In this 4-year open-label, noncomparative, single-center pilot efficacy study, the contraceptive ... more In this 4-year open-label, noncomparative, single-center pilot efficacy study, the contraceptive efficacy, safety, bleeding pattern and acceptability of Implanon was studied in 200 sexually active women of proven fertility in Indonesia. All subjects received the single-rod subdermal implant Implanon, which contains 68 mg etonogestrel (3-keto-desogestrel), with an initial release rate of 67 micrograms etonogestrel/day. Contraceptive efficacy was analyzed by calculation of the pregnancy rate, bleeding patterns were determined by the 90-day reference period method, and acceptability by the discontinuation rate. No in-treatment pregnancies were reported during 658.4 women-years of exposure, resulting in a Pearl Index of 0.0 (95% CI 0.0-0.6). The overall bleeding pattern was acceptable, with no discontinuations because of irregular bleeding. Incidence of irregular bleeding was highest during the first two reference periods and decreased thereafter. Amenorrhea was experienced by 7%-12% of subjects during years 1 and 2, by 5%-7% during year 3, and by 2%-5% during year 4, with one discontinuation because of amenorrhea. No clinically significant changes were reported for systolic and diastolic blood pressure, body mass index, and hemoglobin level. Three adverse experiences were related to treatment and resulted in discontinuation (two headaches and one dyspnea). One difficult implant removal was reported. In conclusion, this pilot efficacy study indicates that Implanon provides excellent contraceptive reliability and an acceptable bleeding pattern. Overall safety and acceptability are good, as suggested by the low incidence of adverse experiences and the low discontinuation rate.
Vascular endothelial growth factor (VEGF) expression and the microvascular density of the endomet... more Vascular endothelial growth factor (VEGF) expression and the microvascular density of the endometrium were studied in Norplant users and normal controls, using immunohistochemistry on formalin-fixed paraffinembedded endometrial sections. The VEGF staining index was quantified using computerized image analysis. The VEGF staining index between stages of the menstrual cycle and between normal and Norplant endometria were compared. Norplant VEGF staining index was analysed for correlation with microvascular density, duration of Norplant use, the number of bleeding/spotting days in the reference period up to 90 days prior to biopsy, and the length of time since the last bleeding/spotting episode. The results showed that immunoreactive VEGF was detected predominantly in endometrial glands but weakly expressed in the stroma throughout the menstrual cycle, and also in Norplant users. Large variation in the VEGF staining index between individuals was observed and no significant difference in the VEGF staining index was detected between stages of the menstrual cycle for the glands and stroma. The glandular and stromal VEGF staining indices were significantly higher in Norplant than in normal endometrium (P <1⍥10 -4 ). No correlation was found between the Norplant VEGF staining index and endometrial microvascular density, duration of Norplant use, the number of bleeding/spotting days in the reference period, and the length of time since the last bleeding/spotting episode. The VEGF staining index was higher in glands than stroma for both normal and Norplant endometrium. The results suggest a differential control of endometrial glandular versus stromal VEGF expression, and possible positive effects of levonorgestrel on VEGF expression.
Inhibins and activins are dimeric hormones which share common subunits and which have diverse end... more Inhibins and activins are dimeric hormones which share common subunits and which have diverse endocrine and paracrine roles in regulating reproductive function. Endometrial expression of inhibin α, βA and βB subunits was examined by immunohistochemistry and in-situ hybridization, across the menstrual cycle and in early pregnancy. All three subunits were found to be expressed in endometrium, primarily by glandular epithelium in the early stages of the cycle. Following the onset of decidualization, expression of α, βA and βB subunits was up-regulated in decidualized stromal cells. A marked down-regulation of α subunit was detected in glandular epithelium, whilst expression of βA and βB subunits was maintained. This pattern was consistent in decidua from early pregnancy and additionally in endometrium from women using progestinonly contraceptives, either subdermal implants (Norplant ® ) or levonorgestrel-releasing intrauterine systems (Lng-IUS). Immunostaining was also observed for both βA and βB subunits in subpopulations of endometrial leukocytes, identified to be distinct subsets of macrophages, neutrophils and mast cells. Potential paracrine roles for activins may be envisaged in facilitating tissue remodelling during decidualization, in tissue repair following menstruation, and additionally in modulating premenstrual inflammatory events.
ABSTRACT Many concerns have been expressed regarding the introduction of a new contraceptive meth... more ABSTRACT Many concerns have been expressed regarding the introduction of a new contraceptive method into family planning programmes. One of the concerns is the return of fertility after discontinuing the method. To evaluate the subsequent fertility status of the Indonesian women after removal of Norplant, a prospective longitudinal study was undertaken in Klinik Radeb Saleh, Jakarta. Fifty-one women whose Norplant were removed because of their wish to become pregnant were followed-up for a period of two years or until pregnancy occurred, whichever was earlier. Two groups of women who had Lippes C IUD removed or discontinued the use of DMPA for planning pregnancy served as control and were followed-up for equal length of time. The cumulative conception rate for ex-Norplant users, ex-IUD users and ex-DMPA users at one year was 76.5, 74.7 and 70.2 per 100 women, respectively. There was no significant difference between the groups (p greater than 0.05). The present study, along with other studies, indicate that the prolonged use of Norplant do not impair the return of fertility.
Norplant, subdermally implanted slow-release levonorgestrel, is an effective and widely used cont... more Norplant, subdermally implanted slow-release levonorgestrel, is an effective and widely used contraceptive agent but has a high rate of discontinuation due to unacceptable abnormal uterine bleeding. Matrix metalloproteinases (MMPs) are expressed in normal cycling endometrium and are postulated to be responsible for the tissue breakdown at menstruation. We have compared the immunolocalization of MMP-9 and migratory cells in endometrium from Indonesian women using Norplant with normal controls. Positive MMP-9 immunostaining was observed intracellularly within stromal and intravascular leukocytes and extracellularly in areas of tissue lysis adjacent to these migratory cells. The MMP-9 positive cells were identified as neutrophils, eosinophils, CD3ϩ T-cells and macrophages. Quantitative assessment revealed that the number of MMP-9 positive cells, neutrophils and eosinophils were significantly increased in those endometrial biopsies from Norplant users displaying a shedding morphology and in normal controls at menstruation. There was no correlation between the number of MMP-9 positive cells and the number of bleeding days reported. Endometrial immunostaining for tissue inhibitor of metalloproteinases was similar in Norplant users and normal controls. These results suggest that MMP-9, an enzyme capable of degrading basement membrane components, may be involved in endometrial breakdown in women using Norplant.
A series of 191 endometrial biopsy procedures were performed on Indonesian women who had received... more A series of 191 endometrial biopsy procedures were performed on Indonesian women who had received between 3 and 12 months exposure to Norplant®. In all, 87 biopsy procedures were attempted with a microhysteroscope using biopsy forceps, and 104 procedures were attempted with either Pipelle or Karman suction curettes. Regardless of the biopsy method, diagnosable endometrium was obtained in only -50% of procedures. Myometrium was often found in microhysteroscope but not in suction biopsies. An analysis of a number of clinical characteristics showed that women from whom diagnosable endometrial tissue was obtained had higher mean peripheral oestrogen concentrations hi the 2 weeks prior to biopsy (439 ± 35 versus 289 ± 33 pmol/1; P = 0.0018) and significantly more days when endometrial bleeding occurred in the 90 days prior to biopsy (26.5 ± 2.1 versus 16.2 ± 1.8; P = 0.0003). These results suggest that after 3-12 months exposure to Norplant -50% of women have an endometrium too thin to sample, and that this group is characterized by lower peripheral oestrogen concentrations and reduced menstrual bleeding.
Leukocytes are critical mediators of endometrial remodeling, but the mechanisms by which leukocyt... more Leukocytes are critical mediators of endometrial remodeling, but the mechanisms by which leukocyte subpopulations enter the uterus are currently unknown. Endometrial leukocytes have no genomic progesterone receptors; thus, we hypothesized that leukocyte migration is induced indirectly by progesterone-regulated chemokines. Fractalkine (CX3CL1), a chemotactic membrane-bound adhesion factor, and its receptor (CX3CR1) were assessed by immunohistochemistry in endometrial samples across the menstrual cycle, in early pregnancy, and in women using progestin-only contraceptives. Fractalkine was localized predominantly to glandular epithelial and decidualized stromal cells, with the highest staining intensity in the secretory phase and early pregnancy. It was also detected in subpopulations of endometrial leukocytes (macrophages and uterine NK cells), with maximal numbers during the proliferative phase and early pregnancy. CX3CR1 was similarly colocalized to the glandular epithelium and decidualized stromal cells, with the highest expression in the secretory phase. CX3CR1-positive leukocytes (macrophages and neutrophils) were in greatest abundance during the menstrual phase. In the endometrium of women using progestin-only contraceptives, immunoreactive fractalkine was markedly reduced in the glandular epithelium, but was increased in decidualized stroma and infiltrating leukocytes. These findings support a number of roles for fractalkine in the endometrium, in the secretory phase, in early pregnancy, and when influenced by progestin-only contraceptives.
The endometrium contains many leukocytes, including macrophages, the numbers varying with the tim... more The endometrium contains many leukocytes, including macrophages, the numbers varying with the time of the menstrual cycle and being maximal peri-menstrually. The long-acting progestogenic contraceptive Norplant ® , has a high rate of discontinuation due to uterine bleeding; this is associated with large numbers of endometrial macrophages. Monocyte chemotactic proteins (MCP) act to recruit and activate monocytes into sites of inflammation. This study compared the cellular localization of endometrial MCP-1 and MCP-2 across the normal menstrual cycle and in users of Norplant ® . Both MCP-1 and MCP-2 were present in normal endometrium, but with very different patterns of cellular location and considerable variability between individuals. MCP-1 of epithelial origin was present in 77% of tissues, while stromal staining was present in 52% and vascular staining in 34% of samples. MCP-1 was also released from both epithelial and stromal cells in culture. MCP-2 staining was predominantly epithelial and was found in 52% of tissues while stromal staining was present in only 3/56 samples. Vascular staining of MCP-2 was found in 2/56 samples. The epithelial staining was mostly punctate and sometimes within uterine secretions. No correlation of staining for MCP-1 or -2 with the phase of the cycle was found in any cellular compartment. Very little immunoreactive MCP-1 or MCP-2 was detected in endometrium from Norplant ® users regardless of morphological subtype. These distributions do not support a role for either MCP-1 or MCP-2 in the migration of macrophages into the endometrium and suggest that these cytokines may have other functions in this tissue.
BACKGROUND: Abnormal uterine bleeding is commonly associated with progestin-only contraceptives, ... more BACKGROUND: Abnormal uterine bleeding is commonly associated with progestin-only contraceptives, including depot medroxyprogesterone acetate (DMPA), and remains the main reason why these agents are discontinued. Matrix metalloproteinases (MMP), enzymes which degrade specific extracellular matrix components, and leukocytes are implicated in menstruation. Alteration in endometrial MMP-9 and leukocytes has been described in users of other progestin-only contraceptives, suggesting a potential role in the pathogenesis of abnormal uterine bleeding. METHODS: This study describes the immunohistochemical localization of MMP-9, the tissue inhibitors of metalloproteinases (TIMP)-1, TIMP-2 and TIMP-3, and leukocytes [CD3ϩ T lymphocytes, CD68ϩ macrophages and CD56ϩ uterine natural killer cells (uNK cells)] in the endometrium of women using DMPA. Comparison is made with perimenstrual endometria from normal cycling women. RESULTS: Similar to the perimenstrual period, an influx of MMP-9 positive cells (identified as neutrophils and CD3ϩ T cells on the basis of dual immunofluorescence), macrophages and uNK cells was observed in the endometrium of DMPA users. However, significantly more endometrial T lymphocytes were observed in DMPA users. Immunoreactive TIMP, present in all endometrial compartments, demonstrated a significantly decreased immunostaining intensity score in endometrial epithelium (TIMP-1 and TIMP-2), stroma (TIMP-1, TIMP-2 and TIMP-3), endothelium (TIMP-1 and TIMP-2) and vascular smooth muscle (TIMP-1) of DMPA users compared with controls. No correlation was observed between the parameters studied and bleeding patterns reported by subjects. CONCLUSIONS: These findings provide additional evidence for the importance of the MMP/TIMP balance in the loss/maintenance of endometrial integrity and in the complex pathological mechanisms involved in the troubling side-effect of menstrual bleeding disturbance.
A previous report has shown that progesterone up-regulates cathepsin D expression in human endome... more A previous report has shown that progesterone up-regulates cathepsin D expression in human endometrial cell culture. In women using the levonorgestrel-releasing implant Norplant®, the plasma levonorgestrel and immunoreactive endometrial progesterone receptor concentrations are elevated. However, the functional status of these receptors is not known. This study used endometrial cathepsin D expression both as an indirect marker for the functional status of endometrial progesterone receptors, and to identify the cell types that express cathepsin D. The results show that cathepsin D is primarily found in glandular epithelia and luminal epithelia in control and Norplant® endometria. There is no significant difference in cathepsin D expression between the control and Norplant endometria, between the various stages of the menstrual cycle, or between Norplant users with varying degrees of breakthrough bleeding. Cathepsin D is also detected in cells scattered in the stroma in both control and Norplant endometria. The majority of these cells are macrophages. These data indicate that there is no evidence for progesterone regulation of cathepsin D in the human endometrium. Cathepsin D thus cannot be used as a marker for the functional status of progesterone receptors found in the Norplant-exposed endometrium. Key words: cathepsin D/endometrium/human/Norplant®/progesterone Staining for cathepsin D Sections of 5 (im thickness were cut, dewaxed and washed in three changes of 100%, 100% and 75% ethanol. The sections were rehydrated and stained for cathepsin D using a commercially available mouse monoclonal antibody (NCL-CDm:
Long-acting progestin contraceptives have been available in many countries for a number of years ... more Long-acting progestin contraceptives have been available in many countries for a number of years with a large number of women now using them. Although some improvements in delivery systems have been made, the major problem with progestin-only contraceptives remains unpredictable endometrial breakthrough bleeding (BTB), which is responsible for more than 50% of drop-outs from this form of contraception. Using hysteroscopy, endometrial petechiae and ecchymoses are a common finding among Norplant users, although these features do not always correlate with BTB. It has been postulated that epithelial and subepithelial tissues may provide a barrier to BTB, as long as epithelial integrity is maintained. The aim of this pilot study is to explore structural changes in the endometrial surface epithelium, and subepithelial collagen III fibres. Endometrial biopsies from noresthisterone-enanthate (Net-En) users (n = 6) and controls (n = 6) were assessed using routine haematoxylin and eosin staining and immunohistochemical staining for cytokeratins 8, 18 and 19, and collagen III. A conventional silver impregnation method was also used to identify subepithelial collagen III fibres. Most of the Net-En tissues showed reduced surface epithelial cell height compared to controls (P = 0.002). Cytokeratin staining was weaker (P = 0.04) and distributed evenly between basal and apical parts of the cell in Net-En tissue, compared to more apically in controls. Both immunohistochemical and con-ventional silver staining methods revealed that the subepithelial collagen III meshwork remained unchanged in Net-En compared to control endometrium. Both staining methods identified collagen fibres with equal sensitivity. In conclusion, atrophic changes remain the dominant appearance for progestin-exposed endometrium, with reduced cytokeratin staining, but apparently there is little change in subepithelial collagen III expression.
Progestin-only contraceptives are associated with menstrual bleeding disturbances; a major reason... more Progestin-only contraceptives are associated with menstrual bleeding disturbances; a major reason why these agents are discontinued. The pathogenesis of such abnormal uterine bleeding associated with progestin-only contraceptives remains ill-defined. Matrix metalloproteinases (MMP)s and mast cells (MC)s are postulated to be involved in endometrial breakdown observed in normal menstruation. In this study comparisons were made of the immunolocalization of MMP-1 and -3 and MC in endometrium from women using Norplant or depot medroxyprogesterone acetate (DMPA) with normal controls. Positive MMP immunostaining was observed focally in stromal cells and adjacent extracellular matrix. Quantitative assessment revealed significantly higher MMP-1 immunostaining associated with the use of Norplant compared with DMPA or menstrual phase controls. Endometrial MMP-1 immunostaining in DMPA users was similar to that in menstrual controls. Positive MMP-3 immunolocalization was observed in a minority of endometrial samples. Activated MC, shown by the presence of extracellular MC tryptase, predominated in the endometrium of Norplant and DMPA users as also observed in menstrual phase controls. There was no correlation between MMP immunostaining, number of MC and number of bleeding days reported. These results indicate that in women using progestin-only contraceptives, endometrial MMP-1, -3 and MC demonstrate similarities to menstrual phase controls but also variation with different progestins.
Cytokeratins 8, 18 and 19 are members of the cytoskeletal intermediate filament protein family. T... more Cytokeratins 8, 18 and 19 are members of the cytoskeletal intermediate filament protein family. They are expressed in all simple epithelial tissues, including endometrium, and are recognised as dynamic structures that can be affected by numerous external factors. The Norplant system is a subdermal slow release levonorgestrel implant commonly used as a long-acting progestogen contraceptive. Norplant implants have been shown to have atrophic effects on endometrial epithelial and stromal cells, and cause a range of endometrial bleeding problems among users. The aim of this study is to describe changes in the immunohistochemical expression and distribution of cytokeratins 8, 18 and 19 in endometrial epithelial cells of Norplant implants users and normal menstrual cycle controls. Endometrial biopsies were collected from 65 control normal cycle women and 37 Norplant implants acceptors. The normal menstrual cycle was classified histologically into 9 stages; one menstrual, five proliferative and three secretory. Norplant implants bleeding patterns were categorised into 6 groups according to current World Health Organisation (WHO) definitions; amenorrhoea, frequent bleeding, infrequent bleeding, irregular bleeding, "'normal" bleeding, and prolonged bleeding. The tissues were fixed in formalin,
A clinical study was conducted to assess the effects of oestrogen in controlling increased endome... more A clinical study was conducted to assess the effects of oestrogen in controlling increased endometrial bleeding problems in the first year of Norplant® use. Three treatment groups were studied: (i) 50 ug ethinyl oestradiol (EE); (ii) a combined pill containing 30 \i% EE and 150 ug levonorgestrel (LNG); and (ill) placebo. Based on menstrual diary records, women with prolonged, frequent or irregular bleeding, as defined by World Health Organization criteria, were randomly allocated to one treatment for 21 days. A first endometrial biopsy was taken before commencing treatment and a second biopsy at either day 14 or 21 of treatment Following treatment, all subjects kept a menstrual diary card for 90 days. In this preliminary study, 48 subjects had completed the full 90 day post-treatment record. Within 21 days of EE treatment, the number of bleeding/spotting days was reduced significantly (P < 0.02). In the 90 days following treatment, the administration of EE and EE+LNG significantly decreased the number of bleeding/spotting days (P < 0.05). There was no reduction in the number of bleeding/spotting episodes in the EE and EE+LNG groups, but the length of each bleeding/spotting episode was significantly shorter (P < 0.05). Histopathologlcal findings of endometrium on day 0 revealed consistent progestogenic effects, and there was no apparent change in response by day 14 or 21 of EE or EE+LNG treatment The results of this study confirm the clinical effectiveness of EE and EE+LNG for the treatment of irregular, frequent and prolonged bleeding in Norplant users.
Women using the progestin-only contraceptive Norplant® often suffer from unpredictable bouts of b... more Women using the progestin-only contraceptive Norplant® often suffer from unpredictable bouts of breakthrough bleeding, which usually occurs from a thin atrophic endometrium. The role of cellular apoptosis in the endometrial response to Norplant has not been investigated. The aim of the present study was to use immunohistochemistry to produce semi-quantitative scores for expression of the apoptosis-related proteins Bcl-2, Fas and caspase 3 in endometrium from 16 controls and 42 women using Norplant with minimal or major breakthrough bleeding problems. The results showed no difference in endometrial immunostaining for any of the three proteins between Norplant users with and without breakthrough bleeding. There was also no evidence of endometrial endothelial cell immunostaining for any of the proteins. Bcl-2 was the only protein to show a cyclical pattern, with higher expression in the proliferative compared to secretory glands. All three proteins showed different expression levels in control functionalis versus basalis, with the survival protein Bcl-2 being higher in basalis, and the death receptor Fas and the proteolytic enzyme caspase 3 being higher in the functionalis. Overall, the results suggest that apoptosis is regulated differently in functionalis compared to basalis, and that atrophic Norplant-exposed endometrium appears more like functionalis than basalis with respect to expression of Fas and caspase 3. There was no evidence for a role for apoptosis in the mechanisms that underlie progestininduced endometrial breakthrough bleeding.
In this 4-year open-label, noncomparative, single-center pilot efficacy study, the contraceptive ... more In this 4-year open-label, noncomparative, single-center pilot efficacy study, the contraceptive efficacy, safety, bleeding pattern and acceptability of Implanon was studied in 200 sexually active women of proven fertility in Indonesia. All subjects received the single-rod subdermal implant Implanon, which contains 68 mg etonogestrel (3-keto-desogestrel), with an initial release rate of 67 micrograms etonogestrel/day. Contraceptive efficacy was analyzed by calculation of the pregnancy rate, bleeding patterns were determined by the 90-day reference period method, and acceptability by the discontinuation rate. No in-treatment pregnancies were reported during 658.4 women-years of exposure, resulting in a Pearl Index of 0.0 (95% CI 0.0-0.6). The overall bleeding pattern was acceptable, with no discontinuations because of irregular bleeding. Incidence of irregular bleeding was highest during the first two reference periods and decreased thereafter. Amenorrhea was experienced by 7%-12% of subjects during years 1 and 2, by 5%-7% during year 3, and by 2%-5% during year 4, with one discontinuation because of amenorrhea. No clinically significant changes were reported for systolic and diastolic blood pressure, body mass index, and hemoglobin level. Three adverse experiences were related to treatment and resulted in discontinuation (two headaches and one dyspnea). One difficult implant removal was reported. In conclusion, this pilot efficacy study indicates that Implanon provides excellent contraceptive reliability and an acceptable bleeding pattern. Overall safety and acceptability are good, as suggested by the low incidence of adverse experiences and the low discontinuation rate.
Vascular endothelial growth factor (VEGF) expression and the microvascular density of the endomet... more Vascular endothelial growth factor (VEGF) expression and the microvascular density of the endometrium were studied in Norplant users and normal controls, using immunohistochemistry on formalin-fixed paraffinembedded endometrial sections. The VEGF staining index was quantified using computerized image analysis. The VEGF staining index between stages of the menstrual cycle and between normal and Norplant endometria were compared. Norplant VEGF staining index was analysed for correlation with microvascular density, duration of Norplant use, the number of bleeding/spotting days in the reference period up to 90 days prior to biopsy, and the length of time since the last bleeding/spotting episode. The results showed that immunoreactive VEGF was detected predominantly in endometrial glands but weakly expressed in the stroma throughout the menstrual cycle, and also in Norplant users. Large variation in the VEGF staining index between individuals was observed and no significant difference in the VEGF staining index was detected between stages of the menstrual cycle for the glands and stroma. The glandular and stromal VEGF staining indices were significantly higher in Norplant than in normal endometrium (P <1⍥10 -4 ). No correlation was found between the Norplant VEGF staining index and endometrial microvascular density, duration of Norplant use, the number of bleeding/spotting days in the reference period, and the length of time since the last bleeding/spotting episode. The VEGF staining index was higher in glands than stroma for both normal and Norplant endometrium. The results suggest a differential control of endometrial glandular versus stromal VEGF expression, and possible positive effects of levonorgestrel on VEGF expression.
Inhibins and activins are dimeric hormones which share common subunits and which have diverse end... more Inhibins and activins are dimeric hormones which share common subunits and which have diverse endocrine and paracrine roles in regulating reproductive function. Endometrial expression of inhibin α, βA and βB subunits was examined by immunohistochemistry and in-situ hybridization, across the menstrual cycle and in early pregnancy. All three subunits were found to be expressed in endometrium, primarily by glandular epithelium in the early stages of the cycle. Following the onset of decidualization, expression of α, βA and βB subunits was up-regulated in decidualized stromal cells. A marked down-regulation of α subunit was detected in glandular epithelium, whilst expression of βA and βB subunits was maintained. This pattern was consistent in decidua from early pregnancy and additionally in endometrium from women using progestinonly contraceptives, either subdermal implants (Norplant ® ) or levonorgestrel-releasing intrauterine systems (Lng-IUS). Immunostaining was also observed for both βA and βB subunits in subpopulations of endometrial leukocytes, identified to be distinct subsets of macrophages, neutrophils and mast cells. Potential paracrine roles for activins may be envisaged in facilitating tissue remodelling during decidualization, in tissue repair following menstruation, and additionally in modulating premenstrual inflammatory events.
ABSTRACT Many concerns have been expressed regarding the introduction of a new contraceptive meth... more ABSTRACT Many concerns have been expressed regarding the introduction of a new contraceptive method into family planning programmes. One of the concerns is the return of fertility after discontinuing the method. To evaluate the subsequent fertility status of the Indonesian women after removal of Norplant, a prospective longitudinal study was undertaken in Klinik Radeb Saleh, Jakarta. Fifty-one women whose Norplant were removed because of their wish to become pregnant were followed-up for a period of two years or until pregnancy occurred, whichever was earlier. Two groups of women who had Lippes C IUD removed or discontinued the use of DMPA for planning pregnancy served as control and were followed-up for equal length of time. The cumulative conception rate for ex-Norplant users, ex-IUD users and ex-DMPA users at one year was 76.5, 74.7 and 70.2 per 100 women, respectively. There was no significant difference between the groups (p greater than 0.05). The present study, along with other studies, indicate that the prolonged use of Norplant do not impair the return of fertility.
Norplant, subdermally implanted slow-release levonorgestrel, is an effective and widely used cont... more Norplant, subdermally implanted slow-release levonorgestrel, is an effective and widely used contraceptive agent but has a high rate of discontinuation due to unacceptable abnormal uterine bleeding. Matrix metalloproteinases (MMPs) are expressed in normal cycling endometrium and are postulated to be responsible for the tissue breakdown at menstruation. We have compared the immunolocalization of MMP-9 and migratory cells in endometrium from Indonesian women using Norplant with normal controls. Positive MMP-9 immunostaining was observed intracellularly within stromal and intravascular leukocytes and extracellularly in areas of tissue lysis adjacent to these migratory cells. The MMP-9 positive cells were identified as neutrophils, eosinophils, CD3ϩ T-cells and macrophages. Quantitative assessment revealed that the number of MMP-9 positive cells, neutrophils and eosinophils were significantly increased in those endometrial biopsies from Norplant users displaying a shedding morphology and in normal controls at menstruation. There was no correlation between the number of MMP-9 positive cells and the number of bleeding days reported. Endometrial immunostaining for tissue inhibitor of metalloproteinases was similar in Norplant users and normal controls. These results suggest that MMP-9, an enzyme capable of degrading basement membrane components, may be involved in endometrial breakdown in women using Norplant.
A series of 191 endometrial biopsy procedures were performed on Indonesian women who had received... more A series of 191 endometrial biopsy procedures were performed on Indonesian women who had received between 3 and 12 months exposure to Norplant®. In all, 87 biopsy procedures were attempted with a microhysteroscope using biopsy forceps, and 104 procedures were attempted with either Pipelle or Karman suction curettes. Regardless of the biopsy method, diagnosable endometrium was obtained in only -50% of procedures. Myometrium was often found in microhysteroscope but not in suction biopsies. An analysis of a number of clinical characteristics showed that women from whom diagnosable endometrial tissue was obtained had higher mean peripheral oestrogen concentrations hi the 2 weeks prior to biopsy (439 ± 35 versus 289 ± 33 pmol/1; P = 0.0018) and significantly more days when endometrial bleeding occurred in the 90 days prior to biopsy (26.5 ± 2.1 versus 16.2 ± 1.8; P = 0.0003). These results suggest that after 3-12 months exposure to Norplant -50% of women have an endometrium too thin to sample, and that this group is characterized by lower peripheral oestrogen concentrations and reduced menstrual bleeding.
Leukocytes are critical mediators of endometrial remodeling, but the mechanisms by which leukocyt... more Leukocytes are critical mediators of endometrial remodeling, but the mechanisms by which leukocyte subpopulations enter the uterus are currently unknown. Endometrial leukocytes have no genomic progesterone receptors; thus, we hypothesized that leukocyte migration is induced indirectly by progesterone-regulated chemokines. Fractalkine (CX3CL1), a chemotactic membrane-bound adhesion factor, and its receptor (CX3CR1) were assessed by immunohistochemistry in endometrial samples across the menstrual cycle, in early pregnancy, and in women using progestin-only contraceptives. Fractalkine was localized predominantly to glandular epithelial and decidualized stromal cells, with the highest staining intensity in the secretory phase and early pregnancy. It was also detected in subpopulations of endometrial leukocytes (macrophages and uterine NK cells), with maximal numbers during the proliferative phase and early pregnancy. CX3CR1 was similarly colocalized to the glandular epithelium and decidualized stromal cells, with the highest expression in the secretory phase. CX3CR1-positive leukocytes (macrophages and neutrophils) were in greatest abundance during the menstrual phase. In the endometrium of women using progestin-only contraceptives, immunoreactive fractalkine was markedly reduced in the glandular epithelium, but was increased in decidualized stroma and infiltrating leukocytes. These findings support a number of roles for fractalkine in the endometrium, in the secretory phase, in early pregnancy, and when influenced by progestin-only contraceptives.
The endometrium contains many leukocytes, including macrophages, the numbers varying with the tim... more The endometrium contains many leukocytes, including macrophages, the numbers varying with the time of the menstrual cycle and being maximal peri-menstrually. The long-acting progestogenic contraceptive Norplant ® , has a high rate of discontinuation due to uterine bleeding; this is associated with large numbers of endometrial macrophages. Monocyte chemotactic proteins (MCP) act to recruit and activate monocytes into sites of inflammation. This study compared the cellular localization of endometrial MCP-1 and MCP-2 across the normal menstrual cycle and in users of Norplant ® . Both MCP-1 and MCP-2 were present in normal endometrium, but with very different patterns of cellular location and considerable variability between individuals. MCP-1 of epithelial origin was present in 77% of tissues, while stromal staining was present in 52% and vascular staining in 34% of samples. MCP-1 was also released from both epithelial and stromal cells in culture. MCP-2 staining was predominantly epithelial and was found in 52% of tissues while stromal staining was present in only 3/56 samples. Vascular staining of MCP-2 was found in 2/56 samples. The epithelial staining was mostly punctate and sometimes within uterine secretions. No correlation of staining for MCP-1 or -2 with the phase of the cycle was found in any cellular compartment. Very little immunoreactive MCP-1 or MCP-2 was detected in endometrium from Norplant ® users regardless of morphological subtype. These distributions do not support a role for either MCP-1 or MCP-2 in the migration of macrophages into the endometrium and suggest that these cytokines may have other functions in this tissue.
BACKGROUND: Abnormal uterine bleeding is commonly associated with progestin-only contraceptives, ... more BACKGROUND: Abnormal uterine bleeding is commonly associated with progestin-only contraceptives, including depot medroxyprogesterone acetate (DMPA), and remains the main reason why these agents are discontinued. Matrix metalloproteinases (MMP), enzymes which degrade specific extracellular matrix components, and leukocytes are implicated in menstruation. Alteration in endometrial MMP-9 and leukocytes has been described in users of other progestin-only contraceptives, suggesting a potential role in the pathogenesis of abnormal uterine bleeding. METHODS: This study describes the immunohistochemical localization of MMP-9, the tissue inhibitors of metalloproteinases (TIMP)-1, TIMP-2 and TIMP-3, and leukocytes [CD3ϩ T lymphocytes, CD68ϩ macrophages and CD56ϩ uterine natural killer cells (uNK cells)] in the endometrium of women using DMPA. Comparison is made with perimenstrual endometria from normal cycling women. RESULTS: Similar to the perimenstrual period, an influx of MMP-9 positive cells (identified as neutrophils and CD3ϩ T cells on the basis of dual immunofluorescence), macrophages and uNK cells was observed in the endometrium of DMPA users. However, significantly more endometrial T lymphocytes were observed in DMPA users. Immunoreactive TIMP, present in all endometrial compartments, demonstrated a significantly decreased immunostaining intensity score in endometrial epithelium (TIMP-1 and TIMP-2), stroma (TIMP-1, TIMP-2 and TIMP-3), endothelium (TIMP-1 and TIMP-2) and vascular smooth muscle (TIMP-1) of DMPA users compared with controls. No correlation was observed between the parameters studied and bleeding patterns reported by subjects. CONCLUSIONS: These findings provide additional evidence for the importance of the MMP/TIMP balance in the loss/maintenance of endometrial integrity and in the complex pathological mechanisms involved in the troubling side-effect of menstrual bleeding disturbance.
A previous report has shown that progesterone up-regulates cathepsin D expression in human endome... more A previous report has shown that progesterone up-regulates cathepsin D expression in human endometrial cell culture. In women using the levonorgestrel-releasing implant Norplant®, the plasma levonorgestrel and immunoreactive endometrial progesterone receptor concentrations are elevated. However, the functional status of these receptors is not known. This study used endometrial cathepsin D expression both as an indirect marker for the functional status of endometrial progesterone receptors, and to identify the cell types that express cathepsin D. The results show that cathepsin D is primarily found in glandular epithelia and luminal epithelia in control and Norplant® endometria. There is no significant difference in cathepsin D expression between the control and Norplant endometria, between the various stages of the menstrual cycle, or between Norplant users with varying degrees of breakthrough bleeding. Cathepsin D is also detected in cells scattered in the stroma in both control and Norplant endometria. The majority of these cells are macrophages. These data indicate that there is no evidence for progesterone regulation of cathepsin D in the human endometrium. Cathepsin D thus cannot be used as a marker for the functional status of progesterone receptors found in the Norplant-exposed endometrium. Key words: cathepsin D/endometrium/human/Norplant®/progesterone Staining for cathepsin D Sections of 5 (im thickness were cut, dewaxed and washed in three changes of 100%, 100% and 75% ethanol. The sections were rehydrated and stained for cathepsin D using a commercially available mouse monoclonal antibody (NCL-CDm:
Long-acting progestin contraceptives have been available in many countries for a number of years ... more Long-acting progestin contraceptives have been available in many countries for a number of years with a large number of women now using them. Although some improvements in delivery systems have been made, the major problem with progestin-only contraceptives remains unpredictable endometrial breakthrough bleeding (BTB), which is responsible for more than 50% of drop-outs from this form of contraception. Using hysteroscopy, endometrial petechiae and ecchymoses are a common finding among Norplant users, although these features do not always correlate with BTB. It has been postulated that epithelial and subepithelial tissues may provide a barrier to BTB, as long as epithelial integrity is maintained. The aim of this pilot study is to explore structural changes in the endometrial surface epithelium, and subepithelial collagen III fibres. Endometrial biopsies from noresthisterone-enanthate (Net-En) users (n = 6) and controls (n = 6) were assessed using routine haematoxylin and eosin staining and immunohistochemical staining for cytokeratins 8, 18 and 19, and collagen III. A conventional silver impregnation method was also used to identify subepithelial collagen III fibres. Most of the Net-En tissues showed reduced surface epithelial cell height compared to controls (P = 0.002). Cytokeratin staining was weaker (P = 0.04) and distributed evenly between basal and apical parts of the cell in Net-En tissue, compared to more apically in controls. Both immunohistochemical and con-ventional silver staining methods revealed that the subepithelial collagen III meshwork remained unchanged in Net-En compared to control endometrium. Both staining methods identified collagen fibres with equal sensitivity. In conclusion, atrophic changes remain the dominant appearance for progestin-exposed endometrium, with reduced cytokeratin staining, but apparently there is little change in subepithelial collagen III expression.
Progestin-only contraceptives are associated with menstrual bleeding disturbances; a major reason... more Progestin-only contraceptives are associated with menstrual bleeding disturbances; a major reason why these agents are discontinued. The pathogenesis of such abnormal uterine bleeding associated with progestin-only contraceptives remains ill-defined. Matrix metalloproteinases (MMP)s and mast cells (MC)s are postulated to be involved in endometrial breakdown observed in normal menstruation. In this study comparisons were made of the immunolocalization of MMP-1 and -3 and MC in endometrium from women using Norplant or depot medroxyprogesterone acetate (DMPA) with normal controls. Positive MMP immunostaining was observed focally in stromal cells and adjacent extracellular matrix. Quantitative assessment revealed significantly higher MMP-1 immunostaining associated with the use of Norplant compared with DMPA or menstrual phase controls. Endometrial MMP-1 immunostaining in DMPA users was similar to that in menstrual controls. Positive MMP-3 immunolocalization was observed in a minority of endometrial samples. Activated MC, shown by the presence of extracellular MC tryptase, predominated in the endometrium of Norplant and DMPA users as also observed in menstrual phase controls. There was no correlation between MMP immunostaining, number of MC and number of bleeding days reported. These results indicate that in women using progestin-only contraceptives, endometrial MMP-1, -3 and MC demonstrate similarities to menstrual phase controls but also variation with different progestins.
Cytokeratins 8, 18 and 19 are members of the cytoskeletal intermediate filament protein family. T... more Cytokeratins 8, 18 and 19 are members of the cytoskeletal intermediate filament protein family. They are expressed in all simple epithelial tissues, including endometrium, and are recognised as dynamic structures that can be affected by numerous external factors. The Norplant system is a subdermal slow release levonorgestrel implant commonly used as a long-acting progestogen contraceptive. Norplant implants have been shown to have atrophic effects on endometrial epithelial and stromal cells, and cause a range of endometrial bleeding problems among users. The aim of this study is to describe changes in the immunohistochemical expression and distribution of cytokeratins 8, 18 and 19 in endometrial epithelial cells of Norplant implants users and normal menstrual cycle controls. Endometrial biopsies were collected from 65 control normal cycle women and 37 Norplant implants acceptors. The normal menstrual cycle was classified histologically into 9 stages; one menstrual, five proliferative and three secretory. Norplant implants bleeding patterns were categorised into 6 groups according to current World Health Organisation (WHO) definitions; amenorrhoea, frequent bleeding, infrequent bleeding, irregular bleeding, "'normal" bleeding, and prolonged bleeding. The tissues were fixed in formalin,
A clinical study was conducted to assess the effects of oestrogen in controlling increased endome... more A clinical study was conducted to assess the effects of oestrogen in controlling increased endometrial bleeding problems in the first year of Norplant® use. Three treatment groups were studied: (i) 50 ug ethinyl oestradiol (EE); (ii) a combined pill containing 30 \i% EE and 150 ug levonorgestrel (LNG); and (ill) placebo. Based on menstrual diary records, women with prolonged, frequent or irregular bleeding, as defined by World Health Organization criteria, were randomly allocated to one treatment for 21 days. A first endometrial biopsy was taken before commencing treatment and a second biopsy at either day 14 or 21 of treatment Following treatment, all subjects kept a menstrual diary card for 90 days. In this preliminary study, 48 subjects had completed the full 90 day post-treatment record. Within 21 days of EE treatment, the number of bleeding/spotting days was reduced significantly (P < 0.02). In the 90 days following treatment, the administration of EE and EE+LNG significantly decreased the number of bleeding/spotting days (P < 0.05). There was no reduction in the number of bleeding/spotting episodes in the EE and EE+LNG groups, but the length of each bleeding/spotting episode was significantly shorter (P < 0.05). Histopathologlcal findings of endometrium on day 0 revealed consistent progestogenic effects, and there was no apparent change in response by day 14 or 21 of EE or EE+LNG treatment The results of this study confirm the clinical effectiveness of EE and EE+LNG for the treatment of irregular, frequent and prolonged bleeding in Norplant users.
Women using the progestin-only contraceptive Norplant® often suffer from unpredictable bouts of b... more Women using the progestin-only contraceptive Norplant® often suffer from unpredictable bouts of breakthrough bleeding, which usually occurs from a thin atrophic endometrium. The role of cellular apoptosis in the endometrial response to Norplant has not been investigated. The aim of the present study was to use immunohistochemistry to produce semi-quantitative scores for expression of the apoptosis-related proteins Bcl-2, Fas and caspase 3 in endometrium from 16 controls and 42 women using Norplant with minimal or major breakthrough bleeding problems. The results showed no difference in endometrial immunostaining for any of the three proteins between Norplant users with and without breakthrough bleeding. There was also no evidence of endometrial endothelial cell immunostaining for any of the proteins. Bcl-2 was the only protein to show a cyclical pattern, with higher expression in the proliferative compared to secretory glands. All three proteins showed different expression levels in control functionalis versus basalis, with the survival protein Bcl-2 being higher in basalis, and the death receptor Fas and the proteolytic enzyme caspase 3 being higher in the functionalis. Overall, the results suggest that apoptosis is regulated differently in functionalis compared to basalis, and that atrophic Norplant-exposed endometrium appears more like functionalis than basalis with respect to expression of Fas and caspase 3. There was no evidence for a role for apoptosis in the mechanisms that underlie progestininduced endometrial breakthrough bleeding.
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