
cal procedure. of curette has been designed for superficial skin
biopsies in dermatology. Its sharp scalpel-like
edge ensures that a sufficient amount of tissue is
7.5.2 Endocervical Curettage obtained. If the cervical canal is narrow, the ring
of the curette can be compressed using a sterile
If the squamocolumnar junction is not visible, clamp (Fig. 7.15).
or if there is cytologic evidence of abnormal Drescher et al. (1983) found a positive endo-
endocervical gland cells, endocervical curet- cervical curettage result in 17.9% of patients
tage (ECC) is performed. Usually, endocervical with a visible squamocolumnar junction (T1/
curettage is done without anesthesia, because the T2), including several patients with a normal-
internal cervical os does not need to be dilated appearing ectocervix. While some colposco-
as with regular D&C. A narrow curette is used, pists regularly perform endocervical curettage,
which can be inserted into the cervical canal others rely on the cytological smear, which is
without resistance. obtained by inserting a brush into the endocervi-
When pulling back the curette, pressure is cal canal. According to Weitzman et al. (1988),
applied. The curette is then washed out in nor- the diagnostic information of both methods is
mal saline, and the procedure is repeated three comparable.

Liu et al. (2017) recommend routine ECC in women aged 45 years or older with HPV 16 infec-
tion. All women aged 30 years or older should receive endocervical curettage, if any of the follow-
ing criteria are met:
• HSIL (or higher) or ASC-H
• Colposcopy: major change
• ASC-US or LSIL, when the entire transformation zone is not visible (T3)
118 7 Colposcopic Examination

Fig. 7.15 If the cervical canal is narrow, the ring of the Fig. 7.16 Photographic documentation through the eye-
Stiefel® curette can be compressed using a sterile clamp piece of the colposcope

7.6 Documentation

To document colposcopic findings, a drawing

can be made outlining the localization and shape
of abnormal areas and illustrating the biopsy
site. An essential component of all documenta-
tion is the localization of the squamocolumnar
junction or a statement as to whether the junc-
tion is completely visible (T1) or can be visual-
ized (T2).
Photographic documentation has many advan-
tages. For continuous learning, the colposcopist
should always compare colposcopic findings and Fig. 7.17 Photographic documentation through the eye-
piece of the colposcope. Major change with irregular
histological results.
punctation and coarse mosaic
For image or video capture, several techni-
cal solutions are available, including the three-
dimensional recording of slides. With most
compact digital cameras, images can be taken
directly through the eyepiece of the optical
colposcope (Figs. 7.16 and 7.17). This works
just as well with a microscope as with any
colposcope.
Figure 7.18 depicts a custom-made smart-
phone adapter, which can be adjusted to both
microscopes and colposcopes.

Fig. 7.18 Smartphone adapter attached to the eyepiece

of a standard colposcope
7.7 Practical Colposcopic Examination Step by Step 119

7.7 Practical Colposcopic

Examination Step by Step

Step #1 Take patient’s history See Table 7.1

Step #2 Inform the patient about the exam Providing information and addressing the patient’s
and obtain informed consent concern (fertility, hysterectomy, cancer) prior to
colposcopy will greatly reduce anxiety
Step #3 Label the specimen containers and Always label containers prior to obtaining the
slides specimens to avoid any mix-up
Step #4 Inspect vulva and anus CIN, HSIL of the vulva, AIN, and condylomata
acuminata are all HPV-associated and can occur
simultaneously
Step #5 Insert the speculum Use a Cusco or Grave speculum that is wide enough
to allow adequate colposcopic assessment. Use a
proper technique for handling the speculum to avoid
unnecessary discomfort
Change gloves. From now on, touch colposcope
and instruments only
Step #6 Adjust the position of the Place the colposcope in a comfortable working
gynecologic exam chair and focus position and adjust the patient’s position on the
the colposcope examination chair accordingly (not vice versa)
Step #7 Inspect cervix and upper vagina Use different magnifications. Use green filter. Start
with photo or video documentation
Step #8 Obtain Pap smear Remove cervical mucus and take Pap smear under
colposcopic guidance
See Sect. 7.4 about indications for Pap test during
colposcopic triage
Step #9 Apply 5% acetic acid solution The optimal time to identify HSIL and LSIL
lesions is 1 min after the application of acetic acid.
Colposcopic observation should not be longer than
about 3 min because of fading of the acetic acid
effect (Hilal et al. 2020)
Step #10 Inspect cervix and upper vagina Step #10 is the most important part of colposcopic
again assessment, as minor and major changes, and
sometimes cancer, may be identified
Spread the cervical canal with Kogan speculum or
other suitable device if required
Step #11 Photo/video documentation Alternatively, a drawing by hand is also acceptable
Step #12 Apply Lugol’s solution After Lugol’s solution, repeat or continue photo/
video documentation
Step #13 Obtain biopsy and/or endocervical If in doubt, obtain tissue samples. Always consider
curettage under colposcopic biopsy of grade 1/minor change lesions
guidance
Step #14 Ensure hemostasis Use Monsel’s solution. In case of heavier bleeding,
insert tampon
Step #15 Inspect the vagina The middle and lower one-third of the vagina is
inspected when slowly retracting and rotating the
speculum. If VAIN is suspected, Lugol’s solution
is applied
120 7 Colposcopic Examination

Step #16 Bimanual transvaginal and This is not a routine part of colposcopic triage
transrectal examination when there is no evidence of malignant disease
Step #17 Check for vasovagal reaction before Ask the patient if she has had a vasovagal reaction
the patient gets out of the before. Watch for symptoms and be prepared
examination chair
Step #18 Document the colposcopic findings This is another important element to decrease
and explain them to the patient patient’s anxiety levels
Step #19 Write final report with further This should be done in a timely fashion. The
management recommendations patient should also receive a copy of the report

acetic acid during colposcopy: a prospective study.

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cytology at the time of first colposcopya retrospective 017-4369-x
analysis of 1,087 cases. Am J Clin Pathol 135(4):628– Lellé RJ (2007) Die Münster-Kolposkopielupe - eine
636. [Link] Alternative zum konventionellen Kolposkop. Geburt-
Hilal Z, Rezniczek GA, Tettenborn Z, Hefler LA, Temp- shilfe Frauenheilkd 67:154–155
fer CB (2016) Efficacy of monsel solution after Liu AH-C, Walker J, Gage JC, Gold MA, Zuna R,
cervical biopsy: a randomized trial. J Low Genit Dunn ST et al (2017) Diagnosis of cervical pre-
Tract Dis 20(4):312–316. [Link] cancers by endocervical curettage at colposcopy
LGT.0000000000000234 of women with abnormal cervical cytology. Obstet
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trial. Obstet Gynecol 130(2):411–419. [Link] bring S, Boehmer G, Gieseking F et al (2015) Util-
org/10.1097/AOG.0000000000002127 ity and reproducibility of the international federation
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Meisels A (1990) Are two smears better than one? Letter cal cancer control. A guide to essential practice, 2nd
34(3):459–460 edn. Springer, New York
Monsel ML (1856) Propriété hémostatique du sul- Young NA, Naryshkin S, Bowman RL (1993) Value
fate de peroxyde de fer. Recueil Mémoirs Méd 17: of repeat cervical smears at the time of colposcopic
424–434 biopsy. Diagn Cytopathol 9(6):646–649
Panos JC, Jones BA, Mazzara PF (2001) Usefulness of Zardawi IM, Rode JW (2002) Clinical value of repeat
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biopsy. Diagn Cytopathol 25(4):270–273 46(3):495–498
Weitzman GA, Korhonen MO, Reeves KO, Irwin JF,
Carter TS, Kaufman RH (1988) Endocervical brush
 Operative Colposcopy
8

Contents
8.1 Risks for Future Pregnancies 124
8.2 Surgical Procedures 125
8.3 Assessment of Complete Removal of Cervical Dysplasia 128
References 131

The use of the colposcope during surgery

The term “operative colposcopy” refers to for cervical intraepithelial lesions has a direct
the use of the colposcope during ablative impact on the size of resection.
and/or destructive treatment of cervical, According to Heineman et al. (2016), col-
vaginal, and vulvar precancerous lesions. poscopic guidance led to significant reduction of
In the following chapter, various proce- the height and diameter of the excised specimen.
dures such as cold-knife conization, LEEP, However, in this retrospective study, no significant
cryosurgery, and CO2 laser vaporization reduction of the rate of treatment failure could
are described. Also, follow-up assess- be demonstrated. A prospective randomized trial
ment of successful removal of squamous (Hilal et al. 2018) confirmed that intraoperative
and glandular precancerous lesions is colposcopy significantly reduces the amount of
discussed. tissue removed. At the same time, margin status
is not compromised, i.e., in the groups with and
without intraoperative colposcopy, R0 resection
The term “operative colposcopy” refers to the was achieved in more than 80% of patients.
use of the colposcope during all procedures for To date, the treatment of precancerous
treatment of precancerous lesions of the lower lesions of the cervix is always done surgically,
genital tract including perianal or anal lesions. as there are no effective drugs for conservative
Under colposcopic magnification, the affected treatment. A therapeutic HPV vaccine is not yet
tissue can be completely and gently removed available.
avoiding unnecessary tissue loss. Ideally, all Imiquimod, which is used for topical treat-
gynecologists performing these procedures, ment of condylomata acuminata, is a potential
which are often regarded as “minor surgery,” candidate for medical treatment of HPV-induced
should be trained in colposcopy. precancer. A study on topical application of

© Springer Nature Switzerland AG 2023 123

R. J. Lellé, V. Küppers, Colposcopy, [Link]
124 8 Operative Colposcopy

imiquimod (Aldara®) showed that 73% of high- results compared to the standard resection group
grade lesions of the cervix responded to treatment (80% versus 78%), although margins were more
compared to 39% in the placebo group (Grimm frequently involved (20% versus 8%).
et al. 2012). However, patients require imiqui-
mod treatment for at least 16 weeks. In com-
parison, a colposcopically guided tissue-sparing 8.1 Risks for Future Pregnancies
cervical resection will take only a few minutes to
perform and is much more effective with a suc- Kyrgiou et al. (2006) came to the conclusion that
cess rate of over 90%. all ablative surgical procedures—with the excep-
Van de Sande et al. (2018) initiated a non- tion of CO2 laser vaporization alone—lead to a sig-
inferiority trial on imiquimod treatment of nificantly increased rate of premature births with
patients with recurrent cervical intraepithelial a relative risk of delivery before the 37th week of
neoplasia in order to avoid potential adverse out- gestation of 2.59 after conization and 1.70 after
comes of future pregnancies caused by multiple loop resection (LEEP). Many clinicians and several
cervical resections. A 5% imiquimod cream was national guidelines, including Germany, consider
used intravaginally three times a week for 16 the classic cold-knife conization obsolete. Arbyn
weeks. Patients in the standard arm were treated et al. (2008) also came to the conclusion that peri-
by LLETZ. Ultimately, this prospective random- natal mortality is increased after cold-knife coniza-
ized trial concept had to be changed as not enough tion and presumably also following laser surgery or
patients agreed to randomization (Koeneman extensive electrosurgical resections.
et al. 2017). Obviously, a strong patient prefer- Especially for treatment of reproductive
ence existed for either modality. Therefore, the age patients, the scalpel should not be used
study had to be continued in a nonrandomized anymore.
open-label design. Treatment was successful in Ortoft et al. (2010) found that conization
95% of patients who opted for loop resection, increases perinatal mortality by a factor of 2.8.
whereas topical imiquimod treatment was suc- This is due to the fact that the likelihood of
cessful in 59% only. Furthermore, all patients extreme prematurity (delivery before the 28th
treated with imiquimod reported side effects, week) is increased by a factor of 4.9. Thirty-
most frequently headaches and fatigue. seven patients underwent two consecutive cervi-
In summary, ablation or destruction of the cal resections. For those, the risk of premature
dysplastic epithelium remains the treatment delivery was tenfold higher than in women with-
of choice for persistent high-grade squamous out previous conization.
intraepithelial neoplasia of the cervix. Cone length appears to play an important role.
It is unclear how much cervical tissue should If the specimen is longer than 1 cm, Simoens
be removed as there are no reliable data available. et al. (2012) found a statistically significant
However, Kolben et al. (2019) tried to evaluate increase of premature births. According to Poon
limited excision of the dysplastic lesion (CIN3) et al. (2012), the risk of premature birth can be
in order to limit the amount of resected tissue. estimated by measuring cervical length between
Limited resection was compared to the standard the 20th and 24th week of gestation. However,
approach, i.e., removal of the entire transforma- obstetrical management of patients with a history
tion zone. Unfortunately, the prospective ran- of surgery for CIN is not clear (Quenby 2010).
domized trial was terminated early after inclusion In contrast to the studies mentioned above,
of 50 patients in each group due to slow accrual. Castanon et al. (2012), who analyzed data from
However, preliminary results seem to favor lim- the UK health system, did not find a significant
ited resection. As was to be expected, cone size increase in preterm deliveries following loop
was significantly lower in the limited resection excision of cervical dysplasia and suggested that
group (1.02 ccm versus 1.97 ccm, respectively). this might be related to British quality manage-
After 6 months of follow-up, this patient group ment and the fact that resection volume was kept
did not have a lower rate of HPV-negative test to a minimum.
8.2 Surgical Procedures 125

A systematic review and meta-analysis involv- The goal of all surgical procedures for prema-
ing 36,954 patients treated during pregnancy and lignant disease of the lower genital tract is the
1,794,174 controls still leaves several questions complete removal of all dysplastic epithelium
unanswered (Jin et al. 2013). LEEP was in fact without excessive damage to healthy tissue. This
associated with a risk of preterm delivery both can be achieved either by resection or tissue
before week 32 and week 28. However, although destruction or a combination of the two.
a cervical length of less than 3 cm occurred sig- According to the recommendations of the
nificantly more frequently in treated patients, a IFCPC (Bornstein et al. 2012), nonstandardized
larger resection volume or depth was not found terms such as “conization” or “loop excision”
to be associated with an increased risk of pre- should be avoided. Instead, excision types 1, 2,
term delivery. Also, perinatal mortality was not and 3 are defined in analogy to the three types of
increased by prior LEEP. transformation zones: T1, T2, and T3.
Preoperative colposcopic diagnosis and col- The goal of surgical treatment of premalig-
poscopically guided surgery are potentially able nant disease of the cervix is to cause as little
to reduce the rate of pregnancy complications. damage as possible to healthy tissue. This
Despite the findings quoted above, it might still is especially important in reproductive age
be useful to document the resection volume. patients, as any type of cervical resection has
The tissue volume can easily be measured by the potential to significantly increase the risk
determining its fluid displacement according to of premature delivery and thus the risk of
Archimedes’ principle immediately following perinatal morbidity and mortality. In young
the procedure (Fig. 8.1). patients, the resection volume should be as low
as possible without compromising complete
removal of disease. Wide resection margins,
which are essential in oncologic surgery, must
be considered overtreatment in precancerous
lesions and should be avoided.

8.2 Surgical Procedures

8.2.1 Cold-Knife Conization

Through cold-knife conization, a more or less

cone-shaped piece of cervical tissue is obtained
by using either a regular scalpel or an angled
conization scalpel. Usually, the goal of cold-
knife conization is the complete removal of the
transformation zone and not just a limited area
of the cervix. This fairly bloody procedure can-
not be performed under colposcopic guidance,
and it is usually done under general or spinal
anesthesia.
Staining with Lugol’s iodine serves as a guide
to the position of the transformation zone. Most
Fig. 8.1 Measurement of the resection volume of LEEP surgeons place lateral cervical sutures at 3 and 9
using the Archimedes’ principle. From a sealed 5 or 10 ml
o’clock to reduce bleeding by ligating the lower
syringe, the plunger is removed and half filled with saline.
The removed tissue is placed into the saline. The increase branches of the uterine artery. Instead of mono-
of the fluid level is equal to the resection volume polar coagulation, the so-called Sturmdorf suture
126 8 Operative Colposcopy

used to be performed for hemostasis. By placing LEEP can and should be performed under
sutures anteriorly and posteriorly, the edges of colposcopic guidance. Hemostasis is achieved
the remaining cervix are folded inward in order by monopolar coagulation. For loop resection,
to compress the cervical surface for hemostasis. the electrical generator should be set on a low
However, the Sturmdorf procedure will make it coagulation current so that the coagulation zone
more difficult to evaluate the cervix during fol- of the tissue margins comprises only a small
low-up colposcopic exams so that it is preferably number of cell layers. Thus, histopathological
to use monopolar coagulation instead of sutures. assessment of resection margins will not be
Kalliala et al. (2007) conducted a retrospec- impaired.
tive study over a 30-year period and determined There is no doubt anymore that loop resec-
that cold-knife conization is the least effective tion is as effective as cold-knife conization for
method compared to all other surgical treatment treatment of cervical intraepithelial neopla-
options with regard to prevention of recurrences sia as shown by a meta-analysis (Jiang et al.
(severe dysplasia or carcinoma). Santesso et al. 2016). This applies to all parameters stud-
(2016) compared data on cold-knife coniza- ied: recurrence, positive margins, postopera-
tion, LEEP, and cryotherapy for the treatment of tive bleeding, and the occurrence of cervical
CIN. The systematic review and meta-analysis stenosis.
were disappointing as mostly low-quality evi- Following tissue removal by LEEP/LLETZ,
dence was identified with a lack of prospective an endocervical curettage is performed. A narrow
randomized studies, most importantly regarding curette is used so that no cervical dilatation is
pregnancy outcomes. Recurrence rate was 5.3% required. Neither inspection of the uterine corpus
12 months after LEEP or cryotherapy compared (hysteroscopy) nor endometrial curettage is nec-
to only 1.4% following cold-knife conization. essary and would only increase the complication
However, complications including long-term rate. However, if the cytopathologist suspects an
pregnancy-related problems appeared to occur endometrial lesion, both endocervical and endo-
more frequently after cold-knife conization. metrial curettage are done.
In addition to cold-knife conization and
LEEP/LLETZ, which preserve the resected tis-
8.2.2 Monopolar Loop Resection
sue for further histopathologic evaluation, local-
(LEEP)
ized tissue destruction with cryosurgery or CO2
laser vaporization is also acceptable, when cer-
With LEEP (Loop Electrosurgical Excision
tain criteria are met.
Procedure) or LLETZ (Large Loop Excision
of the Transformation Zone), resection of
either the entire transformation zone or of a
specific lesion is possible using loops of differ-
8.2.3 Cryosurgery
ent sizes (Fig. 8.2).
For cryosurgery of cervical lesions, a cryo-
probe is chosen that covers the entire transfor-
mation zone. Nitrous oxide or liquid nitrogen
is passed through this probe. Decompression of
the liquid gas leads to very low temperatures.
Freezing of the transformation zone causes tis-
sue destruction, and the patient will notice a
strong vaginal discharge in the days following
the treatment.
Cryosurgery is an inexpensive method
that is performed without general anesthe-
Fig. 8.2 Electrosurgical loops of different sizes for the sia. However, it is not suitable for treatment
“loop electrosurgical excision procedure” (LEEP) of large cervical lesions, although the exact
8.2 Surgical Procedures 127

extent of tissue destruction is difficult to • The entire squamocolumnar junction must be

control. visible (preferably T1).
• There should be no suspicion of invasive dis-
ease, neither on colposcopy, cytopathology,
8.2.4 CO2 Laser Vaporization nor tissue biopsy.

The CO2 laser beam has similar physical prop-

erties as light beams from the visible spectrum.
A system of mirrors is used to direct the laser
beam from the generator tube to the manipulator
device that is attached to the colposcope. With a
joystick-controlled mirror, the laser beam can be
directed onto the target tissue with a high degree
of precision. Since the CO2 laser beam itself is
invisible, a red laser pointer is projected into the
beam path (Fig. 8.3).
If the entire transformation zone is to be
vaporized, it is treated from dorsal to ventral. The
laser beam is set to 15–25 W and defocused as
appropriate. The depth of vaporization should be
7 mm in order to treat all dysplastic tissue within
the glandular crypts (Fig. 8.4).
Treatment modalities such as cryosurgery
or CO2 laser vaporization that destroy tissue
rather than make it available for histopathologic
evaluation are only acceptable, if the following Fig. 8.4 Cervix immediately after CO2 laser vaporization.
prerequisites are met: Destruction of the tissue should be about 7 mm in depth

Fig. 8.3 CO2 laser

vaporization of the
cervix. The laser is
attached to the
colposcope and can be
controlled very precisely
128 8 Operative Colposcopy

• The area to be treated must be clearly identifi- Depending on the location of a precancerous
able und fully visible. squamous epithelial lesion, resection by LEEP
• HSIL/CIN2/CIN3 must have been unequivo- may be combined with localized tissue destruc-
cally confirmed by targeted biopsy. tion using CO2 laser vaporization. When using
• There must be no significant disagreement this approach, histological evaluation of the mar-
between the different diagnostic modalities gin status is pointless.
(cytopathology, colposcopy, histopathology). If a positive resection margin is identified
on histopathologic evaluation of squamous
LEEP resection may be combined with dysplasia, immediate repeat surgery is not
CO2 laser vaporization. The peripheral part indicated and should be avoided, especially in
of the transformation zone can be vaporized, reproductive age patients.
whereas the center is removed with the loop, Repeat resections are associated with a 1:36
as the area close to the cervical canal is more risk of preterm birth before the 28th week of ges-
likely to be affected by high-grade dysplasia. tation in subsequent pregnancies (Ortoft et al.
If the CO2 laser beam is focused, it can also be 2010). On the other hand, 78% of patients will no
used to excise the tissue instead of vaporizing it. longer have high-grade dysplasia on follow-up,
Thus, “laser conization” is another alternative to although specimen margins had been positive for
cold-knife conization. CIN3 (Reich et al. 2002).
If the transformation zone is not fully vis-
ible postoperatively and/or low-grade dysplas-
8.3 Assessment of Complete tic changes are present at the resection margins,
Removal of Cervical this is not an indication for repeat surgery as it
Dysplasia is unlikely that these patients still have CIN2+
disease, and a wait-and-see strategy is recom-
8.3.1 Squamous Dysplasia mended (Day et al. 2008).
A meta-analysis of 97 eligible studies (Arbyn
The primary goal of all surgical procedures et al. 2008) found a recurrence rate of 6.6% for
described above is complete removal of all dys- CIN2+ lesions. The recurrence rate was sig-
plastic epithelium. In invasive cancer treatment, nificantly higher if resection margins had been
histologically free margins are a benchmark for positive.
complete removal (R0). However, cervical dys- However, postoperative HPV positivity
plasia should not be treated like invasive cancer. more accurately predicts recurrence risk than
Whereas CIN1 usually does not require treat- positive margins at the time of surgery.
ment, HSIL (CIN2 and CIN3) should be surgi- In women beyond reproductive years,
cally treated without removing too much of the hysterectomy is sometimes recommended as
surrounding healthy tissue, especially in repro- a definitive treatment of cervical dysplasia.
ductive age patients. However, there is a significant risk of subse-
This can be best achieved by performing the quent vaginal intraepithelial dysplasia.
resection using a colposcope. Under colposcopic Schockaert et al. (2008) conducted a retrospec-
guidance, the abnormal tissue is removed in its tive analysis of women with a diagnosis of CIN2+
entirety, but without a wide safety margin. It is and no previous history of VAIN who underwent
not uncommon that the dysplastic lesion reaches hysterectomy within 6 months of CIN diagnosis.
the resection margins of the histopathological The incidence rate of subsequent VAIN2+ was
specimen. However, this does not mean that the 7.4% including two patients with carcinoma of the
lesion has not been removed completely, as the vagina. Thus, Schockaert et al. came to the con-
cervix needs to be coagulated for hemostasis, so clusion that hysterectomy in patients with CIN2+
that additional tissue will be eliminated. cannot be considered as a definitive treatment.
8.3 Assessment of Complete Removal of Cervical Dysplasia 129

Follow-up of patients treated for high-grade Follow-up recommendations will vary widely
cervical lesions is crucial as there is an increased between different countries, and no definitive
risk of death from cervical or vaginal carcinoma recommendation can be given on follow-up inter-
for patients treated for CIN3. Using the data of vals. The German guideline quoted above recom-
the Swedish Cancer Registry from 1958 to 2008, mends co-testing (Pap smear and HPV) after 6,
Strander et al. (2014) estimated a mortality risk 12, and 24 months, respectively. If either test is
of 2.35 (95% CI 2.11–2.61) compared to the gen- abnormal/positive, colposcopy is done.
eral population.
Rather than using margin status as a predic-
tor of persistent disease, the cervix should be 8.3.2 Adenocarcinoma In Situ (AIS)
reassessed colposcopically after it has healed
completely, for example, after about 3 months. All the above considerations relate to squamous
If the endocervical curettage had been positive cell precancers. However, follow-up for adeno-
for squamous dysplasia at the time of surgery, carcinoma in situ treated by loop resection is
endocervical curettage should be performed. much more challenging. Several considerations,
Alternatively, an intracervical brush cytology especially regarding surgical margin status and
specimen may be obtained. the role of hysterectomy, are different.
Integrating the HPV test into follow-up care is The safest treatment option for patients
also helpful (Kocken et al. 2012), as demonstrated with adenocarcinoma in situ after childbear-
for the UK health-care system (Legood et al. 2012). ing age is still hysterectomy, especially for
The authors of the German guidelines for cervi- extensive and/or multifocal AIS.
cal cancer prevention presented a meta-analysis Of course, for younger women who desire to
on the use of the HPV test for follow-up (https:// preserve their fertility, this is not an acceptable
[Link]/uploads/tx_szleitlinien/015- treatment option.
027OLl_Praevention_Zervixkarzinom_2020-03- In Australia, cold-knife conization is consid-
[Link]; viewed April 2021). Seventeen ered the standard of care for adenocarcinoma
relevant studies were identified, 15 of which were in situ in patients who wish to preserve fertility.
prospective studies. The sensitivity and specificity However, according to a retrospective study by
of Pap smear, HPV test, or combined testing for Munro et al. (2015), loop excision (LEEP) was
the prediction of persistent/recurrent disease were equally effective. The only determinant of persis-
as follows: tence or recurrence of AIS was the presence or
absence of a positive margin.
Pap smear alone
With loop excision, the cervical lesion should
Sensitivity: 72.0% 95% CI: 65.6–77.5%
be removed in its entirety. Ideally, surgical mar-
Specificity: 84.6% 95% CI: 80.7–87.9%
HPV test alone
gins should be free of disease on histopathology
Sensitivity: 94.3% 95% CI: 88.4–97.3% (Hopkins 2000). Excision using an adequately
Specificity: 80.0% 95% CI: 74.2–84.8% sized loop is the treatment of choice, preferably
Pap smear plus HPV test applying a one-pass technique to avoid fragmen-
Sensitivity: 95.3% 95% CI: 88.1–98.2% tation of the specimen (van Hanegem et al. 2012).
Specificity: 69.6% 95% CI: 61.7–78.5% In a meta-analysis of 1278 patients with cervi-
cal adenocarcinoma in situ (Salani et al. 2009),
The German guidelines recommend the com- 19.4% of patients with positive margins devel-
bination of both tests as it traditionally relies oped recurrence compared to 2.6% of women
heavily on cytological testing. However, there is with negative margins. In addition, the authors
no doubt that the much more sensitive of the two found a higher likelihood of invasive adenocarci-
test modalities is the HPV test. noma when margins were positive (5.2% versus
130 8 Operative Colposcopy

0.1%). Costa et al. (2007) report on 42 patients months later, both sensitivity and specificity were
after conservative AIS treatment and a median 100%.
follow-up of 40 months, of whom 17 (40.4%) The Society of Gynecologic Oncology
relapsed. Even if the margins were negative on (SGO), in cooperation with the American
conization, the rate of recurrence was still 19%. Society of Colposcopy and Cervical
On the other hand, Andersen and Nielsen (2002) Pathology (ASCCP), has published guide-
argue against overvaluation of retrospective sur- lines for the diagnosis and management of
gical margin assessment. adenocarcinoma in situ (Teoh et al. 2020).
In any case, the recognizability of recur- According to these recommendations, mar-
rence in AIS and possibly adenocarcinoma is gin status is decisive for the management
significantly worse than in squamous epithelial of patients who wish to preserve the uterus.
lesions. The combination of cytological smear Fertility-sparing treatment is not acceptable
and HPV test is the most effective strategy for if negative margins cannot be achieved, even
these patients, with the HPV test clearly hav- with repeat excisions. Also, hysterectomy is
ing the strongest predictive value. According to recommended after childbearing age, unless
Costa et al. (2012), sensitivity and specificity for HPV tests on follow-up exams are consis-
the combined approach were 90.0% and 50.0%, tently negative.
respectively, for the first follow-up visit after 6 Figure 8.5 summarizes the SGO recommenda-
months. For the second follow-up visit another 6 tions.

Adenocarcinoma in situ
(histological diagnosis)

Excisional procedure
-scalpel or LEEP
-not fragmented
-at least 10 mm long

Positive margins
Negative margins

Repeat excisional procedure

Preservation of fertility desired? feasible and safe?

Yes No No Yes

Surveillance: Simple hysterectomy

Pap + HPV + ECC
every 6 months for 3 years,
then annually until
hysterectomy

Hysterectomy
after completion of
family planning
or
continuous monitoring
if HPV consistently negative

Fig. 8.5 Management of adenocarcinoma in situ of the cervix with or without negative margins after excision.
According to these recommendations, a negative margin status is decisive for uterus-conserving treatment
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Ortoft G, Henriksen T, Hansen E, Petersen L (2010)
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After conisation of the cervix, the perinatal mortal-
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 Colposcopy of the Surgically
Treated Cervix 9

Contents
9.1 State of Preservation of the Cervix 134
9.2 Visibility of the Transformation Zone 135
9.3 Stenosis of the Cervical Canal 137
9.4 Colposcopic Diagnosis of Persistent or Recurrent Cervical Dysplasia 138
References 142

Colposcopy plays a crucial role in the diagno- pregnancies. Therefore, a certain percentage of
sis of persistence or recurrence of intraepithe- patients with recurrent disease is acceptable.
lial neoplasia. As the appearance of the cervix Soutter et al. (Soutter et al. 2006) found a
changes significantly after surgical treatment recurrence rate of 3%. Recurrence was defined
of dysplasia, this presents an additional chal- as CIN2+ disease. The authors recommend post-
lenge for colposcopy. operative colposcopy as an adjunct to cytologi-
cal examination to increase the sensitivity for
the detection of high-grade disease. In this retro-
The surgeon who performs the dysplasia spective study, sensitivity was 91% for patients
treatment should see the patient at least once undergoing both colposcopy and Pap smear
for follow-up colposcopy. compared to only 64% for cytological follow-up
Thus, the surgeon can continuously check only. With the combined approach, specificity
on his own technique, regarding the functional dropped from 95% to 88%. Soutter et al. con-
preservation of the cervix as well as the success clude that the benefit of colposcopy would be
rate of dysplasia treatment. The primary objec- greater in women with a higher risk of treatment
tive is complete removal of the dysplastic lesion. failure, for example, patients with involved tis-
However, for all women of childbearing age, it sue margins.
is equally important to limit the amount of tissue Colposcopic examination following treatment
resection. Naturally, the more tissue is removed, for dysplasia is best done after 10 to 12 weeks. At
the less likely dysplasia will persist or recur. On this time, it can be safely assumed that the cervix
the other hand, a large resection volume will be has healed completely (Fig. 9.1) and can be opti-
more likely to cause complications for future mally assessed colposcopically.

© Springer Nature Switzerland AG 2023 133

R. J. Lellé, V. Küppers, Colposcopy, [Link]
134 9 Colposcopy of the Surgically Treated Cervix

ing surgery had been positive, ECC should be

repeated. All visible abnormalities that are iden-
tified colposcopically, including both major and
minor changes, need to be biopsied.

9.1 State of Preservation

of the Cervix

Postoperatively, the cervix of reproductive age

women should still protrude above the level of
the vagina and be shaped normally. Figure 9.2
shows an example of an acceptable postopera-
tive result following cold-knife conization. In
contrast, in Figs. 9.3 and 9.4, hardly any cer-
vical tissue is visible, and the opening of the
cervical canal is within the plane of the vaginal
wall.

Fig. 9.1 Cervix 7 days after LEEP. The 25-year-old

patient underwent surgery for microcarcinoma of the cer-
vix (FIGO stage Ia1). As suturing of the cervix is avoided,
there is an open wound. Any attempts of colposcopic or
cytological assessment would be misleading

Colposcopic examination after surgical dys-

plasia treatment of the cervix should address the
following questions:

– How well is the cervix preserved?

– Can the transformation zone visualized
completely?
– Is there stenosis of the cervical canal?
– Is there any colposcopic evidence of persistent
cervical dysplasia?
– Is there evidence for vaginal intraepithelial
neoplasia?

If the colposcopic exam is normal, a Pap

Fig. 9.2 Acceptable result after cold-knife conization of
smear including an intracervical brush specimen the cervix. The 39-year-old female had a CIN3 lesion
is obtained. If the endocervical curettage dur- removed 8 weeks ago
9.2 Visibility of the Transformation Zone 135

9.2 Visibility of the

Transformation Zone

Ideally, the entire transformation zone should be

visible postoperatively with or without manipula-
tion of the cervical os (T2 and T1, respectively).
Unfortunately, this is not always possible regard-
less of the surgical technique used. In a Cochrane
meta-analysis, Martin-Hirsch et al. (2000) were
unable to identify a superior surgical technique.
Also, the frequency of cervical stenosis did not
differ. Only with CO2 laser vaporization there
was a tendency of less frequent stenosis and the
more frequent visualization of the entire transfor-
mation zone.
In Figs. 9.5, 9.6, and 9.7, there has been opti-
mal postoperative regeneration of the cervix so
that the squamocolumnar junction is completely
visible.
Fig. 9.3 Cervix after previous LEEP. The patient is only
26 years old and underwent surgery 3 years ago. Most of
the ectocervix had been removed

Fig. 9.4 Cervical stenosis after three previous LEEP in a 36-year-old patient
136 9 Colposcopy of the Surgically Treated Cervix

Fig. 9.5 Visible squamocolumnar junction (T1) 6 months after LEEP

Fig. 9.6 New transformation zone 3 months after coniza-

tion. The 34-year-old woman has been treated for CIN3.
There is a significant tissue defect. The squamocolumnar
junction is visible
9.3 Stenosis of the Cervical Canal 137

Fig. 9.7 Acceptable result 11 months after LEEP for HSIL/CIN3 in a 29-year-old patient. The squamocolumnar junc-
tion can be visualized with the Kogan speculum

9.3 Stenosis of the Cervical

Canal

Narrowing of the cervical canal can be an unwel-

come consequence of cervical tissue resections
(Fig. 9.8).
Per definition, cervical stenosis is present
when a 2.5 mm Hegar dilator cannot be inserted
(Christianson et al. 2008). Thus, the cytological
brush usually cannot pass the cervical os.
If follow-up Pap smears can only be obtained
from the ectocervix, this information should be
relayed to the cytopathologist, and the Pap smear
result may be less accurate.
Retrospective data obtained by Hasegawa
et al. (Hasegawa et al. 2016) show a high rate of
cervical stenosis following conization. Stenosis
occurred significantly more frequently in post-
menopausal than in premenopausal patients
(59.1% versus 8.3%). This may in part be related
to cone size, as cone length was significantly Fig. 9.8 Persistent cervical stenosis after conization. The
greater in postmenopausal patients. 39-year-old patient underwent conization 6 years previously,
and intracervical cytology could not be obtained anymore. A
Christianson et al. (Christianson et al. 2008) small loop resection (LEEP) was performed to reopen the cer-
summarized several articles on the frequency of vical canal. But 7 months later, the cervix is stenotic again
138 9 Colposcopy of the Surgically Treated Cervix

cervical stenosis: 17% of patients following laser ing age. As a rule, HSIL should be confirmed
conization, 4–19% for loop resections, and 10% histologically by colposcopically guided
or more for cold-knife conizations. However, biopsy prior to any surgical intervention.
cervical stenosis that is severe enough that men- Low-grade dysplasia (LSIL/CIN1) is not an
strual blood is blocked inside the uterus leading acceptable reason for repeat surgery and will
to severe dysmenorrhea is quite rare. unnecessarily increase the complication rate
With cervical stenosis, it is difficult or impos- of subsequent pregnancies.
sible to obtain an intracervical cytological sam- The recurrence rate after treatment for cervi-
ple. However, if the screening Pap smear lacks cal intraepithelial neoplasia is between 5% and
an endocervical component, accelerated repeat 7% (Arbyn et al. 2017; Santesso et al. 2016).
examination is not recommended (Polanco The colposcopic assessment criteria after dys-
Jacome et al. 2018), as the subsequent risk of plasia treatment are basically the same as
high-grade dysplasia or cancer is not significantly prior to surgery. Attention should be paid to
higher compared to patients with Pap smears acetowhite and/or Lugol-negative areas, espe-
containing endocervical and/or metaplastic cells cially if these are located close to the squamo-
(Sultana et al. 2014). columnar junction.
Misoprostol, a prostaglandin E1 analog, is Occasionally, acetowhite and Lugol-
sometimes used intravaginally or orally for cer- negative areas are due to postoperative regen-
vical ripening. In a meta-analysis (Pergialiotis eration. Punctation and mosaic resembling
et al. 2015), misoprostol was able to increase the dysplastic changes may also be seen.
rate of colposcopic exams that could visualize the These findings (Figs. 9.9, 9.10, 9.11, and 9.12)
transformation zone. This conversion rate ranged may be colposcopically indistinguishable from
between 55.5% and 78.9%. However, numbers actual recurrence (Figs. 9.13 and 9.14). Targeted
were too small to draw any definite conclusions. biopsy will confirm or rule out recurrent or per-
Some patients, who are at high risk for devel- sistent disease.
oping intracervical CIN2+ disease, may have to If endocervical curettage performed as part of
undergo diagnostic/therapeutic LEEP. Also, hys- the initial surgical treatment had been positive for
terectomy might be an option in individual cases, dysplasia, curettage should be repeated during
as long as the patient knows about the signifi- the first colposcopic follow-up exam, especially
cant risk of subsequent development of vaginal if the squamocolumnar junction is not visible
intraepithelial neoplasia. (T3). In addition, an intracervical Pap smear
In summary, cervical stenosis, especially fol- should be obtained with a brush.
lowing surgical procedures of the cervix, is still In select cases, LEEP may be performed
a challenging problem, and treatment recommen- without prior histological confirmation of HSIL,
dations are unsatisfactory, as no evidence-based when the Pap smear indicates CIN3 disease and
algorithms can be established (Maier et al. 2017). colposcopy confirms a major change or the squa-
mocolumnar junction is not visible. If no dyspla-
sia is detected histologically, the possibility of
9.4 Colposcopic Diagnosis vaginal intraepithelial dysplasia should be con-
of Persistent or Recurrent sidered and the vagina carefully reexamined with
Cervical Dysplasia particular attention to the vaginal wall adjacent
to the cervix. All Lugol-negative areas should be
Immediate or premature repeat surgery biopsied.
for suspected persistent disease should be The high value of HPV testing in the follow-up
avoided, especially in women of childbear- of dysplasia treatment has already been discussed
9.4 Colposcopic Diagnosis of Persistent or Recurrent Cervical Dysplasia 139

Fig. 9.9 Metaplastic changes 7 months after LEEP. After application of acetic acid (center), there is intense acetowhite
staining with mosaic (major change). Biopsy from this area did not reveal dysplasia

Fig. 9.10 New transformation zone/ectopy following CIN3 treatment (LEEP). The opaque acetowhite area at 12
o’clock with coarse mosaic (major change) suggests persistent or recurrent disease. However, biopsy is negative

previously (Chap. 6). Therefore, histopathologic The benefit of routine colposcopic examina-
evaluation as to whether a dysplastic lesion has tion of all patients 12 months after LEEP has been
been removed completely (free margins) has only questioned by Thompson and Marin (Thompson
a limited predictive value regarding persistence and Marin 2013). In a retrospective analysis, they
or recurrence of dysplasia. Rather, the result of came to the conclusion that the sensitivity of col-
the HPV test should be used to select patients poscopy is low whereas the likelihood of false-
who should undergo colposcopic follow-up after negative findings is high causing unnecessary
dysplasia treatment. follow-up exams.
140 9 Colposcopy of the Surgically Treated Cervix

Fig. 9.11 Postoperative cervical mosaic resembling persistent CIN. The 31-year-old patient underwent LEEP twice.
The last surgery for CIN3 was 3 months ago. There is irregular punctation at 12 o’clock. However, the acetowhite area
is still transparent (minor change)

Fig. 9.12 Cervix with postoperative major change in pregnancy resembling HSIL. The 33-year-old patient is 22 weeks
pregnant and has had LEEP resection for ACIS at 14 weeks of gestation. Biopsy of the major change does not confirm
the suspicion of dysplasia, and the HPV test (HC2) is negative
9.4 Colposcopic Diagnosis of Persistent or Recurrent Cervical Dysplasia 141

Fig. 9.13 HSIL/CIN3 recurrence 6 months after conization in a 31-year-old patient. At 12 o’clock, there is a small area
of regular mosaic (minor change). A biopsy using the Stiefel® curette shows HSIL/CIN2 disease

Fig. 9.14 A 38-year-old patient with recurrent HSIL/CIN3 7 months after LEEP. There is a significant tissue defect. At
12 o’clock, there is prominent leukoplakia, visible both before (left) and after (center) acetic acid application. Otherwise,
the colposcopic features are uncharacteristic
142 9 Colposcopy of the Surgically Treated Cervix

References Polanco Jacome EC, Maerki J, Chau K, Akerman M, Saj-

jan S, Klein M et al (2018) Lack of transformation zone
in cervical Pap tests, should it be a concern? A quality
Arbyn M, Redman CWE, Verdoodt F, Kyrgiou M, Tzaf-
assurance initiative. Diagn Cytopathol 46(7):584–588.
etas M, Ghaem-Maghami S et al (2017) Incomplete
[Link]
excision of cervical precancer as a predictor of treat-
Santesso N, Mustafa RA, Wiercioch W, Kehar R, Gandhi
ment failure: a systematic review and meta-analy-
S, Chen Y et al (2016) Systematic reviews and meta-
sis. Lancet Oncol 18(12):1665–1679. [Link]
analyses of benefits and harms of cryotherapy, LEEP,
org/10.1016/S1470-2045(17)30700-3
and cold knife conization to treat cervical intraepithe-
Christianson MS, Barker MA, Lindheim SR (2008) Over-
lial neoplasia. Int J Gynaecol Obstet 132(3):266–271.
coming the challenging cervix: techniques to access
[Link]
the uterine cavity. J Low Genit Tract Dis 12(1):24–31.
Soutter WP, Butler JS, Tipples M (2006) The role of col-
[Link]
poscopy in the follow up of women treated for cervical
Hasegawa K, Torii Y, Kato R, Udagawa Y, Fukasawa
intraepithelial neoplasia. BJOG 113(5):511–514
I (2016) The problems of cervical conization for
Sultana F, English DR, Simpson JA, Canfell K, Gertig
postmenopausal patients. Eur J Gynaecol Oncol
DM, Saville M (2014) High-grade cervical abnormali-
37(3):327–331
ties and cervical cancer in women following a negative
Maier E, Gieseking F, Lellé RJ (2017) Stenose der Cervix
Pap smear with and without an endocervical compo-
uteri. Praktische Gyn 22:1–6
nent: a cohort study with 10 years of follow-up. Int
Martin-Hirsch PL, Paraskevaidis E, Kitchener H (2000)
J Cancer 135(5):1213–1219. [Link]
Surgery for cervical intraepithelial neoplasia.
ijc.28756
Cochrane Database Syst Rev 2:CD001318
Thompson V, Marin R (2013) Is colposcopy necessary at
Pergialiotis V, Vlachos D-E, Pitsouni E, Perrea D, Vlachos
twelve months after large loop excision of the trans-
GD (2015) Vaginal misoprostol for overcoming inad-
formation zone? A clinical audit. Aust N Z J Obstet
equate colposcopies: a meta-analysis of randomized
Gynaecol 53(6):571–573. [Link]
controlled trials. J Low Genit Tract Dis 19(3):257–
ajo.12153
261. [Link]
 Colposcopy During Pregnancy
10

Contents
10.1 Pregnancy-Related Changes of the Cervix 144
10.2 Management of Cervical Dysplasia During Pregnancy 153
10.3 Conization (LEEP) During Pregnancy 155
10.4 Management of HSIL After Delivery 156
References 156

If the cervical smear is abnormal during of preterm delivery compared to women without
pregnancy, colposcopic triage is crucial CIN. Therefore, it cannot be ruled out that HPV
to rule out invasive disease. Due to physi- infection and CIN constitute independent risk
ologic tissue changes due to pregnancy, factors for the pregnant patient apart from any
colposcopy is particularly demanding. In type of surgical intervention of the cervix.
patients who previously have not had regu- Since HSIL has a low probability of malig-
lar cervical screening, a Pap smear and an nant progression within the relatively short
HPV screening test (≥30 years) should be time period of pregnancy, surgical treatment
obtained at the time of the first office visit. of dysplasia should be postponed until after
If test results suggest CIN2+ disease, col- delivery and until pregnancy-related changes
poscopy is required. have regressed. Thus, the primary objective of
colposcopic diagnosis in pregnancy is to rule
out invasive disease.
Colposcopy with targeted biopsy is feasible at It is important to realize that pregnancy itself
any stage of pregnancy and does not involve an is not to be considered a risk factor for malignant
increased risk for the fetus (Hunter et al. 2008). transformation of HSIL. Preliminary data pre-
Also, the diagnosis of cervical precancer should sented by Ciavattini et al. (2017) even suggest a
not influence obstetrical management, i.e., nei- less aggressive behavior of preneoplastic cervical
ther preterm delivery nor cesarean section is lesions in pregnant patients compared to non-
required (Flannelly 2010; Henes et al. 2013). pregnant patients. Whereas in nonpregnant
However, according to a meta-analysis by women both Ki67 and p16 expression increased
Danhof et al. (2015), the presence of CIN, even significantly with CIN grade, this increase could
when not being treated, confers a significant risk not be observed in pregnant patients.

© Springer Nature Switzerland AG 2023 143

R. J. Lellé, V. Küppers, Colposcopy, [Link]
144 10 Colposcopy During Pregnancy

However, even within a setting where colpos-

copy is readily and timely available during preg-
nancy, advanced stage cervical carcinoma still
occurs. The overall prognosis for cervical cancer
diagnosed during pregnancy is not worse com-
pared to cervical cancer not associated with preg-
nancy. Even an intentional delay of oncologic
treatment until the fetus reaches maturity does
not have a negative impact on prognosis (Takushi
et al. 2002).

10.1 Pregnancy-Related Changes Fig. 10.1 CIN3 in the 35th week of pregnancy. The
of the Cervix opaque acetowhite areas are partially obscured by
increased mucus secretion
Colposcopic evaluation of the cervix is more dif-
ficult during pregnancy due to physiological
changes. Therefore, the colposcopist has to be
familiar with both normal and abnormal col-
poscopic images in pregnancy.
Due to increased fluid retention within the
cervix and subsequent increase of volume, the
squamocolumnar junction can be visualized
more frequently during pregnancy. If the
squamocolumnar junction cannot be seen
during the first trimester, it may well be visible
on repeat colposcopy a few weeks later.
The further along the pregnancy, the more dif-
ficult the colposcopic evaluation becomes. After
the 30th week of pregnancy, adequate col-
poscopic assessment may no longer be possible.
Almost all of the observed changes are due to
the increasing level of estrogen during pregnancy
and may lead to overdiagnosis of dysplasia and
possibly cancer.
As the cervix becomes hypertrophic and
edematous, the tissue assumes a bluish hue. In Fig. 10.2 CIN3 in the 31st week of pregnancy. At the
the Anglo-American literature, this is called anterior (ventral) part of the cervix, the partially trans-
“Chadwick’s sign.” In the nineteenth century, formed glandular epithelium is very prominent and polyp-
ous. By colposcopic inspection alone, invasion cannot be
Chadwick recognized the bluish discoloration as
ruled out
a pregnancy-related change (Gleichert 1971).
The cervical glandular epithelium is particu- changes, may be prominently vascularized and
larly active and prominent during pregnancy. The misinterpreted as suspicious for cancer (Figs. 10.2
transformation zone is gradually shifting toward and 10.3).
the ectocervix. The gland cells secrete a large Stromal cell hypertrophy leads to “pseudo-
amount of cervical mucus, which can complicate punctation.” This should not be confused with the
colposcopic assessment (Fig. 10.1). The glandu- irregular punctation within an acetowhite area
lar epithelium, which undergoes metaplastic that is typically caused by dysplasia (Fig. 10.4).
10.1 Pregnancy-Related Changes of the Cervix 145

The application of acetic acid causes a more

intense white reaction (Figs. 10.5, 10.6, 10.7,
10.8, 10.9, and 10.10). Also, the acetowhite epi-
thelium may be raised above the level of the nor-
mal epithelium (Figs. 10.11 and 10.12).
Sometimes, the intense acetowhite staining has a
“gyrated” appearance (Figs. 10.13 and 10.14).
Since the acetowhite reaction is more pronounced
in both dysplasia and metaplasia, it may be mis-
interpreted as major change.
The high glycogen content of the intermediate
cells of the squamous epithelium, which are
called “navicular cells” by the cytopathologist
Fig. 10.3 CIN3 in the tenth week of pregnancy. Within
the mature transformation zone, the gland openings are
due to their boat-shaped morphology, leads to a
surrounded by a coarse-white epithelium (periglandular deep chestnut-brown coloration on Schiller’s
cuffing) with prominent but not atypical vessels iodine test.
It is relatively common for stromal cells of the
cervix to undergo decidualization (decidual trans-
formation). In fact, Schneider and Barnes (1981)
identified cervical decidua in 30.8% of pregnant
uteri. In one-third of these patients, decidual cells
were detected on the cytological smear.
Both the colposcopic appearance (Figs. 10.15,
10.16, 10.17, 10.18, and 10.19) and the cytologi-
cal features of cervical decidualization may be
misdiagnosed as high-grade cervical dysplasia or
squamous cell carcinoma (van Diepen et al.
2015). In cases like that, targeted biopsy will pro-
vide clarification. Differential diagnosis of decid-
ual polyps protruding from the cervical canal
also includes cervical myoma (Fig. 10.20).
Decidual transformation occasionally occurs
Fig. 10.4 “Pseudo-punctation” of a normal cervix in the
26th week of pregnancy. The punctation is regular, and
outside of pregnancy and may also be confused
there is no acetowhite staining with high-grade dysplasia (Fig. 10.21).

Fig. 10.5 HSIL/CIN2 in the 18th week of pregnancy. The cervix is already enlarged due to physiologic changes in
pregnancy. The entire cervix is acetowhite with a slightly irregular mosaic (major change)
146 10 Colposcopy During Pregnancy

Fig. 10.8 CIN3 in the 24th week of pregnancy (see

Fig. 10.7). At higher magnification, an irregular mosaic is
visible (major change)

Fig. 10.6 CIN3 in the 22nd week of pregnancy. The ace-

towhite reaction is very prominent (major change)

Fig. 10.9 CIN3 in the 21st week of gestation. Almost the

entire anterior (ventral) cervix is acetowhite with raised
borders (major change)

Fig. 10.7 CIN3 in the 24th week of pregnancy. In addi-

tion to “pseudo-punctation” anteriorly (ventrally), there is
a raised and sharply delimited acetowhite area at the pos-
terior (dorsal) cervix (major change)

Fig. 10.10 CIN3 in the 27th week of pregnancy. There is

intense acetowhite opaque staining with sharp borders and
slightly raised epithelium (major change)
10.1 Pregnancy-Related Changes of the Cervix 147

Fig. 10.12 CIN3 in the 8th week of pregnancy. The ace-

towhite epithelium shows coarse punctation and an irregu-
Fig. 10.11 CIN3 in the 25th week of pregnancy. The lar mosaic (major change). Also, there are several inner
acetowhite epithelium has an irregular surface. Multiple border lesions
biopsies should be obtained to rule out invasion

In summary, the following pregnancy-related changes of the cervix are observed colposcopically:
– The cervix becomes increasingly hypertrophic due to fluid retention.
– The squamocolumnar junction gradually shifts toward the ectocervix.
– The mucous secretion of the endocervical epithelium increases.
– Partial decidualization (decidual transformation) of the cervix may occur.
– “Pseudo-punctation” may be observed.
– The local blood flow is increased significantly.
– Local blood flow is increased with prominent vessels.
– The acetowhite reaction of both dysplastic epithelium and immature metaplasia is more
pronounced.
– Lugol’s staining is also more intense as more glycogen is stored.
– Deciduosis occurs frequently and may mimic CIN or cancer.

Occasionally, condylomata acuminata occur vagina, and cervix, cesarean section may be indi-
during pregnancy. Typical condylomata do not cated (Fig. 10.22), unless condylomata can be
necessarily need to be confirmed by biopsy. In eliminated prior to delivery (CO2 laser vaporiza-
the event of extensive involvement of vulva, tion around the 32nd week of gestation).
148 10 Colposcopy During Pregnancy

Fig. 10.13 CIN3 in the 16th week of gestation. There is “gyration” of the intensely acetowhite squamous epithelium
(major change)
10.1 Pregnancy-Related Changes of the Cervix 149

Fig. 10.14 CIN in the 27th week of gestation. The intensely acetowhite area is “gyrated” with a ridge sign at 12
o’clock
150 10 Colposcopy During Pregnancy

Fig. 10.15 Decidual polyp in the 32nd week of pregnancy. One year ago, the 31-year-old patient underwent cone
biopsy for HSIL. There is a considerable tissue defect and scarring. The decidual polyp is Lugol-negative

Fig. 10.16 Decidual polyp in the 32nd week of pregnancy (see Fig. 10.15). At 12 o’clock, there is a small exophytic
lesion with very small and finely branched blood vessels

Fig. 10.17 Decidual polyp in the eighth week of preg- Fig. 10.18 Decidual polyp in the 13th week of preg-
nancy. Differential diagnosis includes incipient spontane- nancy. If in doubt, biopsy should be performed
ous abortion and even invasive disease
10.1 Pregnancy-Related Changes of the Cervix 151

Fig. 10.19 HSIL/CIN2 and deciduosis in the 24th week of pregnancy. At 12 o’clock, there is a typical major change
indicating HSIL. Close to the cervical canal, a slightly exophytic lesion is seen that is bleeding on touch. Interestingly,
the image prior to application of acetic acid rather clearly suggests deciduosis. If any doubt remains, invasive disease
should be ruled out by biopsy
152 10 Colposcopy During Pregnancy

9 weeks of gestation 12 weeks of gestation 16 weeks of gestation

Fig. 10.20 A 34-year-old patient with cervical myoma in pregnancy. The patient has had two previous conizations for HSIL

Fig. 10.21 Decidual transformation outside of pregnancy. Fig. 10.22 Condylomata acuminata of cervix and vagina
This can be caused by progestins. The patient uses a in the 33rd week of pregnancy. Due to the extent of warts,
NuvaRing® for contraception, which releases the gestagen cesarean section should be done
etonogestrel in addition to ethinylestradiol
10.2 Management of Cervical Dysplasia During Pregnancy 153

10.2 Management of Cervical and considerable bleeding from the biopsy site
Dysplasia During Pregnancy may occur. However, hemostasis can be achieved
just as effectively as in nonpregnant patients. It is
Colposcopic examination during pregnancy does helpful to apply pressure on the biopsy site
not differ significantly from the nonpregnant immediately after taking the tissue sample using
patient. However, the blades of the speculum a cotton tip. Then Monsel’s solution is applied. If
need to be sufficiently wide so that the vaginal hemostasis is still unsatisfactory, a tampon is
walls do not impede inspection of the cervix. By loosely inserted into the vagina, and the patient is
placing a protective cover or condom—as used instructed to remove it after 3–4 h.
for the vaginal ultrasound probe—over the spec- In pregnant patients, endocervical curet-
ulum and incising its tip, the vaginal walls can be tage is not advisable to avoid injury of the
further pushed aside. amniotic sac. If the transformation zone is not
Figure 10.23 suggests an algorithm on how to visible, colposcopy is repeated 6–8 weeks later.
proceed during pregnancy, when the Pap smear is If the diagnosis of dysplasia is confirmed,
abnormal. repeat colposcopies are done depending on both
In most cases, a tissue biopsy should be the colposcopic diagnosis and the biopsy result.
obtained during colposcopy as colposcopic A high-grade lesion should be reevaluated every
assessment is less reliable due to tissue changes 8 weeks.
related to pregnancy. Cervical biopsies are During pregnancy, any suspicion of inva-
feasible at any time of gestation. sive disease based on cytopathology and/or
Compared to nonpregnant patients, the proce- colposcopy (Figs. 10.24, 10.25, and 10.26) must
dure is less likely to cause discomfort or pain. be either confirmed or ruled out through tar-
However, the cervix is much better vascularized, geted biopsy or biopsies.

Abnormal Pap smear during pregnancy

colposcopy and biopsy

no dysplasia HSIL microinvasion invasive disease

repeat colposcopy individual treatment

during pregnancy conization (LEEP)
decision
every 8 weeks

colposcopy and biopsy

8 - 12 weeks
postpartum

treatment as usual
outside of pregnancy

Fig. 10.23 Algorithm for triage of an abnormal Pap smear during pregnancy
154 10 Colposcopy During Pregnancy

Fig. 10.24 CIN3 in the 23rd week of gestation. The patient is 29 years old. The surface of the large acetowhite and
Lugol-negative transformation zone is irregular and bleeding on touch. Invasion should be ruled out by (repeat) biopsy

Fig. 10.25 Squamous cell carcinoma in the 11th week of

pregnancy. The 36-year-old patient, gravida I, is free of
symptoms. The Pap smear had been normal. A biopsy
revealed squamous cell carcinoma. The patient opted for
termination of the pregnancy with surgical treatment (rad-
ical hysterectomy). Histological examination revealed a
3.5 cm poorly differentiated squamous cell carcinoma
with lymph-vascular space invasion
10.3 Conization (LEEP) During Pregnancy 155

Fig. 10.26 Squamous cell carcinoma in the 11th week of gestation (see Fig. 10.25). Especially when viewed with the
green filter, vessels are grossly atypical. Also, there is focal keratinization

10.3 Conization (LEEP) During

Pregnancy

Cold-knife conizations or loop resections during

pregnancy are usually not indicated for preinva-
sive disease and should be avoided whenever pos-
sible. However, if invasive disease is suspected
and the diagnosis cannot be clarified through tar-
geted biopsy, a more comprehensive tissue
removal from the cervix may be necessary.
Conization in pregnancy is mainly diagnos-
tic and not therapeutic, as it is usually not pos-
sible to obtain free margins due to pregnancy
changes.
As an alternative to cold-knife conization,
loop resection (LEEP) is also acceptable during
pregnancy (Mitsuhashi and Sekiya 2000). First,
a cerclage suture is placed but not tied. Then the Fig. 10.27 Cervix immediately after conization and cer-
clage during pregnancy
posterior (dorsal) cervical tissue is removed
with an appropriately sized loop followed by
removal of the anterior (ventral) tissue. Finally, is significantly increased by CIN surgery during
the cerclage suture is tied. This will significantly pregnancy, not however when CIN had been pre-
reduce bleeding. Finally, the wound surface is viously treated outside of pregnancy, thus contra-
coagulated (Fig. 10.27). If hemostasis is still not dicting numerous articles quoted above that
satisfactory, absorbable sutures are placed. demonstrate an increased risk after any type of
A meta-analysis by Danhof et al. (2015) came previous cervical resection for preinvasive
to the conclusion that the risk of preterm delivery disease.
156 10 Colposcopy During Pregnancy

10.4 Management of HSIL After References

Delivery
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reevaluated colposcopically. Any abnormal areas nancy status with p16 and Ki-67 protein expression.
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Danhof NA, Kamphuis EI, Limpens J, van Lonkhuijzen
be confirmed again histopathologically after
LRCW, Pajkrt E, Mol BWJ (2015) The risk of preterm
delivery before surgical treatment is performed, birth of treated versus untreated cervical intraepithe-
as CIN2 and CIN3 have a tendency to regress lial neoplasia (CIN): a systematic review and meta-
after birth, although the majority of patients will analysis. Eur J Obstet Gynecol Reprod Biol 188:24–33.
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Gleichert JE (1971) Etienne Joseph Jacquemin, discoverer
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