Status of Estrogen Receptor Beta Gene Polymorphisms rs1256049 and rs4986938 in a cohort of South Indian women with Primary ovarian insuf(cid:2510)iciency

The current study is aimed to investigate the role of estrogen receptor beta gene polymorphisms for causation of Primary Ovarian insuf(cid:977)iciency (POI) in south Indian women. Primary Ovarian insuf(cid:977)iciency (POI) is a menstrual disorder presenting with Primary and secondary amenorrhea, decreased estradiol levels and increased gonadotrophin levels. Most cases of POI remain unre-solved even after exhaustive investigations and is a signi(cid:977)icant cause of infertility in women in the reproductive age. In this study a total of 276 subjects, of which 138 POI patients and 138 controls were analysed for biochemical pro-(cid:977)iles of FSH, LH and Estradiol. Genotypic frequencies were calculated for different models of the SNP rs1256049 and rs4986938 in the Estrogen receptor Beta gene by PCR-RFLP (Restriction length polymorphism) analysis. Statistically, signi(cid:977)icant relation is found for FSH, LH and Estradiol pro(cid:977)iles of a different group of Premature ovarian insuf(cid:977)iciency and controls (p<0.0001). Genotypic frequencies between patients and controls were compared, and strength of the interdependence of genotypes and POI is con(cid:977)irmed by the odds ratio and 95% con(cid:977)idence intervals. Incidence of 1082 G>A (rs1256049) polymorphisms and 1730 G>A (rs4986938) were not signi(cid:977)icantly different in both patient and control group ( P =0.21 and P =0.4). The presence of 1082 G>A (rs1256049) polymorphisms and 1730 G>A (rs4986938) is not associated with causation of Premature ovarian insuf(cid:977)iciency in south Indian women.

The current study is aimed to investigate the role of estrogen receptor beta gene polymorphisms for causation of Primary Ovarian insuf iciency (POI) in south Indian women. Primary Ovarian insuf iciency (POI) is a menstrual disorder presenting with Primary and secondary amenorrhea, decreased estradiol levels and increased gonadotrophin levels. Most cases of POI remain unresolved even after exhaustive investigations and is a signi icant cause of infertility in women in the reproductive age. In this study a total of 276 subjects, of which 138 POI patients and 138 controls were analysed for biochemical proiles of FSH, LH and Estradiol. Genotypic frequencies were calculated for different models of the SNP rs1256049 and rs4986938 in the Estrogen receptor Beta gene by PCR-RFLP (Restriction length polymorphism) analysis. Statistically, signi icant relation is found for FSH, LH and Estradiol pro iles of a different group of Premature ovarian insuf iciency and controls (p<0.0001). Genotypic frequencies between patients and controls were compared, and strength of the interdependence of genotypes and POI is con irmed by the odds ratio and 95% con idence intervals. Incidence of 1082 G>A (rs1256049) polymorphisms and 1730 G>A (rs4986938) were not signi icantly different in both patient and control group (P=0.21 and P=0.4). The presence of 1082 G>A (rs1256049) polymorphisms and 1730 G>A (rs4986938) is not associated with causation of Premature ovarian insuf iciency in south Indian women.

INTRODUCTION
POI could be a common functional disorder of the ovaries within which accelerated loss of functional follicle occurs. The condition is characterised by amenorrhoea and oligoamenorrhoea with raised gonadotrophin levels (FSH >25 IU/L) and attenuated ovarian production of estrogen (estradiol <50 pmol/L) and progesterone. (Casson, 2000;Vujovic et al., 2010). POI is the leading cause of infertility in actively reproducing group as loss of function of the ovary occurs before the age of 40 years. (de Moraes-Ruehsen and Jones, 1967) Depending on biochemical parameters and menstrual irregularities, three different forms of POI are categorised. Patients with regular menstrual cycles and normal biochemical pro iles are considered occult. The second category that is biochemical POI is a disorder with a range of rising FSH from 10 to 40IU/L and amenorrhoea. The third category is overt POI, with elevated levels of FSH above 40IU/L and cessation of periods (Welt, 2008). POI is a multifactorial defect of several predisposing alleles. Metabolic disorders, chemotherapy, chromosomal abnormalities, autoimmune disorders, radiation and surgery, are the common etiological causes of POI. Incidence of premature ovarian insuf iciency account to 1% (Tartagni et al., 2007).
Estrogen is a Gonadal steroid hormone which is produced by the developing follicle. Estrogen imparts a crucial role in the proper functioning of the reproductive tract, cardiovascular system, progesterone induction, endometrial proliferation and maintenance of bone mass, fetoplacental function and maturation. Estrogen appears to enact a pivotal role in the development of the primordial follicle and ovulation. Estrogen imparts both positive and negative feedback regulation of the production of FSH. During the follicular phase developing follicle produces more amounts of estrogen which inhibits FSH secretion. This inhibition is a negative feedback regulation which prevents the growth of resting pool of primordial follicles restricting the loss of follicular pool, contrarily during the ovulatory phase estrogen stimulates the anterior pituitary for an outsized surge of luteinising hormone and a lesser surge of FSH (Stocco, 2001;Sharma et al., 1999) which stimulates the growth of follicles for the next menstrual cycle.
Progression of various diseases is additionally associated with estrogen including breast, colorectal, endometrial, Prostate, ovarian cancers, insulin resistance, cardiovascular diseases, lupus erythematosus, endometriosis, obesity and neurodegenerative disorders (Beck-Peccoz and Persani, 2006;Cramer, 1990;van der Schouw et al., 1996). The biological effect of estrogens is mediated through the activation of its receptors ER-α and ER-β, belonging to a nuclear receptor family and functioning as ligand-dependent transcription factors (Enmark and Gustafsson, 1999). In 1996 ESR2 was irst identi ied in the rat prostate and ovary (Kuiper et al., 1996). In the ESR2, two typical SNPs were selected to be analysed, rs1256049 (G1082A) and (G1730A) a A1730G the ligand-binding domain in exon ive and an untranslated 3 ′ -area in exon eight respectively were selected.

Patients and controls
The study group includes cases diagnosed with POI recruited from different hospitals in Hyderabad, and Institute of Genetics and hospital for Genetic disor-ders (n=138) and their age and sex-matched controls (n=138). All patients were selected based on at least four months of amenorrhea and two FSH serum measurements > 25 IU/L before 40 years of age. Blood samples were collected only after the informed consent proforma accepted by the institutes' ethical committee was duly signed by the subjects.
Both from patients and controls 5 ml of Peripheral blood was collected and stored in vacutainers coated with EDTA as an anticoagulant. Using the phenol-chloroform method according to changes in Blin & Stafford (1976) genomic DNA was extracted from peripheral blood lymphocytes. E2, FSH, luteinising hormone (LH), were quantitatively estimated in human serum samples using enzymelinked immunosorbent assay (ELISA) kits. All values are presented as means ± standard deviation. Deviations were considered signi icant at p < 0.05

Statistical analysis
Allele and genotype frequencies between groups are compared with the chi-square test to estimate the Hardy-Weinberg equilibrium. Using SNP STAT software. Signi icance of statistical test and χ2 analysis were carried out. P-values were two-tailed, and 95% con idence intervals (CIs) were calculated. Statistical signi icance is considered with a p-value < 0.05. To identify the association of individual polymorphisms haplotype inference were performed with Gabriel's rule applied in Haploview software (version 4.1; Broad Institute of MIT and Harvard University, Boston, MA).

Demographic analysis
Total samples were categorised into three groups for biochemical analysis of FSH, LH and Estrogen hormones. Group I for premature ovarian failure cases, group 2 secondary amenorrhea, and group 3 for primary amenorrhea.

DISCUSSION
Estrogens put their biological effects into action by activating their receptors ER-α and ER-β. ERβ gene (ESR2) spans of eight exons localised to chromosome 14q23-24.1. (Enmark et al., 1997) and is the main receptor in ovarian tissue. ERα and ERβ mRNA expression patterns with RT-PCR of Human fetal ovaries revealed the importance of estrogen and its receptors in ovaries (Revelli et al., 1996;Chiang et al., 2000). Many animal studies in adult hamster (Yang et al., 2002), rat (Palter et al., 2001;Bao et al., 2000), monkey (Pau et al., 1998), reported low levels of ERα and high levels of ERβ expression in the granulosa cells attributing its important role in the development and arrest of the resting primordial follicle pool.
Patients in the POI community were predicted to have substantially higher levels of serum FSH and LH and lower levels of E2 relative to healthy controls. High FSH in females indicates low ovarian reserve, and women with high FSH have signi icantly lower pregnancy chances than women with normal FSH levels. Exogenous estrogens were hypothesised to function by decreasing serum FSH, restoring follicles sensitivity or directly enhancing granulose cell sensitive to the impact of FSH.
For con irmed diagnosis of premature ovarian failure, FSH levels > 40 IU/l, with an interval of 4-6 weeks and recorded twice, is considered (Rebar et al., 1982). The average mean FSH levels in the patient's groups 1 (65.35) and group 3 (38.17) are 11 and 7 times higher that of control mean (5.60) whereas the average mean FSH value of group 2 (5.49) is comparable with that of control mean value. The LH mean of group1 and group 2 are determined to be 34.97 and 26.06, which are 4.8 and 3.6 times higher than the normal range in comparison to control mean (7.22). The mean LH value of group 2 is almost equal to that of control mean. Normal development of oocytes is critically dependent upon the pituitary hormones, luteinising hormone (LH) and follicle-stimulating hormone (FSH). Many of ovarian follicles fail to function normally because they become luteinised prematurely due to the associated chronically elevated serum LH levels (Nelson, 1994). The usual range of LH determined in women at reproductive age with the regular menstrual cycle is LH 1-5 IU/l. In POF, defects in LH receptor are typically associated with a serum LH elevation (> 10 U/L) more pronounced than that of serum FSH (Seeman et al., 1988). The mean estradiol levels in group1, group2 and group3, are 18.20, 51.51 and 15.87 respectively. These levels are 7.5, 2.6 and 8.6 times less than that of control mean value (136.97) ( Table 1). Examination of FSH levels in women with hypogonadotropic hypogonadism in the absence of follicle development, during follicle development, and ovulatory cycles, evidenced that estradiol, inhibin A, and inhibin B play a role in the dynamic negative feedback control of FSH secretion across the normal menstrual cycle (Welt et al., 2003). The absence of estradiol suggests aromatase defects (Bulun, 1996) or LH receptor abnormalities (Latronico and Segaloff, 1999); however, these congenital defects are associated with primary amenorrhea and are not acquired defects as in the hypogonadotropic woman studied. Plasma levels of E 2 , inhibin B, and AMH are of limited value in predicting the presence of an ovarian reserve in patients with POF (Bachelot et al., 2009).
Paired t-test for FSH and LH between control & group 1 and control & group 3 shows that the difference between the control mean and group mean signi icant whereas in case of control and group 2 there is no signi icant difference between the control mean and group mean. The paired test for estradiol exhibits a signi icant difference between the means of control and the three groups, p<0.0001 (Table 2).
Welch's test when performed for FSH and LH, the calculated p-value is much higher between control & group 1 and control & group 3, indicating the difference between the control mean, and group means are signi icant. Whereas in case of control and group 2 for FSH and LH, there is an insigni icant difference between the means of control and groups. In the case of estradiol, Welch's Test showed higher pvalue indicating a signi icant difference between the means of control and groups (Table 3).
Correlation matrix of FSH indicates a moderate negative degree of correlation between the Control and Group 1, negative reasonably high degree of correlation between control and group 2, and low negative degree of correlation between control and group 3. Further, a positive, meagre degree of correlation between group 1 and group 3. In the case of LH, there is a low negative degree to moderate negative degree of correlation between control and group1, 2 and 3. In the case of estradiol, a meagre degree of negative correlation between control and groups (1, 2, 3). It indicates that the secretion of estradiol has been decreased in group 1, group 2 and group 3 in comparison to control Table 4.
In (Krege et al., 1998) developed female ERβ knockout mice to study the role of estrogen beta in follicular development. The follicular growth retardation of these mice con irmed its essential role in normal ovulation, but not in lactation, sexual differentiation and fertility. Tsukamoto et al. irst identi ied a silent G1082A transition in exon 5 of the ERβ gene and association were reported by Arko et al. in a small cohort of postmenopausal Slovenian women with RsaI polymorphism (Arko et al., 2002). The same kind of association was reported in Italian postmenopausal women with the G1082A single nucleotide polymorphism on BMD and vertebral fractures (Becherini et al., 2001). (Sundarrajan, 2001) reported a positive association with ovulatory dysfunction including primary amenorrhoea, secondary amenorrhoea, POF and PCOS for the G1730A polymorphism in exon 8 of the ERβ gene.
Increased risk of breast cancer in postmenopausal women not in premenopausal women has been associated with polymorphisms of ESR2 (Zheng et al., 2003). Bianco and cols (Bianco et al., 2009) the effect of +1730 G/A polymorphism in ERβ gene (rs4986938) was investigated in infertile women with endometriosis and control and the risk of endometriosis, regardless the stage of the disease was increased. A positive relationship was established irst by Aschim et al. (2005) between genetic variants of the ERβ gene and male infertility, showing that the frequency of Rsa1 AG genotype in infertile men has been increased compared with the reference group. In the present study, genotype distributions of single SNPs1082 G>A (rs1256049) polymorphisms and rs4986938 (G1730A) are not significantly differing in the patients and control group. Even though RsaI and AluI polymorphisms in the ERb gene are silent mutations, they might confer susceptibility to a diseased condition when they are in linkage disequilibrium with other gene polymorphisms. (Noble et al., 1996)

CONCLUSIONS
Estrogen exerts bene icial effects on the development and regulation of follicular development. An early loss of ovarian function puts a woman at higher risk for cardiovascular diseases, osteoporosis, and ovarian cancer, increases the risk of mortality as estrogen receptor-mediated signalling pathway plays an essential role. In the present study, it was observed that polymorphisms of estrogen receptors ER-β) were not signi icantly associated with primary ovarian insuf iciency. Our inding is in discordance with previous studies that were conducted to explore the association between SNPs of ER -beta gene and POI in Singapore population, where it was found that these SNP's were signi icantly associated with POI. While the individual contribution of these polymorphisms to POI pathogenesis need to be carried out in a large number of association studies, remains to be veri ied and con irmed.

Con lict of Interest
The authors declare that they have no con licts of interest.