1 Biomedical Research Unit, Jessop Hospital for Women, Sheffield and 2 Division of Biomedical Sciences/Health Research Institute, Sheffield Hallam University, Sheffield, UK
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Abstract |
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Key words: CA 125/cyclical sequential oestrogen/gestodene therapy/endometrial morphology/placental protein 14/postmenopausal women
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Introduction |
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Oestrogen plays the central role of priming the endometrium, and allows progesterone to exert its effects (Li et al., 1992a). In the presence of atrophic endometrium due to insufficient oestrogen priming, the addition of a progestogen may have no effect or render the endometrium unstable and lead to abnormal bleeding. Bleeding may also depend on the type of progestogen used, as this could influence the endometrial vascular changes (Casanas-Roux et al., 1996
). Levonorgestrel, in particular, may have an additional effect on endometrial vasculature to produce well-developed arterioles and dilated sinusoids, which may account for the higher incidence of breakthrough bleeding with this progestogen (Graham and Fraser, 1982
).
Currently, the most commonly used progestogen in HRT preparations is the 19-nortestosterone, norethisterone, and this has significant androgenic activity. In recent years newer progestogens with less androgenic effect and with better cycle control have been developed and used for oral contraception. Such new progestogens include desogestrel, gestodene and norgestimate (Speroff and DeCherney, 1993).
Gestodene is a 19-nortestosterone progestogen that has a number of theoretical advantages. One of its most important properties is efficient cycle control. In view of its potency, lower doses will reduce androgenic activity whilst producing secretory endometrium, particularly in women with hirsutism and acne. Previous pilot safety evaluations of gestodene in postmenopausal HRT have suggested that 25 and 50 µg of gestodene in cyclical regimens for 12 days both caused a secretory endometrial transformation, without untoward changes such as endometrial hyperplasia during the period of follow-up (Schering Reports 91056, 9291 and 8946). Previous systematic clinical studies of dose-related, progesterone-related endometrial effects at constant oestrogen doses in artificial cycles showed significant differences in the mitotic activity, volume fraction of gland occupied by gland cells and volume fraction of endometrium occupied by glands, together with a significant reduction of the number of supra- and subnuclear secretory vacuoles (Li et al., 1992b).
The study of endometrial function has been by histological examination of endometrial biopsy specimens using either simple light microscopy or more refined histological techniques such as morphometry (Li et al., 1988), histochemistry (Klentzeris et al., 1991
), immunohistochemistry (Bell et al., 1985
) and computer-assisted three-dimensional evaluation (Casanas-Roux et al., 1996
). These techniques have permitted a more detailed study of the various components of the endometrium. A recent development has been the study of endometrial physiology by analysis of uterine protein content (Li et al., 1993a
) and patterns of distribution of these proteins (Beier-Hellwig et al., 1989
) in endometrial secretions obtained by direct aspiration or by the technique of uterine flushing (Li et al., 1993b
).
One of the endometrial proteins which reflects the secretory activity of the endometrium in premenopausal women is placental protein 14 (PP14). Concentrations of this glycoprotein, which is secreted by the endometrium, start to rise in the luteal phase and reach their highest concentrations in the plasma and uterine fluids in the luteal phase (Joshi et al., 1986; Julkunen et al., 1986
; Li et al., 1993a
). Another glycoprotein produced by the endometrium is CA 125 (Jacobs and Bast, 1989
), concentrations of which in uterine flushings have been shown to correlate with uterine PP14 (Dalton et al., 1995
). It has been shown previously that plasma PP14 concentrations rise in women receiving HRT (Li et al., 1992c
), but to date few data are available regarding the concentrations of these two proteins in uterine flushings from women receiving HRT.
The objective of this study was to compare two different doses of gestodene in a sequential oestrogen/progestogen combination using morphological and biochemical methods. In addition, the morphometric and biochemical changes were compared to determine how well the changes correlated to each other. This study formed part of a double-blind, multicentre trial evaluating the safety of 25 and 50 µg gestodene in combination with 2 mg oestradiol-17ß as a novel HRT; however, the data on PP14 and CA 125 were collected only at this centre.
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Materials and methods |
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Uterine flushing
This was performed as an out-patient procedure on day 25 or 26 of the treatment cycle using the Pipelle endometrial sampler (Laboratoire CCD, Paris, France) (Figure 1). None of the women had started bleeding before this procedure was performed. The method previously described (Okon et al., 1998
) was used. With the volunteer in the dorsal position, the thighs and legs in flexion, pelvic examination was performed to exclude any gross pelvic pathology and to determine the position and size of the uterus. Following asepsis of the external genitalia using Savlon® (0.015% w/v chlorhexidine gluconate, 0.15% w/v cetrimide; Zeneca, Wimslow, Cheshire, UK), a bivalve speculum was inserted into the vagina, exposing the cervix, which was then cleaned with Savlon. A Pipelle endometrial sampler that had been preloaded with 1 ml of sterile physiological saline (0.9% NaCl) was slowly introduced into the uterus. After about 30 s, the tip of the sampler was withdrawn by about 2 cm into the lower part of the uterine cavity, after which the saline solution was aspirated slowly through the vacuum mechanism of the Pipelle sampler. The plunger was withdrawn in increments of ~2 cm over a period of ~30 s. A volume of between 0.8 and 1 ml was consistently aspirated. The aspirate was immediately centrifuged at 2200 g for 5 min and the supernatant stored at 20°C at the Biomedical Research Unit at the Jessop Hospital for Women, for assay of PP14 and CA 125.
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The morphometric features measured were: (i) number of mitoses per 1000 gland cells; (ii) amount of secretion in gland lumen (score 0 to 3); (iii) volume fraction of gland occupied by gland cell; (iv) volume fraction of endometrium occupied by gland; (v) volume fraction of gland cell occupied by nucleus; (vi) number of supranuclear secretory vacuoles per 100 gland cells; (vii) number of subnuclear secretory vacuoles per 100 gland cells; and (viii) number of mitoses per 1000 stromal cells. For each measurement a minimum of 20 fields were examined from each slide.
PP14 assay
PP14 was measured by radioimmunoassay using the method described previously (Bolton et al., 1987). PP14 was iodinated by the chloramine-T method, and the resulting tracer was purified using a column of ConA Sepharose. For the assay, 100 µl of 1 ng/ml PP14 tracer and 100 µl of standards or sample were incubated at room temperature for 24 h with 100 µl antiserum, at a dilution to bind 45% of the added tracer. The antibody-bound tracer was separated from the unbound using Amerlex-MMT magnetic separating reagent (Amersham International, Little Chalfont, Bucks, UK). The lower sensitivity of the assay was 3 ng/ml, and the intra- and inter-assay coefficients of variation were below 10% at a concentration of 3 ng/ml.
CA 125 assay
CA 125 concentrations in serum and uterine flushing were measured at the Immunology Department of the Northern General Hospital, Sheffield using an enzyme-linked immunosorbent assay (ELISA)-CA 125 11 kit (CIS Bio International, Cedex, France), which is a two-site immunoradiometric assay. The CA 125 was adsorbed onto the solid phase by antibody ELISA-CA 125 11 and then quantified using radioiodinated OC125 antibody as tracer. All assays were carried out in duplicate and performed according to the manufacturer's instructions. The lower and upper limits of detection were 0.5 and 500 IU/ml respectively, and the inter- and intra-assay coefficients of variation were <5% at these concentrations.
Study follow-up and menstrual calendar
The volunteers were requested to record tablet taking and bleeding (if any) on a daily basis on a diary card provided. The women were reviewed every 3 months, and at the end of each 12 months the uterine flushing and the endometrial biopsy were repeated and analysis performed as described previously.
Analysis of data
In view of the variability in concentrations of PP14 and CA 125, the data were log transformed before analysis. Concentrations of PP14 and CA 125 and the morphometric indices in the groups were compared using the MannWhitney (non-parametric) U-test.
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Results |
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The median serum concentrations of CA 125 pre and post 12 cycles of treatment in the 25 µg gestodene group were 16.0 and 15.5 IU/l respectively, while the corresponding values for the 50 µg gestodene group were 15.5 and 14.0 IU/l. There was no significant change in post-treatment plasma concentrations of CA 125 within either group, or between the treatment groups. The median uterine flushing concentrations of CA 125 pre and post 12 cycles of treatment in the 25 µg gestodene group were 45 600 and 41 360 IU/l respectively. No significant rise was observed between the pre- and post-treatment uterine flushing concentrations of CA 125 in either the 25 or 50 µg gestodene groups, or between treatment groups.
Bleeding patterns
Vaginal bleeding was defined as any appearance of blood in the vagina (regardless of amount), or any requirement for sanitary towels. Bleeding during treatment with HRT was classified as light (less than bleeding associated with normal menstruation), moderate (like normal menstruation) and heavy (more than normal menstruation). Bleeding was regular if it occurred between day 23 of the current cycle and day 4 of the subsequent cycle, and irregular for all other episodes of vaginal bleeding with or without regular bleeding. Bleeding data were not complete for two women in the 50 µg group; hence these were excluded from the analysis. The nature of bleeding before the menopause was based on patient recall. On average, bleeding was light to normal in the two groups of HRT regimen compared with the nature of bleeding before the menopause. There was no difference in the pattern of bleeding between the two preparations.
Figure 3 shows the pattern of menstruation in women taking 25 and 50 µg gestodene HRT preparations. The mean number of days of withdrawal bleeding in women taking 25 µg gestodene was 3.8 days, while that for women taking 50 µg was 4.2 days. There was no statistically significant difference between the two groups. Most of the bleeding occurred between day 28 and day 4 of the subsequent cycle. One subject who was taking the 25 µg preparation experienced an episode of irregular bleeding that occurred within the first 6 months of the trial. The overall multicentre regular or no bleeding was 80% for 25 µg gestodene and 74% for 50 µg, whilst the rest were irregular for each group.
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Discussion |
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Biochemistry
In the current study, the uterine flushing concentrations of PP14 appear to show significantly greater variability than were observed in previous studies using period-free preparations (Okon et al., 1998). The reason for this variability may be measurement error, but this is unlikely as the PP14 assay was performed in paired samples. A more likely reason is the considerable inter-subject variations in pharmacokinetics for synthetic progestational compounds such as gestodene. Third, there may be a difference in the pharmacokinetics of the concurrently administered oestradiol 17ß. Additionally, individual variations in the spectrum of oestrogenic, androgenic, antioestrogenic, glucocorticoid and the antimineralocorticoid effects of gestodene (Edgren, 1986
) may have affected the PP14 concentrations. This difference in bioavailability at the endometrial level, though not reflected in the histological biopsies, may account for the observed variation in uterine washing concentrations of PP14. To what extent these variations in endometrial level bioavailability and receptor-mediated responses affect endometrial PP14 production and secretion are speculative. Finally, it is possible that the postmenopausal endometrial secretory response may vary among women. It could be argued that the sampling technique could introduce this variation, but there was no change either in technique or in the person taking the samples (M.A.O.). There was no correlation between the pattern of bleeding and the concentrations of PP14 in the uterine washings, and this is consistent with our previous findings (Okon et al., 1998
) and those of others (Habiba and Al-Azzawi, 1994
).
There was no significant change in the concentration of CA 125 in uterine flushing during treatment. This supports earlier findings that there was no significant variation in the uterine flushing concentrations of this protein either during the menstrual cycle or during treatment with two different forms of period-free HRT (Okon et al., 1998).
Morphology
On the basis of the current results, no significant difference was observed between the various morphological measurements in the 25 and 50 µg gestodene groups, with both regimens producing secretory transformation in the majority of biopsies.
First, the differences in the endometrium constitute a spectrum, the elements blend very closely and may be difficult to discern. However, the chances of error were reduced by the use of the theoretically objective method of morphometric analysis. Nonetheless, to demonstrate a significant difference, the use of this method in a small number of patients requires it to be further improved. The only limitation of the observation is the fact that a relatively small number of biopsies were studied (10 in each group). However, it is still possible that there is indeed a difference between the two groups, which could be detected by a larger sample size; however, the differenceeven if it is presentis likely to be small. It was not possible to show any significant correlation between endometrial morphology and uterine flushings concentrations of PP14, most likely due to the relatively small number of subjects in the study or to the variability of PP14. Thus, larger subject numbers may be required to determine this relationship on a statistical basis.
Hence it appears that gestodene, in doses of 25 and 50 µg, produced a desirable endometrial response in the majority of subjects. The finding that 25 µg gestodene produced results similar to those achieved with 50 µg, means that the former dose level could be used to significant clinical advantages as it would produce fewer androgenic side effects.
Bleeding
Both preparations conferred good cycle control with scheduled withdrawal bleeds. The degree and duration of loss did not differ from premenopausal levels; however, both the current subjects and those from other centres in this study menstruated for fewer days compared with the previously reported pattern of a mean of seven days (Habiba et al., 1996). Fewer women in our centre had irregular bleeding, and this difference may be the result of the smaller sample size compared with the overall result rather than the difference in bleeding patterns of the women at other centres. Irregular bleeding is one of the most important reasons given for withdrawal from HRT (Okon et al., 1996
). This study shows desirable cycle control in the majority of subjects. Gestodene in preparations used for oral contraception is known to produce some of the best cycle control; however, its association in some studies with higher risks of thromboembolic phenomena (Bloemenkamp et al., 1995
; Jick et al., 1995
; WHO Collaborative Study of Cardiovascular Disease and Steroid Hormone Contraception, 1995) has curtailed the prescription of gestodene-containing preparations in recent times.
In conclusion, both preparations of gestodene/oestradiol 17ß regimens produced a significant rise in uterine flushing concentrations of PP14, but not of CA 125. There was no significant difference in endometrial morphometry and biochemistry (PP14 and CA 125) between the two doses (25 and 50 µg) of gestodene used in the samples examined in the current study. As in previous studies, it seems that PP14 is a sensitive biochemical marker in the assessment of endometrial response to HRT in postmenopausal women.
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Acknowledgements |
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Notes |
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References |
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Submitted on October 31, 2000; accepted on March 12, 2001.