Molecular Human Reproduction, Vol. 6, No. 11, 1013-1018,
November 2000
© 2000 European Society of Human Reproduction and Embryology
Uterine physiology |
Expression of sex steroid receptors and Ki-67 in the endometria of menorrhagic women: effects of intrauterine levonorgestrel
1 Department of Obstetrics and Gynaecology, Helsinki University Central Hospital, PO Box 140, 00029 HUCH and 2 Health Services Research Unit, STAKES, Siltasaarenkatu 18, 00531 Helsinki, Finland
Abstract
The levonorgestrel-releasing intrauterine system (LNG-IUS) has proven to be the most effective medical treatment in reducing the amount of menstrual blood loss. However, the molecular mechanisms underlying menorrhagia and/or accounting for the therapeutic effect of the LNG-IUS are still obscure. In this study, we used immunohistochemistry to compare the distribution of sex steroid receptors and the proliferation marker Ki-67 in the endometria of women with and without menorrhagia before and after 6 and 12 months of treatment with an LNG-IUS. The study sample included 67 women (aged 35–49 years) who had spontaneous ovulatory cycles. In women with menorrhagia, secretory phase endometrium exhibited more proliferative activity than in women without menorrhagia. No significant differences were found in the immunoreactivity of the oestrogen or progesterone receptors in women either with or without menorrhagia suggesting that, in addition to endocrine hormones, other factors are involved in the regulation of endometrial proliferation and menstrual blood loss. A total of 35 women were treated with LNG-IUS. After 6 months use of an LNG-IUS, the immunoreactivity of both epithelial and stromal progesterone receptors, as well as those of epithelial Ki-67 declined, and no differences were detectable between the women in the menorrhagia and control groups. Breakthrough bleeding remained a problem for nine (26%) LNG-IUS users, with no association with the pre-treatment amount of bleeding. No significant differences were found in the parameters studied between the women with and without breakthrough bleeding 6 months after insertion of an LNG-IUS.
endometrium/Ki-67/levonorgestrel-releasing IUS/menorrhagia/sex steroid receptors
Introduction
Excessive menstrual bleeding (
80 ml per period) is one of the most common problems in gynaecology (Coulter et al., 1989
). Although new diagnostic methods have improved the clinical triage of menorrhagia, ~50% of the cases display no underlying pathology (Rees, 1987). The mechanisms of dysfunctional menorrhagia are complex and poorly understood. Steroid hormones are the major endocrine regulators of endometrial function, exerting their effects through cellular receptors which, when activated, induce gene expression of local regulatory factors. The interplay between oestrogen, progesterone, their receptors and local factors in endometrial function remains unclear. Contraceptive pills and the levonorgestrel-releasing intrauterine system (LNG-IUS) reduce the amount of menstrual bleeding (Andersson and Rybo, 1990; Milsom et al., 1991), suggesting that hormones are involved in the regulation of menstrual blood loss. On the other hand, cyclic progestogen has no effect on menorrhagia (Higham and Shaw, 1991), indicating that the underlying mechanism is not progesterone deficiency alone. Reports on the expression of sex steroid receptors in the endometria of women with menorrhagia are controversial.
The 19-nortestosterone derivative, levonorgestrel, has a strong progestin effect on the endometrium. The LNG-IUS, which releases 20 µg of levonorgestrel over a 24 h period, was originally designed for contraception but it has now proven to be effective in the treatment of menorrhagia (Andersson et al., 1990; Luukkainen and Toivonen, 1995). Data from studies regarding use of the LNG-IUS for contraception suggest that its mechanism of action is associated with atrophy of epithelial cells (Silverberg et al., 1986). Functional alterations have been shown to include down-regulation of endometrial progesterone receptors (PR) and proliferation (Critchley et al., 1998a; Salmi et al., 1998a) as well as suppression of insulin-like growth factor 1 (IGF-I) mRNA, with concomitant stimulation of IGF-II and IGF-binding protein 1 (IGFBP-1) mRNAs (Rutanen et al., 1997). However, no studies have been conducted on the effect of the LNG-IUS on endometrial steroid receptors and the proliferation marker Ki-67 in women with menorrhagia.
All progestogen-only contraceptives, including the LNG-IUS, are associated with the problem of breakthrough bleeding (Kovacs, 1996). This suggests that an imbalance in either sex steroid concentrations or their receptors may disturb the control of mechanisms responsible for the prevention of aberrations in normal bleeding patterns (Critchley et al., 1998b). To our knowledge, there have been no studies on sex steroid receptors in the endometria of women with and without breakthrough bleeding during use of the LNG-IUS.
The aim of our study was to examine the effect of the LNG-IUS on the distribution of sex steroid receptors and Ki-67 in the endometria of women with menorrhagia. In addition, we wanted to study the same variables in women with breakthrough bleeding during use of the LNG-IUS.
Materials and methods
A randomized multicentre trial, comparing hysterectomy with the LNG-IUS for the treatment of menorrhagia, was started at all five university hospitals in Finland in September 1994. The study population consisted of women aged 35–49 years, who were referred to the gynaecology outpatient clinics because of menorrhagia. Patients with endometrial or ovarian pathology (examined physically and by vaginal ultrasonography) were excluded, as well as those who had previous malignancies, urinary or bowel symptoms, or pain due to large fibroids. Those women who had taken hormonal preparations during the previous month, who had anovulatory cycles or a pathological finding in endometrial biopsy, who were menopausal (FSH >30 IU/l) or who failed to measure their menstrual blood loss (MBL) were also excluded. A total of 67 women, 41 women with menorrhagia and 26 without menorrhagia (controls) were enrolled at Helsinki University Central Hospital between September 1994 and September 1997. This study was approved by the Ethics Committee of Helsinki University Hospital and all women gave their informed consent.
MBL was measured by an alkaline haematin method as described previously (Hallberg et al., 1964; Hurskainen et al., 1998). In order to demonstrate the validity of our MBL measurement, serum ferritin concentrations were measured by direct chemiluminescent immunoassay (Chiron Diagnostics, Halstead, UK). True menorrhagia was defined as MBL of 80 ml per period. Women with a measured MBL of <80 ml were taken to be controls.
Endometrial samples were obtained from all 67 women, using a disposable biopsy curette (Pipelle; Laboratoire CCD, Paris, France), during the first out-patient visit, either in the proliferative or secretory phase of the cycle. The phase of the menstrual cycle was determined on the basis of menstrual history and confirmed by histological dating carried out by the pathologist according to established criteria (Noyes et al., 1975). A LNG-IUS was inserted into the uterus of 24 women with menorrhagia and 11 controls. Endometrial samples were collected for this study from 19 women with menorrhagia and from nine controls 6 months after the insertion, and from 17 women (11 with and six without menorrhagia) 12 months after insertion. Since the date of the menstrual cycle cannot be determined by histological criteria during use of the LNG-IUS, the data obtained after insertion were pooled, regardless of the phase of the menstrual cycle.
Bleeding over 5 days apart from cyclical menstrual bleeding was considered as breakthrough bleeding. Five women from menorrhagia group and four women from controls suffered breakthrough bleeding during the use of the LNG-IUS.
To check ovarian function, we measured serum FSH concentrations by an immunofluorometric method (Wallac, Turku, Finland), and serum oestradiol and progesterone concentrations by using [125I] radioimmunoassay kits (DPC Corporation, Los Angeles, CA, USA) before insertion of the LNG-IUS, and 6 and 12 months after the procedure.
The expression of sex steroid receptors and the proliferation marker Ki-67 in the endometrium was studied by immunohistochemistry, using monoclonal mouse antibodies to human. Oestrogen receptor (ER) (Daco ER; Dako, Glostrup, Denmark), progesterone receptor (PR) (Novocastra Laboratories Ltd, Newcastle-upon-Tyne, UK) and Ki-67 (Pharmigen, San Diego, CA, USA). The antibody against PR detects A + B isoforms of PR, and the antibody against ER reacts with ER
, but not with ERß. Avidin–biotin–peroxidase (ABC) staining was carried out by using Strept ABComplex/HRP Duet, Mouse/Rabbit (Dako) for both sex steroid receptors and Vectastain ABC kits (Vector Laboratories Inc., Burlingame, CA, USA) for Ki-67 as shown in our previous studies (Salmi et al., 1996, 1998a,Salmi et al., b). The formalin-fixed, paraffin wax-embedded tissue sections (5 µm) were deparaffinized in xylene and rehydrated by sequential incubation in decreasing concentrations of ethanol. The deparaffinized tissue sections were pre-treated with 10 mmol/l citric acid (pH 6.0) in a microwave oven for 2x5 min and left in the buffer at room temperature for 20–60 min before the staining procedure. All washings were performed using 0.01 mol/l phosphate-buffered saline (PBS), pH 7.4. The sections were treated with 0.8% hydrogen peroxide in methanol for 5 min to quench the endogenous peroxidase activity, and blocked with normal goat (ER, PR) or horse (Ki-67) serum (1:20) (Dako) for 20 min to reduce non-specific binding of antibodies. The primary antibody diluted in PBS (ER 1:800, PR 1:500, Ki-67 1:300) was added and the sections were incubated overnight at 4°C. After rinsing, biotinylated second-step goat anti-mouse immunoglobulins were added (ER, PR 1:100, Ki-67 1:200) (Dako), followed 30 min later by an ABC reagent (1:100). After 30 min the sites of antibody binding were demonstrated by developing the peroxidase reaction with 3-amino-9-ethyl-carbazole in 50 mmol/l acetate buffer (pH 5.0) containing 0.03% hydrogen peroxide, for 15 min. The sections were rinsed with water and counterstained with diluted haematoxylin. For each tissue section, a negative control was stained with mouse immunoglobulin (Vector Laboratories Inc.), used at the same concentration as the first antibody.
Insulin-like growth factor-binding protein 1 (IGFBP-1 ) immunohistochemistry was used to verify the endometrial response to levonorgestrel. Immunoreactive IGFBP-1 was detected by using monoclonal mouse antibodies to human IGFBP-1 (Mab 6303; Medix Biochemica, Kauniainen, Finland) (Rutanen et al., 1988) as previously described (Rutanen et al., 1991).
The immunohistochemical data was analysed by three independent observers. Ten fields of cells of a tissue section (magnification x200) were examined, and only nuclear staining was considered specific to ER, PR and Ki-67, and cytoplasmic staining specific to IGFBP-1. The immunostaining intensity of all tissue sections as regards sex steroid receptors and Ki-67 was scored on a four-point scale, where 0 = no staining; 1 = mild staining; 2 = moderate staining; and 3 = intense staining. IGFBP-1 was evaluated as being present or absent.
Statistical analysis
The data were analysed using non-parametric tests; the Mann–Whitney test for non-dependent variables and the paired Wilcoxon Signed Ranks test for data comparing women using a LNG-IUS. The associations between serum oestradiol and progesterone concentrations, and sex steroid receptors and Ki-67 were assessed by calculation of Pearson's product-moment correlation coefficients. P < 0.05 was considered to be statistically significant.
Results
Immunoreactive sex steroid receptors and Ki-67 in the endometria of women with and without menorrhagia
Characteristics of the subjects are shown in Table I. The scores for immunoreactivity of sex steroid receptors and Ki-67 in the endometrial samples obtained from women with and without menorrhagia are shown in Table II. When both study groups were analysed together, the scores of immunoreactivity of sex steroid receptors and Ki-67 were higher in proliferative phase than in secretory phase endometrium (ER, P = 0.02; PR, P = 0.0001; Ki-67, P = 0.04) (Mann–Whitney test).
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When comparing endometrial samples obtained from women with menorrhagia with those from controls, a significant difference was found in the immunoreactivity of epithelial Ki-67 in secretory phase endometrium (P = 0.04) (Table II
); women with objective menorrhagia had higher scores for Ki-67 than women with normal bleeding. No significant difference was detected in the immunoreactivity scores of either sex steroid receptors between the study groups (Mann–Whitney test).
Effect of the LNG-IUS
A total of 35 women were treated with the LNG-IUS (24 with menorrhagia, 11 controls). Insertion of a LNG-IUS resulted in widespread morphological changes in endometrial histology. After 6 months use, a decidual reaction in stromal cells and epithelial atrophy were seen in all endometrial samples available (19 menorrhagic, nine controls). The strength of decidualization varied from one region to another, throughout the functional layer. All samples showed immunoreactive IGFBP-1 in decidual cells as a sign of a decidual reaction (Rutanen et al., 1997). The scores for immunostaining of epithelial PR (P = 0.003), stromal PR (P = 0.003) and epithelial Ki-67 (P = 0.02) were significantly lower after 6 months' use of the LNG-IUS compared with proliferative endometrium prior to insertion (Wilcoxon test). A significant decrease was also detected in the immunoreactivity of epithelial PR (P = 0.03) and stromal PR (P = 0.02) when biopsies obtained during LNG-IUS use were compared with biopsies collected prior to insertion from secretory endometrium. Immunostaining scores for ER, PR and Ki-67 after 12 months use of the LNG-IUS were similar to those at 6 months, and no significant difference was detected at either timepoint between the women with and without menorrhagia (Table III).
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After 6 months use of the LNG-IUS, no significant differences were found in immunoreactivity of endometrial sex steroid receptors and Ki-67 between women with (n = 9) and without breakthrough bleeding (n = 19). There was no correlation between serum FSH, oestradiol or progesterone concentrations and immunoreactive ER, PR or Ki-67 (data not shown).
Discussion
Our finding of higher immunoreactivity of sex steroid receptors in proliferative phase than in secretory phase endometrium is in accordance with the results of previous studies (Lessey et al., 1988; Snijders et al., 1992; Critchley et al., 1993; Salmi et al., 1998a).
This study demonstrates, for the first time, higher immunoreactivity of the proliferation marker Ki-67 in the epithelium of secretory phase endometrium of menorrhagic women compared with the endometrium of controls. No significant differences were found in the immunoreactive scores of either ER or PR (A + B subtypes) between the endometria of women with menorrhagia and those without menorrhagia. Increased proliferation activity, which reflects oestrogen action, without any major change in either ER or PR in the endometria of women with menorrhagia suggests that either ER or PR in these endometria may exhibit functional alterations; some ER-mediated signals, e.g. those stimulating cell proliferation, may be stronger in women with menorrhagia than in women with normal bleeding. Since no differences were found in either serum oestradiol concentrations or immunoreactive ER in the endometrium, it is possible that errors in autocrine/paracrine factors that mediate and modulate the actions of hormones in the endometrium may account for both increased proliferation and increased blood loss in women with menorhagia (Giudice, 1994). However, it has to be considered that only ER is included in our evaluations.
To our knowledge, there is only one immunohistochemical study on endometrial sex steroid receptors in women with normal and excessive MBL (>80 ml per period) (Critchley et al., 1994). In that study, no differences were found between the two groups and this was confirmed by the present study. In another study (Gleeson et al., 1993), solid phase immunoassays were used to examine endometrial sex steroid receptors in menorrhagic women, and demonstrated an increased concentration of oestrogen receptors in premenstrual endometria of women with menorrhagia.
Use of the LNG-IUS for 6 months resulted in a significant decrease in the amount of menstrual blood loss (unpublished data), and simultaneously there was a decline in immunostaining of epithelial and stromal PRs compared with proliferative and secretory endometria before treatment. This is consistent with the results of previous studies (Critchley et al., 1998a; Salmi et al., 1998a). In addition, there was some decline (but not significant) in ER immunoreactivity in both the epithelial and stromal compartments. This is also in keeping with data from a recent study (Salmi et al., 1998a), which showed constant immunoreactivity of ER in women using the LNG-IUS for contraception. In spite of the presence of ER and endogenous oestrogen production, not only expression of PR but also other ER-mediated cellular functions appear to be suppressed in LNG-exposed endometrium (Rutanen et al., 1997; Salmi et al., 1998b). This implies that the molecular mechanisms of levonorgestrel action may be more complex than has been assumed. Indeed, the two distinct isoforms of the human PR (A and B) (Clark et al., 1987; Critchley et al., 1998b) have been shown to be differentially affected by levonorgestrel (Critchley et al., 1998b). Subtype A was actually found to be increased at 12 months post-LNG-IUS insertion and was therefore thought to be the subtype of PR that mediates long-term levonorgestrel action in the endometrium (Critchley et al., 1998b). Similarly, the oestrogen receptor (ER) subtypes and ß (Mosselman et al., 1996; Kuiper et al., 1997) may be differentially expressed in levonorgestrel-exposed endometrium.
Levonorgestrel is known to be a very potent progestogen in the endometrium, and the concentration in the endometrium is high during the use of LNG-IUS (Nilsson et al., 1982). In keeping with this, epithelial atrophy was detected in all endometrial samples. The small difference in Ki-67 immunoreactivity in endometrial epithelium observed between women with menorrhagia and those without menorrhagia before insertion of a LNG-IUS disappeared in 6 months. In addition, the blood loss-reducing effect of the LNG-IUS was similar in women with and without menorrhagia.
Disturbances in the endometrial bleeding pattern remained a major problem associated with the LNG-IUS (26% in this study). Because all women were treated with an equal dose of levonorgestrel and because no differences were found in either serum oestradiol concentrations or ER expression between women with and without breakthrough bleeding, it is likely that local factors rather than systemic hormones are finally responsible for the control of endometrial bleeding. However, the effect of endogenous oestrogen and progesterone on the appearance of breakthrough bleeding cannot be completely excluded. In some of our samples, decidualization in the endometrium varied from one region to another. It is possible that endogenous sex steroids can have an effect in regions where decidualization is weak, leading to breakthrough bleeding.
When interpreting the results of studies such as this, caution must be exercised because of the great degree of inter-individual variability in sex steroid receptors, observed in this and other studies (Snijders et al., 1992; Critchley et al., 1994). It is possible that subtle differences in receptor immunostaining may have been masked.
In summary, our results showed a significant difference in the distribution of the proliferation marker Ki-67 in the secretory phase endometrial epithelium between women with and without objective menorrhagia. No differences were found in immunoreactive scores of either steroid receptors in women with and without menorrhagia or in the women with and without breakthrough bleeding. This suggests that, in addition to endocrine hormones, local factors are involved in the regulation of endometrial bleeding pattern and menstrual blood loss. After 6 and 12 months treatment with LNG-IUS, no significant difference was found in the immunoreactivity of either sex steroid receptors or Ki-67 between the study groups. Immunostaining of epithelial and stromal PR and epithelial Ki-67 was decreased in both groups after use of LNG-IUS.
Acknowledgments
We thank Kristiina Nokelainen and Ritva Kömppä-Laitinen for their skilful technical assistance, Teija Karkkulainen for co-ordination of the women involved, Olli Laitinen for statistical consultation and Seija Puro for the Tables. This work was supported by grants from the University Central Hospital of Helsinki and the Academy of Finland.
Notes
3 To whom correspondence should be addressed at: Siltasaarenkatu 18, 00531 Helsinki, Finland. E-mail: Ritva.Hurskainen{at}stakes.fi 
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Submitted on May 18, 2000; accepted on August 21, 2000.
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