Expression of progesterone receptor mRNA in the endometrium during the normal menstrual cycle and in Norplant users.

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 microns 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.

PIP: At Monash Medical Center in Victoria, Australia, and at the University of Indonesia in Jakarta, researchers used digoxigenin-labeled ribonucleic probes for in-situ hybridization in the endometrium of 53 women across the normal menstrual cycle (controls) and of 39 Norplant users (cases), respectively, to examine the expression of progesterone receptor mRNA in the endometrium during the normal menstrual cycle and in Norplant users. They detected progesterone receptor mRNA in the glandular epithelium and stromal cells at all stages of the menstrual cycle. Concentrations of progesterone receptor mRNA staining in the glands increased from stage 1 (the menstrual to early proliferative stage), when there was little progesterone receptor mRNA staining, to stage 2 (early-to-mid proliferative to late proliferative stage) (1.35 vs. 2.25 staining intensity score; p 0.01). Thereafter, they fell steadily, but not significantly so. On the other hand, concentrations of progesterone receptor mRNA staining in the stroma did not vary during the menstrual cycle. Most endometria of Norplant users had detectable progesterone receptor mRNA in both the glandular epithelia and stromal cells. The endometria of Norplant users had less progesterone receptor mRNA staining in glandular epithelia than stage 2 of the cycle of the controls (1.5 vs. 2.25 score; p 0.01). The Norplant stroma also had less progesterone receptor mRNA than stages 2 (1.75 vs. 2.5 score; p 0.05) and 3 (1.75 vs. 2.5 score; p 0.01). Among Norplant users, the amenorrhea group had more progesterone receptor mRNA staining in the stroma than the bleeding group (2.5 vs. 1.75 score; p 0.05). It had lower plasma concentrations of estrogen than the bleeding group (200.8 vs. 523.9 pmol/l; p 0.05). There were no differences in the plasma progesterone concentrations, however. These findings confirm that there is a significant relationship between reduced endogenous estrogen concentrations and reduced breakthrough bleeding in users of progestin-only contraceptives. They show that glandular and stromal progesterone receptors have different sensitivity to steroid regulation.

Endometrial biopsy collection from women receiving Norplant.

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 approximately 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 approximately 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.

PIP: Research on the causes of progestogen-induced breakthrough bleeding in Norplant users depends on the availability of endometrial biopsy samples. In this study, 3 biopsy techniques were utilized in 191 Indonesian women with 3-12 months of exposure to Norplant: microhysteroscopy with biopsy forceps (n = 87), Pipelle suction curette (n = 52), and Karman cannula (n = 52). Diagnosable endometrium were obtained in 51%, 42%, and 58% of these procedures, respectively. Use of the microhysteroscope often resulted in the collection of full thickness endometrium, including a small amount of myometrial tissue, but the endometrium was always very thin or absent. In contrast, both the suction curette techniques collected endometrial tissue only. Women from whom successful endometrial biopsies were obtained tended to have significantly more days of endometrial bleeding in the 90 days preceding biopsy (26.5 +or- 2.1 versus 16.2 +or- 1.8) and higher mean peripheral estrogen concentrations in the 2 weeks preceding biopsy (439 +or- 35 versus 289 +or- 33 pmol/l). The fact that the group from which successful biopsies are obtained may not be representative of all Norplant users should be considered in the analysis of clinical research.

Progesterone receptor in Norplant endometrium.

Endometrial progesterone receptor plays an important role in determining the biological activity of progestogens in fertility regulation. Studies during the normal menstrual cycle have shown that the concentrations of endometrial progesterone receptor and its mRNA vary in glandular epithelia but remain steady in stromal cells. There is general agreement between progesterone receptor mRNA and protein levels during the normal menstrual cycle. Norplant endometrium had an increase in immunoreactive progesterone receptor concentration but a reduction in progesterone receptor mRNA levels compared with controls. An immunohistochemical study, using the expression of the lysosomal protease cathepsin D as a marker for the functional status of progesterone receptors, failed to confirm the functionality of the receptors in Norplant endometrium. Together, these results suggest that (i) there is a differential sensitivity of glandular and stromal progesterone receptors to steroid regulation during the normal menstrual cycle; (ii) there appears to be a dissociation between the concentrations of progesterone receptor and its mRNA in Norplant endometrium; and (iii) there was significantly more progesterone receptor mRNA and lower plasma oestrogen concentrations in Norplant subjects with amenorrhoea than with endometrial bleeding. The clinical significance of the differences in progesterone receptor mRNA levels and plasma oestrogen concentrations between the amenorrhoea group and the bleeding group requires further investigation.

PIP: Research on the effects of levonorgestrel on endometrial progesterone receptors in Norplant users is critical to reducing the menstrual disturbances associated with this contraceptive method. Studies conducted during the normal menstrual cycle have indicated that the concentrations of endometrial progesterone receptor and its mRNA vary in glandular epithelia but remain steady in stromal cells. The endometrium in Norplant users shows an increase in immunoreactive progesterone receptor concentration but a reduction, compared to controls, in progesterone receptor mRNA levels. Overall, the clinical research suggests: 1) there is a differential sensitivity of glandular and stromal progesterone receptors to steroid regulation during the normal menstrual cycle; 2) there appears to be a dissociation between the concentrations of progesterone receptor and its mRNA in Norplant endometrium; and 3) there are significantly more progesterone receptor mRNA and lower plasma estrogen concentrations in Norplant users with amenorrhea than in those with normal menstrual bleeding.

Oestrogen treatment for increased bleeding in Norplant users: preliminary results.

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 micrograms ethinyl oestradiol (EE); (ii) a combined pill containing 30 micrograms EE and 150 micrograms 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.

PIP: A clinical study conducted in Indonesia confirmed the effectiveness of ethinyl estradiol and ethinyl estradiol plus levonorgestrel for the treatment of the frequent, prolonged, and irregular bleeding associated with Norplant use. The 91 subjects were randomly allocated to receive, for 3 weeks, 50 mcg of ethinyl estradiol, a combined pill containing 30 mcg of ethinyl estradiol and 150 mcg of levonorgestrel, or a placebo. To date, 48 subjects have completed a 90-day post-treatment menstrual diary. Among this subgroup, only ethinyl estradiol alone reduced significantly (p 0.02) the number of bleeding/spotting days during the 21-day treatment period. In the 90 days after treatment, both ethinyl estradiol and the combined pill significantly (p 0.05) reduced bleeding/spotting compared to the 90 days preceding treatment; moreover, the length of each bleeding/spotting episode was significantly (p 0.05) shorter. Although 84 women completed 2 biopsies (before treatment and at day 14 or 21), adequate endometrial tissue at both time points was obtained from only 33 women. Histopathologic analysis revealed no obvious effect of either ethinyl estradiol or the combination pill on endometrium exposed to the levonorgestrel subdermal implant for an average of 8 months.