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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.

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 paraffin- embedded endometrial sections. The VEGF staining index was quantified using computerized image analysis. The VEGF staining index between stage 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 x 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.

The mechanisms that underlie progestogen-induced endometrial breakthrough bleeding are poorly understood. The aim of the present study was to quantify endometrial microvascular density in 54 controls and 42 women with 3-12 months' exposure to Norplant (levonorgestrel subdermal contraceptive implant) and to correlate it with bleeding pattern, endometrial histology, and peripheral plasma oestradiol and progesterone concentrations. Endometrial biopsies were processed routinely and sections immunostained using anti-CD34 antibody to identify vascular endothelial cells. Menstrual record card data were analysed using World Health Organization definitions. The mean microvascular density (+/- SEM) for control samples was 186 +/- 8 vessels/mm2, and there were no significant differences across the cycle. Norplant user's endometrial microvascular density was significantly elevated above controls (294 +/- 18 vessels/mm2, P = 3.36 x 10-8). Endometrial microvascular density in Norplant users did not correlate with oestrogen concentrations prior to biopsy, bleeding patterns or endometrial histology. The results from this study show that women receiving Norplant have significantly increased endometrial microvascular density compared to controls. Another finding from this study was that bleeding in Norplant users often occurred from thin atrophic endometrium. These results provide new insights into the physiological mechanisms that may be involved in progestogen-induced endometrial bleeding.

The mechanisms responsible for Norplant implants-induced menstrual irregularities remain poorly understood. It is unclear whether local changes in endometrial haemostasis are involved. The aim of the present study was to examine the immunohistochemical staining for von Willebrand factor (vWF) in endometrial biopsies taken from 37 women exposed to Norplant implants for 3-22 months and to compare it with 73 controls at various phases of the normal menstrual cycle. The vWF staining intensity was quantified by subjective scoring and by objective computerised colour image analysis. Results from the Norplant implants group were additionally correlated with their bleeding patterns, endometrial histology, and plasma oestradiol (E2) and progesterone (P4) levels. No differences were found between control and Norplant implants subjects in the localization of vWF staining, which was specifically confined to the endothelium of endometrial blood vessels. vWF staining intensity in Norplant implants endometrium was significantly lower than in controls during mid cycle, and reached a mean (+/-SE) level (subjective staining score 2.05 +/- 0.13, n = 37) in the range of the early proliferative and mid secretory phase normal endometrium; nevertheless, it remained significantly higher than that of menstrual and late secretory phase normal endometrium. No significant variations in vWF staining could be related to either the histology of the endometrium or the bleeding pattern of the users. Correlation of vWF staining with either serum E, or serum P, prior to biopsy, or to the number of days of Norplant implants exposure revealed no significant relationships. However, vWF staining was positively correlated (r= 0.419, P < 0.02) to the number of bleeding/spotting days within a 90-day reference period prior to biopsies being taken. These results demonstrate that there are major differences in the mechanism responsible for normal menstruation and Norplant implants-induced intermenstrual bleedi

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 was to quantify endometrial perivascular smooth muscle alpha-actin (alphaSMA) 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 alphaSMA 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 +/- 4%, 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 +/- 4%, P = 0.0667). These results demonstrate that perivascular alphaSMA 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.

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 progestin-induced endometrial breakthrough bleeding.

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 in the 2 weeks prior to biopsy (439 +/- 35 versus 289 +/- 33 pmol/l; 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.

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 mum paraffin embedded endometrial sections. The staining intensity was scored blind semi-quantitatively. 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.

The aim of the present study was to quantify endothelial cell proliferation (a component of angiogenesis) using immunohistochemistry, in the endometrium of users of subdermal levonorgestrel (Norplant). It was postulated that the increased endometrial microvascular density seen in Norplant users, compared to normally cycling women, was associated with an increased rate of endothelial cell proliferation. The results, however, showed that the endometrial endothelial cell proliferative index of Norplant users (0.39 +/- 0.16%; mean +/- SEM) was significantly reduced compared to that seen in normally cycling women (8.99 +/- 1.64). At the same time, total numbers of endometrial endothelial cells per mm2 in Norplant users (317.40 +/- 13.88) were significantly higher than in normally cycling women (223.25 +/- 10.31). It is possible that in the endometrium with levonorgestrel use, there is either a reduced rate of regression of the blood vessels relative to the rest of the tissue, or there is a reduced rate of endothelial cell death or turnover. Peripheral oestrogen and progesterone concentrations, bleeding pattern over the previous 90 days, and the histological appearance of the endometrium did not appear to be associated with the endothelial cell proliferative index. The results suggest that subdermal levonorgestrel use affects the mechanisms that dictate the normal relationship between endometrial blood vessel growth and regression, and the surrounding non-vascular tissue.