Role of FSHR rs6165 and ESR2 rs4986938 polymorphisms in ovarian stimulation of Iranian women who underwent assisted reproduction treatment

Abstract

BACKGROUND:

Gonadotropin therapy was used to stimulate the ovary in infertile women who underwent assisted reproduction treatment (ART). Numerous studies indicated that infertile women showed different responses to gonadotropin therapy. Follicle stimulating hormone receptor (FSHR) and Oestrogen receptor (ER) polymorphisms have been reported to involve induction of folliculogenesis and ovarian response to treatment.

METHODS:

In the present study, two polymorphisms, namely FSHR rs6165 and ESR2 rs4986938, were investigated in 198 Iranian infertile women aged less than 39 years who underwent ART. After DNA extraction, these polymorphisms were genotyped by TaqMan genotyping assay. According to the number of eggs released during ovulation, the patients were categorized into poor responders (PR) and good responders (GR). The results indicated that the good responders showed lower levels of FSH and LH than poor responders. Results: No association was observed between ESR2 rs4986938 and poor response in Iranian women patients. Instead, FSHR rs6165 showed a strong association with ovarian response to ART ( $p<$ 0.05).

CONCLUSION:

The obtained results indicated that FSHR rs6165, not ESR2 rs4986938, could be suggested as a candidate marker to predict poor ovarian response.

Keywords

FSHR ESR2 polymorphism TaqMan genotyping assay poor responders

1. Introduction

The most widely practised assisted reproductive techniques is in vitro fertilization (IVF). This approach is known to be an effective and safe tool in the treatment of infertility. It has opened a new window of hope for infertile couples. The most important indications of IVF included male factor infertility, tubal factor infertility, endometriosis, ovulation disorders, unexplained infertility, ovarian failure,and diminished ovarian reserve (DOR) [1].The success rate is directly correlated with the number of oocytes retrieved, and, in turn, it affected the number of embryos available to transfer. Furthermore, the success of IVF treatment largely depends on controlled ovarian hyperstimulation (COH). This technique involved using injection of exogenous gonadotropins to induce multiple follicles. The purpose of using controlled ovarian stimulation (COH) during IVF cycles was to grow and develop of several follicles simultaneously [2, 3]. In COH, different gonadotropins were used to induce ovulation including HMG (e.g. Repronex, menopur) and urinary or recombinant follicle-stimulating hormone (e.g. Follistim, Bravelle, Gonal-F). Before COH, inhibition of pituitary functionmust be done using agonists (e.g. Lupron) or antagonists of GnRH (e.g. Cetrotide and Ganirelix). These drugs have been demonstrated to prevent premature luteinization during folliculogenesis.

Follicle-stimulating hormone (FSH) played an ovarian function through major effects on granulosa cell proliferation, maturation of eggs, and oestrogen synthesis [2]. Previous studies indicated that decreased concentration of FSH followed by high concentration of oestrogen plays an important function in the selection of the dominant follicle. The physiological action of FSH depends on the activation of its receptor (FSHR). The FSH receptor was expressed in the Granulosa cells [4]. The chromosomal location of the FSHR gene is 2p21-p16, and the most frequent SNP in FSHR is Thr307Ala (rs6165) The polymorphism at position 307, which can cause an amino acid exchange, is located in exon 10 and the extracellular domain of FSHR, which is the hormone-binding region. Threonine is replaced by alanine via rs6165, which also leads to the removal of a potential O-linked glycosylation site [5].

Normal functioning of FSHR is important for folliculogenesis and production of oestradiol. Its inactivating mutations and polymorphisms could impair the function of the receptor, resulting in the disruption of development and maturation of the follicles [6].

Furthermore, oestrogen also plays a major role in follicle formation and maturation of oocytes. Oestrogen controls many physiological responses via binding to its receptor, the oestrogen receptor (ESR). Previous studies indicated that genetic variations in oestrogen receptor could be used as a suitable tool in diagnosis and treatment of infertility.

Two oestrogen receptors have been identified in humans – ESR1 (ER $\alpha$ ) and ESR2 (ER $\beta$ ). The latter is located on chromosome 14q23.2 and, located on 3’ UTR of its exon 8, is the AluI (rs4986938) gene polymorphism, which could affect mRNA stability and translatability [7].

Considering the role of FSH and oestrogen in ovulation and response to treatment, the mutations or polymorphisms in their receptor could affect the patient’s response to COH.

The unpredictable variability in response to gonadotropins were one of the most important problems of IVF treatment, resulting in poor response to ovarian hyper stimulation syndrome (OHSS) [2]. In infertile patients with poor response, the main issue was the limited number of eggs, resulting in muted retrieval of oocytes. The aim of this study was to investigate whether the FSHR (rs6165) and ESR2 (rs4986938) polymorphisms were associated with induction of poor ovulation during ART treatment in Iranian infertile women.

2. Materials and methods

2.1 Subjects

A total of 198 infertile women who underwent ART were recruited in the present study. After using the ovulation-inducing drugs, the number of eggs released during ovulation was evaluated. The patients were categorized into poor responders (PR; $\leqslant$ 5 mature eggs) and good responders (GR; 6–15 mature eggs). The inclusion criteria were infertile women under the age of 39 without coagulation disorders, immunological disorders, chromosomal abnormalities, hormonal disorders, polycystic ovarian syndrome, and endometriosis. Infertility caused by the male factor was not included in this study. Infertile women with hyper ovarian response and ovarian hyper stimulation syndrome (OHSS) were also excluded from the study. COH was conducted based on the long protocol agonist. The dose of FSH was determined at a range between 150–300 IU.

Table 1
Biological and clinical characteristics of 198 infertile women who underwent IVF

Variable	Poor responders ( $n=$ 92)	Good responders ( $n=$ 106)	P value
	Mean $\pm$ SD	Mean $\pm$ SD
Age (Years)	34.75 $\pm$ 3.69	32.18 $\pm$ 3.94	0.500
Total oocytes	5.09 $\pm$ 2.81	12.80 $\pm$ 4.19	0.000*
MI	0.64 $\pm$ 0.94	1.23 $\pm$ 1.73	0.003*
MII	3.16 $\pm$ 1.42	9.95 $\pm$ 3.16	0.000*
GV	1.16 $\pm$ 1.95	1.41 $\pm$ 2.13	0.497
Embryo	2.93 $\pm$ 1.60	8.04 $\pm$ 2.83	0.000*
FSH (lU/L)	7.90 $\pm$ 3.34	6.38 $\pm$ 2.17	0.028*
LH (lU/L)	5.84 $\pm$ 3.81	4.86 $\pm$ 2.35	0.043*
AMH (ng/ml)	1.52 $\pm$ 1.24	2.70 $\pm$ 1.71	0.042*

MI: Metaphase I; MII: Metaphase II; GV: Germinal vesicle; FSH: Follicle-Stimulating Hormone; LH: Luteinizing Hormone; AMH: Anti-Müllerian hormone.

Table 2

Association between FSHR rs6165 and ESR2 rs4986938 polymorphisms and response to treatment in Iranian infertile women

FSHR rs6165 genotype
Polymorphisms	Poor responders (%)	Good responders (%)	Odds ratio (95% CI)	P value
GG	26 (29%)	38 (37%)	1.00 (reference)	—
AG	35 (39%)	48 (46%)	1.06 (0.54–2.06)	0.850
AA	29 (32%)	18 (17%)	2.35 (1.08–5.09)	0.029*
FSHR rs6165 allele
G	87 (48%)	124 (60%)	1.00 (reference)	—
A	93 (52%)	84 (40%)	1.57 (1.05–2.36)	0.026*
ESR2 rs4986938 genotype
GG	37 (40%)	50 (47%)	1.00 (reference)	—
AG	40 (44%)	46 (44%)	1.17 (0.64–2.14)	0.598
AA	15 (16%)	10 (9%)	2.02 (0.81–5.01)	0.126
ESR2 rs4986938 allele
G	114 (62%)	146 (69%)	1.00 (reference)	–
A	70 (38%)	66 (31%)	1.35 (0.89–2.05)	0.149

According to the hormone assay, FSH and LH levels on Day 3 of the menstrual cycle were assessed by electrochemiluminescence (ECL), and AMH levels were measured by means of the enzyme-linked immunosorbent assay (ELISA).

2.2 Genotyping of FSHR rs6165 and ESR2 rs4986938 polymorphisms

Genomic DNA was extracted from peripheral blood using GeneAll Kit (Korea) according to the manufacturer’s instructions [21]. TaqMan assay was performed to determine the genotypes of FSHR rs6165 and ESR2 rs4986938 polymorphisms using 96-well plates on real-time PCR, Light Cycler 96 (Roche Company). The primer-probe sets were made using the Applied Biosystems design service (ID: C_2676873_30, C_11462726_10, PN4351379, Applied Biosystem, California). Two different alleles of each locus were distinguished using the dyes 6-carboxyfluorescein (FAM) and VIC. The amplification reaction was performed in a final volume 10 $\mu$ l including 2 $\mu$ l of DNA, 5 $\mu$ l of TaqMan Universal PCR Master Mix, 0.5 $\mu$ l of primer-probe, and 2.5 $\mu$ l of DNAase-free water. The cycling conditions were as follows: initial denaturation at 95 ${}^{\circ}$ C for 15 min, 40 cycles consisting of denaturation at 95 ${}^{\circ}$ C for 15 sec, and annealing/extension at 60 ${}^{\circ}$ C at 60 sec. The genotype of some PCR products was validated by DNA sequencing.

3. Statistical analysis

T-test was used to assess the statistical differences between PR and GR groups in biological and clinical characteristics. The obtained data were presented as mean $\pm$ standard (SD) deviation. $P<$ 0.05 was considered statistically significant. Odds ratios (OR) were also calculated with 95% confidence interval (95% CI).

Table 3
Hardy-Weinberg equilibrium

Studied population			Normal responders
Genotype	Observed	Expected	Observed	Expected
ESR2
Homozygote reference GG	87	85.4	50	50.3
Heterozygote AG	86	89.3	46	45.5
Homozygote variant AA	25	23.4	10	10.3
Variation allele frequency	0.34	–	0.31	–
	$x^{2}=$ 0.269	0.603	$x^{2}=$ 0.0153	0.901
	$<$ 3.84	with 1 degree of freedom	$<$ 3.84	with 1 degree of freedom
FSHR
Homozygote reference GG	64	57.4	38	37
Heterozygote AG	83	96.3	48	50.1
Homozygote variant AA	47	40.4	18	17
Variation allele frequency	0.46	–
	$x^{2}=$ 3.678	0.055	$x^{2}=$ 0.178	0.672
	$<$ 3.84	with 1 degree of freedom	$<$ 3.84	with 1 degree of freedom

3.1 Results

The 198 infertile women who referred to the IVF unit of the Laleh and Erfan hospitals were selected in the present study. Patients with five or less mature (MII) oocytes after ovulation induction were considered as poor responders. Therefore, 92 patients were identified as poor responders (PR) while there were 106 good responders (GR). In our study, 44 patients had less than five retrieved oocytes and 59 had five or less retrieved oocytes.

Clinical and biological characteristics of the women patients were shown in Table 1. The results showed no significant differences in age and the number of germinal vesicles (GV) between poor responders and good responders. In contrast, these two groups showed a statistically significant difference in terms of the total number of oocytes, MI, MII, and embryos, as well as the levels of hormones LH, FSH, and AMH ( $p<$ 0.05). The average number of total oocytes, MI, MII, embryo, and the AMH levels were higher in good responder patients (GR) than in poor responders (PR). In contrast, the good responders showed lower levels of FSH and LH than poor responders.

Genotype frequencies of FSHR rs6165 and ESR2 rs4986938 polymorphisms were presented in Table 2. There was no significant difference between expected and observed genotype frequencies of FSHR rs6165 as well as ESR2 rs4986938, representing that the genotype distribution of two polymorphisms was in Hardy-Weinberg equilibrium ( $p>$ 0.05) in the studied population and good (normal) responders (as displayed in Table 3).

Statistical analysis showed that ESR2 rs4986938 polymorphism was not associated with response to treatment of Iranian women patients undergoing IVF ( $p>$ 0.05). In contrast, the FSHR rs6165 polymorphism showed a strong association with treatment response ( $p<$ 0.05).There was no significant correlation between the clinical characteristics and FSHR rs6165 or ESR2 rs4986938 genotypes in both groups of responders. However, the number of MI eggs and the AMH level were higher in the poor responders with AA genotype of rs4986938 polymorphism than in those with other genotypes (GG and AG).

4. Discussion

Infertility is recognized as a failure to achieve a successful pregnancy after more than 12 months of regular unprotected intercourse. Infertility has been reported to affect almost 10–15% of couples. IVF is the most common assisted reproductive techniques. Before being used in IVF, the exogenous gonadotropins were used to induce multiple follicle development. Unpredictable variability was observed in response of patients to gonadotropins. The poor response to IVF could lead to failure of treatment. Different environmental and genetic factors have been known to involve in the aetiology of poor response to gonadotropins. The understanding of the ovarian response to stimulation has been identified to play an important role in the success of IVF treatment. Previous studies indicated that some polymorphisms were associated with the severity of symptoms in patients with OHSS and response to FSH in IVF treatment [8, 9].

In the present study, two polymorphisms including FSHR (rs6165) and ESR2 (rs4986938) were assessed in two responder groups of infertile women referred to the IVF unit.

Women below the age of 39 were chosen for this study because the prevalence of poor ovarian response increases with age and women above the age of 40 have a $>$ 50% possibility of poor ovarian response. Follicle depletion increases six times once the individual passes the age of 39, and a great number of clinical experiments have found important fluctuations regarding the ovarian response after the age of 38 [10]. In our study, the average age and the number of GV oocytes did not show any significant differences in both GR and PR. However, there was a significant difference in the total number of eggs, MI, MII, and embryo as well as the levels of AMH, LH, and FSH in the way that all cases except LH and FSH levels were in patients with poor response (PR) compared to those with good response (GR). The levels of LH and FSH were higher in poor responders than in patients with good response. In a previous study, Farimani et al.found that the average age was not statistically different between poor responders and good responders in IVF treatment, confirming this fact that age could not be used as an index for patients’ response to treatment [11]. In another study, the women referred to IVF treatment were divided into three groups – poor (less than 4 follicles), normal (4–14 follicles), and strong (15 $<$ follicles) responders. After that, the possible risk factors for the ovarian response were assessed. The results obtained from this study showed that, in patients with poor and normal response, the average age was higher than in the high responders. However, no difference was observed in the average age of poor and normal responders. Confirming our observations, this study also indicated that the average number of oocytes in PR was lower than in two other groups. Esmaeilzadeh and Faramarz also found no significant difference in the levels of LH and FSH between these groups of responders. These observations were different from our finding [12]. In other study, the levels of FSH and LH did not show any significant difference between responders to IVF treatment. However, there was a strong correlation between the level of AMH, the oocyte number, and patients’ age. The level of AMH was different between poor responders and normal responders, which was consistent with the results obtained from our study [14]. Bordin et al. also showed that the highest success rate of ART was observed in women with low FSH ( $<$ 6.7 units/L) and high LH ( $>$ 4.9 units/L) [13].

Our results also showed that the frequency of AA genotype (rs6165) was significantly higher in the poor responders than in the good responders to IVF treatment (Table 2). Furthermore, women with poor response showed a higher allele A (rs6165) frequency than good responders did. However, there was no significant difference in clinical characteristics (e.g. age, the number of oocytes at stages MI and MII, and embryo, besides levels FSH, LH, and AMH) based on the genotypes of rs6165 polymorphism.

These observations were consistent with the results obtained by Yan et al. [15]. The study on 450 Chinese infertile women indicated that FSHR (rs6165) was associated with poor response to ovarian stimulation. However, they did not show any significant differences in clinical parameters including age, BMI, the level of oestradiol, LH and FSH required for ovulation based on polymorphism rs6165 [15]. Renzi et al. also found that the rs6165 polymorphism was associated with the dose of FSH used by women who had underwent ART [16].

A cross-sectional study, which was conducted in 2014 and included 149 infertile women in Brazil, concluded that Ala307Thr and Asn680ser did not contribute to the determination of FSH and oestradiol serum levels, as well as ovarian response in ART. Ala307Thr, however, was recognized as having some effect on the number of produced embryos [17].

In another study, a need for low amounts of FSH for ovarian stimulation and a heightened danger of OHSS were shown by Indian women who had AA genotypes [18].

The genotype frequencies of rs4986938 did not show any difference between women with poor response and good responders. Our results were contrary to findings obtained from some other studies [19, 20].

Another study, conducted by de Mattos et al., which included 136 infertile women below 39 years, revealed a connection between ESR1 and ESR2 polymorphisms and the results of assisted reproduction for Brazilian women [10].

Based on the genotypes of rs4986938, no differences were observed in clinical characteristics of infertile women referred to IVF in our study. However, the average number of oocytes at the MI stage and the level of AMH were higher in genotype AA than in the two other genotypes in poor responders, but this correlation was not found in women with good response to IVF treatment.

In summary, our results indicated that FSHR rs6165, not ESR2 rs4986938, was associated with poor response to ovarian stimulation in Iranian infertile women, implying that FSHR rs6165 might influence the success of IVF. FSHR rs6165 could be suggested as a predicting marker of ovarian response to stimulation in Iranian women going for IVF.

Footnotes

Conflict of interest

All authors have no conflicts of interests to declare.

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