Molecular Human Reproduction, Vol. 5, No. 9, 797-802,
September 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular endocrinology |
Association of oestrogen receptor gene polymorphisms with outcome of ovarian stimulation in patients undergoing IVF
Department of Obstetrics and Gynaecology, National University of Singapore, National University Hospital, Lower Kent Ridge Road, Singapore 119074
Abstract
Oestrogen plays an important role in follicular formation and oocyte maturation via its receptor (ER). Many studies have shown association of the ER gene polymorphisms with a variety of pathological conditions. In this study we have examined the relationship of a common PvuII and a rare BstUI polymorphism in the ER gene to the mean numbers of follicles and oocytes, their mean ratios, mean number of embryos, mean oestrogen concentrations, mean size of the follicles and pregnancy rates. Analyses were carried out in 200 local Chinese patients undergoing in-vitro fertilization (IVF) and embryo transfer in three consecutive cycles. The mean follicular number, oocyte number, embryo number, follicular size and pregnancy rate were significantly smaller in patients homozygous for PvuII polymorphism (P < 0.001). These results indicate that PvuII polymorphism may be associated with ovarian follicular development and subsequently with the pregnancy rate. This study supports the view that genetic variability in the ER gene may have a role in the quality of the ovarian follicles in stimulation, which may affect implantation. However BstUI polymorphism was not found in either the IVF or control groups, suggesting that it has no role in the local Chinese population.
IVF/oestrogen receptor gene/ovarian stimulation/pregnancy rate/PvuII polymorphism
Introduction
Oestrogen is one of the two important female sex steroid hormones secreted by the ovary. Most ovarian control of pituitary gonadotrophin release is exerted by the steroid hormones: oestrogen and progesterone. Oestrogen plays a crucial role in embryonic and fetal development to influence female secondary sexual characteristics, reproductive cycle, fertility, and maintenance of pregnancy (George and Wilson, 1988
). It also plays a regulatory role in endometrial cell growth and differentiation through the induction of a redox-active protein (Maruyama et al., 1999). Various aspects of female reproduction are related to oestrogen and its functions.
Formation of follicles and maturation of oocytes are complex processes that require interrelated actions of luteinizing hormone (LH), follicle stimulating hormone (FSH) and oestrogen. A negative feedback controller of its own production, oestrogen exerts both autocrine and paracrine actions at the level of the follicle–oocyte unit (Zhuang et al., 1982).
The female sex hormone synthesized from androgen by LH, contributes to oocyte maturation and plays a relevant role in optimizing fertilization and embryo quality (Filicori, 1999). FSH and oestrogen stimulate antral and pre-antral follicular growth which is believed to be mediated by insulin-like growth factors (Yuan and Giudice, 1999).
Oestrogen triggers a broad array of physiological responses, which are tissue and organ specific, by binding to a nuclear receptor protein, the oestrogen receptor ER, of which two subtypes are known: ER-
and ER-ß (Kuiper et al., 1996). Both ER- and ER-ß genes are expressed in the human ovary (EnMark et al., 1997). Oestrogen receptors have been identified within the oocyte (Wu et al., 1993), in granulosa cells (Hurst et al., 1995) and in ovarian epithelial cells (Hillier et al., 1998).
The gene encoding ER- localized on chromosome 6q24-27 appears to be present as a single copy (Walter et al., 1985). Blastocysts and two cell stage embryos express ER- mRNA, which supports the possibility that oestrogen plays a role in early embryonic development (Hou and Gorski, 1993).
The ER- knock-out study in mice resulted in haemorrhagic cystic ovaries and follicular arrest in females. Both male and female mice were infertile. This study infers that ER- plays some role in infertility.
A number of ER- gene polymorphisms and mutations have been identified in patients with breast cancer, spontaneous abortion, osteoporosis and anti-oestrogen therapy resistance (Hill et al., 1989; Lehrer et al., 1990, 1993, Schmutzler et al., 1991; Berkowitz et al., 1994; Wang and Miksicek, 1994; Han et al., 1997; Mizunuma et al., 1997). The PvuII and BstUI polymorphisms in particular have been found to have a strong association with breast cancer and spontaneous abortions (Lehrer et al., 1993; Anderson et al., 1994).
The purpose of this study was to find an association of PvuII and BstUI polymorphisms in the ER- gene with follicular and oocyte quality and pregnancy outcome in patients from Singapore's Chinese population undergoing in-vitro fertilization (IVF) .
Materials and methods
Normally ovulating infertile women (n = 200), ranging from 24–39 years (mean ± SD, 29 ± 3.2 years) of age, with at least a 3 year history of failing to become pregnant and undergoing IVF/embryo transfer in our assisted reproductive technique unit were recruited for this study. Only patients who had normal cycles with unexplained infertility, and with no pathological conditions that would affect their ovarian response, e.g. endometriosis and polycystic ovarian syndrome, were included. There was one patient who had a past history of benign tubal cyst. However, this cyst, of unknown aetiology, was not the cause of her infertility and had been removed surgically 5 years previously. Infertility associated with tubal pathologies usually would not interfere with ovarian response to stimulation and was, therefore, included in the study group. In all 200 patients, the cause of their infertility could not be diagnosed in spite of extensive evaluation, e.g. post-coital tests, hysterosalpingography, laproscopy, basal body temperature, endometrial biopsy, urinary LH, ultrasound, etc. In order to conserve the homogeneity of our study groups and to be able to make reliable comparisons, all the patients were exposed to three consecutive cycles and cases which showed >50% variation in ovarian response to stimulation between successive cycles, e.g. poor response or ovarian hyperstimulation syndrome (OHSS) and single ovary, were excluded. A total of 200 randomly selected normal menstruating women aged 24–39 years (mean ± SD, 29 ± 3.2 years), having at least one spontaneous pregnancy served as controls. In each of the 200 controls, the pregnancy was natural without any induction therapy or stimulation. Approval by ethical committee was obtained.
The following long protocol was followed for follicular growth stimulation. Buserelin (Hoechst, Allemagne, Frankfurt, Germany) at a dose of 0.5 ml every morning was started on day 21 of the preceding cycle and continued for 14 days. Metrodin® (Serono, Aubonne, Switzerland) was given at a dose of 225 IU /day with buserelin reduced to 0.2 ml for 5 days. The dose was maintained if follicular size and numbers were adequate and if the serum oestradiol concentration was satisfactory. Otherwise, Metrodin was increased to 300 IU daily from day 6. When the dominant follicle reached 17 mm, human chorionic gonadotrophin (HCG, Profasi; Serono) 5000 IU was administered and oocyte recovery was carried out 36 h later. The luteal phase was supported by the administration of progesterone (Cyclogest, vaginal pessary; Hoechst) 400 mg per day.
Serum oestradiol values were measured by radioimmunoassay and were recorded 2 days prior to oocyte recovery. Follicular number, mature oocyte number and follicular size were measured by transvaginal ultrasonography. Follicular sizes were recorded on the day of oocyte aspiration. The sizes of leading follicles were calculated as the average of the two dimensions in mm as seen in ultrasonography. Follicular numbers and oocyte numbers were recorded immediately after aspiration. Only oocytes that showed features of maturity by microscopic analysis were selected. The number of embryos obtained and replaced in each of the three groups was recorded. Clinical pregnancies were evaluated by HCG measurement and abdominal ultrasonography. Serum HCG values were measured by enzyme immunoassay. Peripheral blood was drawn from the patients on the day of oocyte aspiration. Nuclear DNA was extracted from the peripheral leukocytes by a standard procedure.
Polymerase chain reaction (PCR) amplifications for the detection of PvuII and BstUI polymorphisms were carried out according to previously described methods (Yaich et al., 1992; Anderson et al., 1994) respectively. The PCR product (10–20 µl) was digested with PvuII and BstUI restriction enzymes as described previously (Liao et al. 1998). The restriction fragments were separated by electrophoresis on 2% agarose gel. The patients were classified into three groups according to their ER polymorphisms.
Statistical analysis
Statistical comparisons of the frequencies of ER genotypes between patients and controls, clinical pregnancies between genotypes, and comparison of frequencies of the polymorphism in the Singapore Chinese and Greek population were carried out using the 
2 test. One-way analysis of variance (ANOVA) was used for the statistical comparison of the mean follicular numbers, mean oocyte numbers, mean ratios of follicular to oocyte numbers, mean embryo numbers, mean oestrogen values and mean follicular sizes between genotypes. P < 0.05 was considered to be significant.
Results
The occurrence of PvuII polymorphism did not differ significantly between the patients and controls (P = 0.663, 2 = 0.0821). Among the 200 patients, 36 had no polymorphism, 68 were homozygous and 96 were heterozygous. Among the 200 controls, 43 had no polymorphism, 67 were homozygous and 90 were heterozygous (Table I). On the other hand, the BstUI polymorphism was seen neither in the cases nor in the controls studied.
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The mean number of follicles in the no-polymorphism group was 17.8, in the heterozygous group was 18.2 and in the homozygous group was 15.4 (Table II
). The mean follicular numbers between the no-polymorphism group and the heterozygous group were not significantly different (P = 0.921). However, the mean follicular number in the homozygous group was significantly smaller than that in the heterozygous group and no-polymorphism group (P < 0.001 and P = 0.015 respectively).
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The mean number of mature oocytes in the no-polymorphism group was 9.8, in the heterozygous group was 7.6 and in the homozygous group was 6.19 (Table II
). There were highly significant negative correlations between the mean mature oocyte numbers and severity of PvuII genotypes (P < 0.001).
Significant differences were found between the ratios of follicle to oocyte numbers in the three groups of PvuII genotypes (P < 0.001), the lowest value was in the no-polymorphism group and the highest in the homozygous group (Table II).
The mean size of leading follicles for the no-polymorphism group was 25.9 mm, that of the heterozygous group was 25.2 mm and the homozygous 23.8 mm (Table II). The difference in mean follicular sizes between the no-polymorphism group and heterozygous group was not significant (P = 0.381). However, the mean follicular size in the homozygous group was significantly smaller than that in the heterozygous group and the no-polymorphism group (P < 0.001).
Significant differences were found between the mean serum oestradiol values in each of the three PvuII genotypes (P < 0.001), the lowest value was in the no-polymorphism group and the highest in the homozygous group (Table II).
The total number of IVF/embryo transfer patients resulting in pregnancy was 72 (Table III). Of these, 32 pregnancies were in the no-polymorphism group (88.9%, 32 out of 36), 30 pregnancies in the heterozygous group (31.2%, 30 out of 96) and only 10 pregnancies in the homozygous group (14.7%, 10 out of 68). The pregnancy rate showed highly significant differences between the three genotypes (pp and Pp: P < 0.001 and 2 < 34.921; Pp and PP: P < 0.001 and 2 = 5.908). It was highest in the no-polymorphism group and lowest in the homozygous group. Hence, the pregnancy rate also showed a strong negative correlation to the severity of PvuII polymorphism.
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In the 72 pregnant patients, the number of embryos obtained and replaced in each of the three PvuII genotypes showed a highly significant negative correlation with the severity of the polymorphism. The mean number of embryos obtained in the no-polymorphism group was 9.4, in the heterozygous group was 4.9 and in the homozygous group was 2.2. The mean number of embryos replaced in the no-polymorphism group was 3.3, in the heterozygous group was 2.4, and in the homozygous group was 1.4. All showed a significant difference (P < 0.001; Table III
). Discussion
IVF involves artificial stimulation of the follicles, aspiration of mature follicles, oocyte retrieval, fertilization of the ova in vitro and transfer of the fertilized embryo inside the uterus. Although all the patients were exposed to the same protocol of follicular stimulation, follicular responses among the patients differed significantly. Numerous factors have been postulated to influence the success of IVF for alleviating human infertility (Edwards et al., 1984). The outcome of this programme depends very much on the factors affecting follicular growth, steroidogenesis and maturation of oocytes (Testart et al., 1983).
FSH plays a crucial role in affecting the growth of follicles. It stimulates proliferation of granulosa cells, aromatizes androgens to oestrogens, augments FSH receptors and induces LH receptors. On the other hand, oestrogen has a local regulatory effect. It augments the action of FSH, promotes granulosa cell proliferation and increases the number of oestrogen and FSH receptors. Oestrogen and FSH thus act in synergism in the ovary to increase the number of FSH receptors in the granulosa cells, resulting in follicular growth and maturation (Ireland and Richards, 1978).
Oestrogen has a direct affect on granulosa cell function. It exerts direct trophic effects on folliculogenesis (Greenwald and Roy, 1994) and protects the growing follicle from androgen-induced atresia (Filicori, 1999).
The hormone affects maturation of oocytes. Its action is required for optimal oocyte cytoplasm and oolemma maturation (Filicori, 1999). Thus, it plays a crucial role in determining the quality of oocytes (Kreiner et al., 1987). It has been documented that, in oestrogen-poor follicles, the quality of oocytes may be adversely affected (Fishel et al., 1983; Botero-Ruiz et al., 1984).
A good quality/matured oocyte is defined as one in which the re-initiation and division of first meiotic phase is complete, and has progressed to metaphase II with completion of the accompanying cytoplasmic process essential for fertilization and early embryonic development. Fertilization rates were proved to be higher with mature oocytes (Edwards et al., 1984).
In our study, follicular and mature oocyte numbers showed a significant negative association with the severity of the PvuII polymorphism. It is possible that this polymorphism down-regulates the ER gene, interfering with the effective mediation of oestrogen and its functions, on the follicle–oocyte unit. This down-regulation could vary with the severity of the polymorphism, leading to variation in follicle and oocyte numbers in each of the three polymorphic groups.
In a recent study in a Greek population, two polymorphisms PvuII and BstUI in the ER- gene that were known to be associated with breast cancer and spontaneous abortion in particular were analysed in IVF patients to elucidate the role of ER- in ovulation induction and implantation (Georgiou et al., 1997). It was found that these polymorphisms were associated only with the ratio of follicles to oocytes and not with the number of follicles or number of oocytes. The polymorphisms appeared to affect the ovarian response by changing the final number of mature oocytes.
In the present study, however, strong negative associations were found between severity of PvuII polymorphism in the ER gene, with both the follicular and mature oocyte numbers, and their ratios. A major contributing factor to the high follicular number and oocyte number in our study and their significant association with PvuII polymorphism could be due to the difference in stimulation protocol, the criteria used to select follicular size for HCG administration, and for oocyte aspiration. Difference in ethnicity of the population group studied could also play some role in contributing to the variation. Screening of this polymorphism in other populations and their responses to stimulation may have to be carried out to substantiate these findings.
The ratio of mean follicle number to mean mature oocyte number was lowest in the no-polymorphism group indicating that this group had the highest number of mature oocytes per follicle. Moreover, the homozygous polymorphic group showed highest ratios with the lowest number of mature oocytes per follicle. ER polymorphisms may thus affect the ovarian response by changing the final number of mature oocytes in relation to the number of follicles.
In our study, a significant negative association was also seen between follicular sizes and severity of the polymorphism. Oestrogen enhances FSH-stimulated aromatase activity, progestin and cAMP biosynthesis in cultured granulosa cells (Adashi and Hsueh, 1982; Welsh et al., 1983). Oestrogen receptors are present in human granulosa cells and play an important role in human follicular development (Hurst and Leslie, 1997).
Ovarian surface epithelial (OSE) cells participate in the formation of ovarian cortex and granulosa cells in early embryonic life (Auersperg et al., 1995). These cells continually proliferate, re-colonize the ovarian surface in the wake of each ovulation and participate in cyclic rupture of the Graffian follicle and formation of the corpus luteum in adult life (Murdoch, 1996). Cultured human OSE cells express both ER- and ER-ß mRNA, consistent with a role for oestrogen in the regulation of OSE cell functions. Oestrogen has also a crucial local positive feedback role in promoting the development of the follicle (Korach, 1994).
The PvuII polymorphism could disrupt the effectiveness of the oestrogen receptor in mediating the functions of oestrogen. This might affect the development or growth of the follicles, resulting in smaller sizes. The degree of disruptiveness could vary with the severity of the polymorphism which might be the underlying cause of the association of homozygous group with small follicular sizes and no-polymorphism group with large follicular sizes.
Large follicular sizes have been documented to have a beneficial effect on the outcome of IVF. Embryo quality as reflected by decreased fragmentation, increased cleavage, and increased implantation rate appears to be improved when HCG is delayed until two or more follicles reach at least 20 mm in diameter (Miller et al., 1996). It has been proven that in follicles of large sizes, the follicular fluid (FF) concentration of oestradiol (suggesting an increased aromatase activity) is high and high oestradiol values in the FF were associated with the recovery of fertilizable oocytes (Fishel et al., 1983). Oestradiol concentrations are related inversely to zona pellucida thickness and a thicker zona pellucida is present in unfertilized oocytes (Bertrand et al., 1996).
Further, many studies have shown that in patients undergoing IVF, FF oestradiol:androstenedione ratios are correlated with pregnancy potential (Botero-Ruiz et al., 1984; Kreiner et al., 1987; Anderson, 1993). It was found that addition of oestradiol to human oocyte maturation medium increased the fertilization and cleavage rates of in-vitro matured oocytes (Tesarik et al., 1995).
In our study, the number of embryos fertilized and the pregnancy rate were remarkably different in the three genotypes. The lowest rate was in the homozygous polymorphic group and the highest in the no-polymorphism group. Thus, our study shows that genetic variability of ER gene could play an important role in affecting pregnancy rates through their mediation of follicular sizes.
In oestrogen resistance, oestrogen receptors in the follicles fail to respond appreciably to the gonadotrophin stimulation with subsequent reduction in the negative feedback response (Chakravorty et al., 1991). In this study, the serum oestradiol concentrations were lowest in the no-polymorphism group and highest in the homozygous polymorphic group. It is possible that the no-polymorphism group, in terms of better efficacy of its receptor, shows more resistance and the homozygous group shows lower resistance. This could contribute to the varying serum oestradiol concentrations. Resistance to hormonal therapy has been seen in breast cancer patients carrying the PvuII polymorphism in ER gene (Yaich et al., 1992).
In a recent study, a nonsense mutation of ER gene was identified in a man (Smith et al., 1994). He showed elevated serum oestrogen concentrations, and no target tissue responses to oestrogen therapy. Similar syndromes of hormone resistance have been reported in mutations of glucocorticoid, androgen, thyroid hormone, and vitamin D receptors (Brooks et al., 1978; Brown et al., 1990; Chrousos et al., 1993; McDermott and Ridgeway, 1993).
Analysis of all findings from our study implies that polymorphism can affect the outcome of IVF by affecting folliculogenesis, oocyte maturation, embryo quality and endometrial receptivity.
In our study, there was no difference in the frequency of PvuII polymorphism between patients and controls (P = 0.663, Table I). Neither was any significance seen in the occurence of PvuII polymorphism between homozygous and heterozygous groups in the patients and controls (0.226 < P <1.000). Though the polymorphism was seen in the same proportion in both patients and controls, the women in the control group had at least one natural pregnancy. This shows that the mere presence of this polymorphism does not lead to infertility. This polymorphism might only alter the ovarian response to stimulation; the more severe the polymorphism, the poorer the quality of ovarian follicles and oocytes. Thus this polymorphism may play an indirect role in affecting the pregnancy rates of patients undergoing stimulation.
Moreover, the frequency of PvuII polymorphism was significantly higher in the local population than in the NorthWestern Greek population (P = 0.007). The occurrence of the homozygous group was significantly higher and the no-polymorphism group was significantly lower in the local Chinese population than in the Greek population (P = 0.024 and 0.006 respectively). However, the prevalence of the heterozygous group was not significantly different in these populations (P = 0.862); this shows that the PvuII polymorphism is more common in Singapore Chinese than in the Greek population.
The PvuII polymorphism, localized in intron 1, 0.4 kb upstream of exon 2 (Castagnoli et al., 1987), results from a point mutation (T
C) at the fifth position of the restriction site (CATCTG) and does not cause amino acid change. The location of the polymorphism in the intron makes it unlikely that the polymorphism may affect ER expression. However, the possibility that the polymorphism is in linkage disequilibrium with other ER mutations which do affect ER expression or function cannot be ruled out (Yaich et al., 1992). Some human infertility may arise from ER gene mutations (Korach, 1994), but the rare BstUI polymorphism was not found either in the patients or in the control subjects studied.
In conclusion, genetic variability of the ER gene may exert an indirect effect on the pregnancy outcome of IVF patients by affecting the development of the follicles, oocytes and embryos. Certain ER genotypes may show their side-effects more on the patients exposed to ovarian stimulation regimes, as seen by the reduction of pregnancy rate only in IVF patients. The PvuII polymorphism may serve as a marker in predicting ovarian responses and pregnancy rates in patients undergoing IVF/embryo transfer.
Acknowledgments
The authors wish to thank members of the ART team for collaboration, Associate Professor Victor Goh for providing the blood samples, and Dr Dong Fang, for statistical analysis.
Notes
1 To whom correspondence should be addressed 
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Submitted on February 17, 1999; accepted on June 22, 1999.
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  M. Simoni, C.B. Tempfer, B. Destenaves, and B.C.J.M. Fauser Functional genetic polymorphisms and female reproductive disorders: Part I: polycystic ovary syndrome and ovarian response Hum. Reprod. Update, September 1, 2008; 14(5): 459 - 484. [Abstract] [Full Text] [PDF]
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 A. Morandi Alessio, L. H. Siqueira, E. C. Couto de Carvalho, R. Barini, A. de Padua Mansur, N. Fenalti Hoehr, and J. M. Annichino-Bizzacchi Estrogen Receptor Alpha and Beta Gene Polymorphisms Are Not Risk Factors for Recurrent Miscarriage in a Brazilian Population Clinical and Applied Thrombosis/Hemostasis, April 1, 2008; 14(2): 180 - 185. [Abstract] [PDF]
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 S. Altmae, K. Haller, M. Peters, O. Hovatta, A. Stavreus-Evers, H. Karro, A. Metspalu, and A. Salumets Allelic estrogen receptor 1 (ESR1) gene variants predict the outcome of ovarian stimulation in in vitro fertilization Mol. Hum. Reprod., August 1, 2007; 13(8): 521 - 526. [Abstract] [Full Text] [PDF]
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R.M. Corbo, L. Ulizzi, L. Piombo, C. Martinez-Labarga, G.F. De Stefano, and R. Scacchi Estrogen receptor alpha polymorphisms and fertility in populations with different reproductive patterns Mol. Hum. Reprod., August 1, 2007; 13(8): 537 - 540. [Abstract] [Full Text] [PDF]
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 H. O. D. Critchley, T. A. Henderson, R. W. Kelly, G. S. Scobie, L. R. Evans, N. P. Groome, and P. T. K. Saunders Wild-Type Estrogen Receptor (ER{beta}1) and the Splice Variant (ER{beta}cx/{beta}2) Are Both Expressed within the Human Endometrium throughout the Normal Menstrual Cycle J. Clin. Endocrinol. Metab., November 1, 2002; 87(11): 5265 - 5273. [Abstract] [Full Text] [PDF]
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W. X. Liao and A. C. Roy Lack of association between polymorphisms in the testis-specific angiotensin converting enzyme gene and male infertility in an Asian population Mol. Hum. Reprod., March 1, 2002; 8(3): 299 - 303. [Abstract] [Full Text] [PDF]
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C. Sundarrajan, W. X. Liao, A. C. Roy, and S. C. Ng Association between Estrogen Receptor-{beta} Gene Polymorphisms and Ovulatory Dysfunctions in Patients with Menstrual Disorders J. Clin. Endocrinol. Metab., January 1, 2001; 86(1): 135 - 139. [Abstract] [Full Text]
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 J. Kitawaki, H. Obayashi, H. Ishihara, H. Koshiba, I. Kusuki, N. Kado, K. Tsukamoto, G. Hasegawa, N. Nakamura, and H. Honjo Oestrogen receptor-alpha gene polymorphism is associated with endometriosis, adenomyosis and leiomyomata Hum. Reprod., January 1, 2001; 16(1): 51 - 55. [Abstract] [Full Text] [PDF]
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