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It has been shown that the endometrium of women using progestin-only contraceptives has increased vascular fragility, although the structural basis for this weakness is unknown, as is its role in breakthrough bleeding (BTB). Perivascular cells such as pericytes and vascular smooth muscle cells surround capillaries during the maturation process following angiogenesis,and act to strengthen and stabilize the vessels. The aim of the present study wasto quantify endometrial perivascular smooth muscle α-actin (αSMA) expression in women using Norplant® with and without BTB problems, and compare it to controls. Using immunohistochemical techniques, vessels were classified as level 0, 1 or 2 depending on whether perivascular αSMA was absent, discontinuous or continuous. In 15 controls the subepithelial plexus had significantly more level 0 vessels than either the functionalis or basalis (61 ± 4 versus 31 ± 6 and 37 ± 4%, P = 0.0006 and P = 0.0007 respectively). In contrast the functionalis and basalis had significantly more level 2 vessels than the subepithelial plexus (20 ± 3 and 23 ± 2 compared to 4 ± 1%, P = 0.0005 and P − 0.000 respectively). The major finding of the study was that in Norplant users, where the relatively atrophic endometrium cannot be divided into different regions, women with BTB problems (n = 20) had significantly more level 0 vessels than those with reduced bleeding (n = 17) (60 ± 4 versus 46 ± 4percnt;, P = 0.0302). Norplant users with BTB problems also had a non-significant reduction in level 2 vessels compared to women without bleeding problems (4 ± 2 versus 11 ± 4percnt;, P equals; 0.0667). These results demonstrate that perivascular αSMA is reduced around the endometrial vessels of Norplant users with BTB compared to those with no bleeding problems, and strongly support the concept that reduced vascular structural integrity plays a key role in endometrial BTB. [ABSTRACT FROM PUBLISHER]

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 conventional conventional 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. [ABSTRACT FROM PUBLISHER]

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-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. [ABSTRACT FROM PUBLISHER]

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 <1Χ 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. [ABSTRACT FROM AUTHOR]

Cytokeratins 8, 18 and 19 are members of the cytoskeletal intermediate filament family found in all simple epithelia. Intermediate filaments are dynamic intracytoplasmic structures that can be influenced by a number of external factors. Norethisterone enanthate (NET-EN) is a long-acting progestogen contraceptive that has been found to arrest endometrial growth in the rat Both Norplant® and NET-EN cause bleeding problems among users which are responsible for >50% of withdrawals with these methods. The aim of this study was to explore changes in the expression and distribution of cytokeratins 8, 18 and 19 in NET-EN- and Norplantexposed endometrial epithelium which could be related to bleeding disturbances. Seven NET-EN and 37 Norplant endometrial biopsies were paraffin-embedded and stained immunohistochemically to evaluate cytokeratin expression and distribution. The results showed that women who had received NET-EN for 3-4 months had a cytokeratin distribution similar to that seen in the normal menstrual cycle. This is in contrast to endometrium from Norplant users in which cytokeratin expression was reduced and the epithelial cells were more rounded. No relationship between cytokeratin expression and breakthrough bleeding pattern was found. NET-EN and Norplant may act differently on endometrial epithelial cytokeratin. [ABSTRACT FROM PUBLISHER]

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 μg ethinyl oestradiol (EE); (ii) a combined pill containing 30 μg EE and 150 μg levonorgestrel (LNG); and (iii) 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). Histopathological 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. [ABSTRACT FROM PUBLISHER]

The expression of endometrial progesterone receptor mRNA during the human menstrual cycle and in Norplant users was studied using digoxigenin-labelled ribonucleic probes for in-situ hybridization on 6 μm paraffin embedded endometrial sections. The staining intensity was scored blind semi-quantitatlvely. Blood ovarian steroid concentrations were measured in Norplant users. All data were analysed by analysis of variance. Glandular progesterone receptor mRNA concentrations were low during the menstrual-to-early proliferative stage but increased during the early-to-mid to late-proliferative stage then declined non-significantly over the secretory stage. No such variation was observed in stromal cells. Progesterone receptor mRNA concentrations were lower in Norplant than controls during early-to-mid to late-proliferative stages (in glandular epithelium and stroma) and during secretory stage (in stroma only). Norplant subjects with amenorrhoea had higher concentrations of stromal progesterone receptor mRNA but lower plasma oestrogen concentrations than subjects with breakthrough bleeding. The pattern of variation in progesterone receptor mRNA concentrations during the normal menstrual cycle resembles the published pattern for the receptor protein. The results demonstrate: (i) a differential sensitivity of glandular and stromal progesterone receptors to steroid regulation; (ii) in contrast to previous findings of an increase in immunoreactive progesterone receptor protein in Norplant endometrium, progesterone receptor mRNA concentrations in these tissues were reduced; and (iii) there was significantly more progesterone receptor mRNA in subjects with amenorrhoea than in those with breakthrough bleeding. [ABSTRACT FROM PUBLISHER]