Involvement of single nucleotide polymorphisms in ovarian poor response.

Purpose Unpredictability in acquiring an adequate number of high-quality oocytes following ovarian stimulation is one of the major complications in controlled ovarian hyperstimulation (COH). Genetic predispositions of variations could alter the immunological profiles and consequently be implicated in the variability of ovarian response to the stimulation. Design Uncovering the influence of variations in AMHR2, LHCGR, MTHFR, PGR, and SERPINE1 genes with ovarian response to gonadotrophin stimulation in COH of infertile women.

Blood samples of the women with a good ovarian response (GOR) or with a poor ovarian response (POR) were collected. Genomic DNA was extracted, and gene variations were genotyped by TaqMan SNP Genotyping Assays using primer-probe sets or real-time PCR Kit.

Except for PGR (rs10895068), allele distributions demonstrate that the majority of POR patients carried minor alleles of AMHR2 (rs2002555, G-allele), LHCGR (rs2293275, G-allele), MTHFR (rs1801131, C-allele, and rs1801133, T-allele), and SERPINE1 (rs1799889, 4G allele) genes compared to the GOR. Similarly, genotypes with a minor allele in AMHR2, LHCGR, MTHFR, and SERPINE1 genes had a higher prevalence among POR patients with the polymorphic genotypes. However, further genotype stratification indicated that the minor alleles of these genes are not associated with poor response. Multivariate logistic analysis of clinical−demographic factors and polymorphic genotypes demonstrated a correlation between FSH levels and polymorphic genotypes of SERPINE1 in poor response status.

Despite a higher prevalence of AMHR2, LHCGR, MTHFR, and SERPINE1 variations in the patients with poor ovarian response, it seems that these variations are not associated with the ovarian response.

Ovarian poor response, Genetic variations, Infertility, Assisted reproductive technology

In vitro fertilization (IVF) is a complicated multistep process with controlled ovarian stimulation (COS) to safely obtain the number of eggs acquired following hormonal stimulation [1]. IVF outcomes can be influenced by qualitative and quantitative factors in oocyte production and collection. Unpredictability and significant inter-individual variability of ovarian response to the stimulation is one of the most challenges in IVF treatment. Herein, various variables contribute to the probability of successful pregnancy in the treatment procedure [2]. Insufficient or poor ovarian response (POR) is one of the major complications in which an inadequate number of oocytes retrieved after stimulation results in cycle cancellation or IVF failure and consequently a significantly lower chance of pregnancy [3]. The prevalence of POR is estimated between 9 and 24% in IVF treatment [4], and ovarian stimulation interventions had minimum effect on POR patients [5, 6]. Although the POR is associated with maternal age, the precise causes of POR are not very well known. Characterization of the ovarian response in COS and IVF can be influenced by genetic variability, and in this context, the genetic background of the patients undergo IVF plays a role in the quality of the response to the medications [7]. The stimulatory effect of follicle-stimulating hormone (FSH) might be different due to the diversity of the human genome and therewith affecting the ovarian response in COS. Several genes implicate in the reproduction system [1], and variations involved in altering ovarian response have been so far identified by gene association studies. Besides the FSH receptor gene (FSHR) which is one of the most studied gene polymorphisms [8], lines of evidence indicate the involvement of various hormonal genes in women enrolled in COS programs, which have not so far been well investigated in reduced ovarian response to the therapy. Herein, an increased risk of developing polycystic ovary syndrome has been found in patients with single nucleotide polymorphism (SNP) rs2293275 in the luteinizing hormone/choriogonadotropin receptor (LHCGR) gene [9]. Progesterone receptor (PGR) is also associated with decreased pregnancy outcomes or primary ovarian insufficiency [10]. However, in contrast to these polymorphisms, no association has so far been reported between anti-mullerian hormone type II receptor (AMHR2) SNPs and low or high response in controlled ovarian hyperstimulation [11]. Also, variations in methylenetetrahydrofolate reductase (MTHFR) are associated with follicle-stimulating hormone levels as well as diminished ovarian response to FSH stimulation and failing cycles of IVF in COS [12–15]. A link has also been shown between estrogen agonist and levels of endothelial plasminogen activator inhibitor (SERPINE1) [16]. The polymorphisms of SERPINE1 are also associated with decreased pregnancy outcomes or primary ovarian insufficiency [17]. Given the importance of variations in the pathogenesis of reproductive failure by altering the immunological profiles, due to the heterogeneity of the studied populations, investigation of candidate genes requires further analysis. To our knowledge, the influence of genetic variability on diminished ovarian response has not been well investigated, and no independent replication of these studies has so far been reported. Additionally, no consistent data exist on a possible association between POR and these SNPs in the Iranian population. Considering the significant impact of gene polymorphisms on ovarian response to stimulation, especially those directly or indirectly are responsible for the hormonal regulation of ovarian response, investigating variations in AMHR2, MTHFR, LHCGR, PGR, and SERPINE1 genes in Iranian patients with POR and women with the good ovarian response (GOR) is the main focus of this study.

Study samples Samples were collected from the women with infertility who referred to the Erfan, Laleh, and Taleghani hospitals in Tehran between 2014 and 2016. All consideration and approval were performed by the Institutional Review Board of Shahid Beheshti University of Medical Sciences, Tehran, Iran, following the 1975 Helsinki Declaration as revised in 2000. After the injection of ovulation-inducing drugs, two groups of women were selected based on their ovarian response to the stimulation. The first group (n=72; mean age 32.6±0.39 years) was the patients with the poor ovarian response (POR), providing ≤ 5 mature eggs. Women (n=147; mean age 31.7±0.28 years) with a good ovarian response (GOR), providing 6 to 20 eggs were categorized as the second group. Participants were informed, and provided written consent was obtained. Relevant clinical information was acquired by questionnaires. Patients included in the present study were subjects with primary infertility who showed a normal or low response to a standard dose of exogenous recombinant FSH (rFSH). Patients with a normal response to the same medication served as controls. The clinical features such as complete blood counts (CBCs) or body mass index of both groups were compared, and sample homogeneity was confirmed. Exclusion criteria were infertile women with risk factors such as high blood pressure, diabetes, hyper-ovarian response, ovarian hyperstimulation syndrome (OHSS), and age of more than 39 years. Moreover, samples with the presence of coagulation and immunological disorders, chromosomal abnormalities, hormonal diseases, polycystic ovary, or endometriosis, and infertility caused by the male subject were excluded from the study. With performing uterosalpingography, anatomical problems were characterized. Levels of FSH and luteinizing hormone (LH) were identified by electrochemiluminescence, and anti-mullerian hormone (AMH) levels were analyzed by enzyme-linked immunosorbent assay (ELISA). DNA extraction and genotyping On the day of receiving the ovule, blood samples of all participants were collected and followed by DNA extraction using the Exgene Cell SV kit (GeneAll, Songpa-gu, Korea). Genotyping of AMHR2 rs2002555 (-482 A>G, non-coding promoter region), LHCGR rs2293275 (N312S, Exon 10), MTHFR rs1801131 (1298C>A, Exon 8), and rs1801133 (677C>T, Exon 5), and PGR rs10895068 (331G/A, 5′-untranslated region) genes was performed by TaqMan SNP Genotyping Assays using primer-probe sets (Applied Biosystems, Foster City, CA). Real-time PCR Kit was used for genotyping of SERPINE1 rs1799889 (−675 4G/5G, coding promoter region) (SNP Biotechnology R&D Ltd., Ankara, Turkey). PCR was carried out on a Roche LightCycler® 96 PCR Machine (Roche, Mannheim, Germany), according to the manufacturer protocol. Briefly, 10 ng of DNA, 5 μl TaqMan Universal PCR Master Mix, 0.5 μl primers and probes (20×), and DNAase-free distilled water were mixed in a final volume of 10 μl, and the appropriate negative controls were included. Statistical analysis Sample size and power analysis were performed using G*Power (version 3.1) (http://www.gpower.hhu.de/). SPSS statistical software program (version 23) was used to execute all data comparisons and statistical analysis. Distribution of genotype and allele frequencies, as well as consistency with the Hardy–Weinberg equilibrium, was acquired by chi-square (χ2) test. Several tests, χ2, analysis of variance (ANOVA), or t-test, were used to compare the clinical and demographic characterizations between patients and their respective controls. Data were shown as mean ± SD or median with interquartile range, and a P value less than 0.05 was considered statistically significant. The P-value was corrected (Pc) for multiple analyses using the Bonferroni method.

Characteristics of studied subjects Infertile patients under 40 years old were recruited into the investigation who referred to the infertility centers in Tehran and received ovarian stimulation during IVF. The power calculation and sample size of the present study were determined considering the prevalence of the variants as reported by NCBI (http://www.ncbi.nlm.nih.gov/snp). With taking a medium effect size of 0.3 into account, our sample size is adequate to reach 90% power at a significance level of 0.05. Table 1 summarizes the clinical and demographic information of the patients in the GOR and POR groups. In this study, 219 participants were included once they meet the inclusion criteria. No significant difference was noticed between patients and controls regarding age (P=0.066). While the levels of LH remained similar between GOR and POR groups, significant differences were found in the total number of oocytes (P=0.000), as well as levels of estradiol (ES) (P=0.000), FSH (P=0.001), and AMH (P=0.028). Genotype frequencies in all groups were within Hardy–Weinberg equilibrium, except genotype frequencies of PGR and SERPINE1 genes which departed from the equilibrium. Table 1. | Characteristics | Patients; Mean ± SEM | P value | | |---|---|---|---| | POR (n=72) | GOR (n=147) | || | Age (mean, years) | 32.6±3.34 | 31.7±3.43 | 0.066 | | AMH | 1.97±1.50 | 2.43±1.41 | 0.028 | | ES | 46.3±24.4 | 69.5±28.0 | 0.000 | | FSH | 7.57±2.69 | 6.42±2.13 | 0.001 | | LH | 4.91±2.09 | 4.87±2.39 | 0.912 | | Total oocytes* | 3 (2–4) | 12 (9 – 15) | 0.000 | P values were performed based on the chi-square or t-test, and a value of <0.05 was considered statistically significant. Significant P values are shown in bold. *Total oocytes are presented as median (interquartile range) POR poor ovarian response, GOR good ovarian response, AMH anti mullerian hormone, ES estradiol, FSH follicle-stimulating hormone, LH luteinizing hormone, n number Polymorphism analysis The allelic frequency and genotype distribution of six polymorphisms in five genes were analyzed in the present study which is summarized in Table 2. To uncover whether genomic alteration could affect the number of oocytes retrieved following ovarian stimulation in infertile patients, variations of AMHR2 (rs2002555), LHCGR (rs2293275), MTHFR (rs1801131 and rs1801133), PGR (rs10895068), and SERPINE1 (rs1799889) was investigated in POR patients with a low number of oocytes and their GOR group with good oocyte retrieval. Analysis of alleles demonstrates that the majority of POR patients carried minor alleles of AMHR2 (rs2002555, G-allele), LHCGR (rs2293275, G-allele), MTHFR (rs1801131, C-allele, and rs1801133, T-allele), and SERPINE1 (rs1799889, 4G allele) genes; calculated odds ratios (OR) with 95% confidence interval (CI) were 2.07 (CI: 1.26–3.40), 3.04 (CI: 1.91–4.84), 3.58 (CI: 2.18–5.88), 4.39 (CI: 2.85–6.76), and 6.39 (CI: 4.11–9.94), respectively. Logistic regression calculation confirmed a higher prevalence of these minor alleles in POR patients with diminished levels of total oocytes. In contrast, POR patients had a lower distribution of PGR (rs10895068) minor A-allele than GOR patients, but this difference was not statistically significant (P = 0.414). Table 2. | Gene | Genotype | POR (n = 72) n (%) | GOR (n = 147) n (%) | OR (95% CI) | P value | Pc | |---|---|---|---|---|---|---| | AMHR2 rs2002555 | AA | 41 (57) | 110 (74.8) | 1.93 (1.19–3.13) | 0.007 | 0.021 | | AG | 25 (34.7) | 32 (21.7) | |||| | GG | 6 (8.3) | 5 (3.4) | |||| | A | 107 (74.3) | 252 (85.7) | 2.07 (1.26–3.40) | 0.004 | 0.008 | | | G | 37 (25.7) | 42 (14.3) | |||| | LHCGR rs2293275 | AA | 31 (43) | 110 (74.8) | 2.52 (1.63–3.92) | 0.000 | 0.000 | | AG | 30 (41.7) | 28 (19.1) | |||| | GG | 11 (15.3) | 9 (6.1) | |||| | A | 92 (63.9) | 248 (84.3) | 3.04 (1.91–4.84) | 0.000 | 0.000 | | | G | 52 (36.1) | 46 (15.7) | |||| | MTHFR rs1801131 | AA | 33 (45.8) | 116 (78.9) | 3.29 (1.98–5.46) | 0.000 | 0.000 | | AC | 31 (43.1) | 27 (18.4) | |||| | CC | 8 (11.1) | 4 (2.7) | |||| | A | 97 (67.4) | 259 (88.1) | 3.58 (2.18–5.88) | 0.000 | 0.000 | | | C | 47 (32.6) | 35 (11.9) | |||| | MTHFR rs1801133 | CC | 17 (23.6) | 95 (64.6) | 3.81 (2.43–5.97) | 0.000 | 0.000 | | CT | 33 (45.8) | 43 (29.3) | |||| | TT | 22 (30.6) | 9 (6.1) | |||| | C | 67 (46.5) | 233 (79.2) | 4.39 (2.85–6.76) | 0.000 | 0.000 | | | T | 77 (53.5) | 61 (20.8) | |||| | PGR rs10895068 | GG | 26 (36.1) | 49 (33.3) | 1.12 (0.80–1.58) | 0.490 | - | | GA | 15 (20.8) | 51 (34.7) | |||| | AA | 31 (43.1) | 47 (32) | |||| | G | 67 (46.5) | 149 (50.7) | 1.18 (0.79–1.76) | 0.414 | - | | | A | 77 (53.5) | 145 (49.3) | |||| | SERPINE1 rs1799889 | 5G5G | 17 (23.6) | 105 (71.4) | 3.65 (2.46–5.39) | 0.000 | 0.000 | | 4G5G | 22 (30.6) | 26 (17.7) | |||| | 4G4G | 33 (45.8) | 16 (10.9) | |||| | 5G | 56 (38.9) | 236 (80.3) | 6.39 (4.11–9.94) | 0.000 | 0.000 | | | 4G | 88 (61.1) | 52 (19.7) | P values were performed based on the chi-square or t-test, and a value of <0.05 was considered statistically significant. Significant P values are shown in bold POR poor ovarian response, GOR good ovarian response, n number Having investigated genotype and allele distributions between GOR and POR groups, patients were further stratified to wild- and polymorphic genotypes to compare the prevalence of these genotypes (Table 3). Taking together all genotypes with minor-allele in AMHR2 (rs2002555, AG + GG), LHCGR (rs2293275, AG + GG), MTHFR (rs1801131, AC + CC, and rs1801133, CT + TT), and SERPINE1 (rs1799889, 4G5G + 4G4G) genes, a higher OR regarding the prevalence of genotypes with the minor allele were observed among POR patients with the polymorphic genotypes when compared to the GOR patients; calculated OR (CI) were 2.24 (1.23–4.08), 3.93 (2.16–7.14), 4.42 (2.40–8.13), 5.91 (3.11–11.2), and 8.08 (4.21–15.5), respectively. As far as the polymorphic genotypes in PGR variation were analyzed, a similar prevalence of genotypes with minor-allele PGR (rs10895068, 4G5G + 4G4G) was observed in the POR population and GOR group. Table 3. | Gene | Allele | POR (n = 72) n (%) | GOR (n = 147) n (%) | OR (95% CI) | P value | Pc | |---|---|---|---|---|---|---| | AMHR2 rs2002555 | AG + GG | 31 (43) | 37 (25.1) | 2.24 (1.23–4.08) | 0.008 | 0.024 | | LHCGR rs2293275 | AG + GG | 41 (56.9) | 37 (25.1) | 3.93 (2.16–7.14) | 0.000 | 0.000 | | MTHFR rs1801131 | AC + CC | 39 (54.2) | 31 (21.1) | 4.42 (2.40–8.13) | 0.000 | 0.000 | | MTHFR rs1801133 | CT + TT | 55 (76.4) | 52 (35.4) | 5.91 (3.11–11.2) | 0.000 | 0.000 | | PGR rs10895068 | GA+ AA | 46 (63.9) | 98 (66.7) | 0.88 (0.49–1.59) | 0.684 | - | | SERPINE1 rs1799889 | 4G5G + 4G4G | 55 (76.4) | 42 (28.6) | 8.08 (4.21–15.5) | 0.000 | 0.000 | P values were performed based on the chi-square or t-test, and a value of <0.05 was considered statistically significant. Significant P values are shown in bold POR poor ovarian response, GOR good ovarian response, n number Association of clinical−demographic characteristics and genotype of the patients with poor ovarian response Given the significantly higher prevalence of minor alleles in POR patients, clinical−demographical characteristics were taken into analysis with genotypes to unveil any relationships with regard to the wild- and polymorphic genotypes (Table 4). POR patients with polymorphic genotypes (4G5G + 4G4G) in SERPINE1 rs1799889 were significantly younger than those with wild genotype (5G5G) (P = 0.02). Although a lower FSH level or a higher oocyte number was observed in POR women with minor allele-C in MTHFR rs1801131 and allele-G in AMHR2 rs2002555, these differences did not reach the levels of significance (P = 0.05). Regarding oocyte number, minor alleles of the variations did not affect the ovarian response, neither in POR nor GOR patients. Similarly, no further correlation was found between other genotypes and clinical−demographical characteristics in Iranian patients with regard to the response to treatment in IVF. Table 4. | Gene/variation | Genotypes | Age | AMH | Estradiol | FSH | LH | Total oocytes* | |||||| |---|---|---|---|---|---|---|---|---|---|---|---|---|---| | POR | GOR | POR | GOR | POR | GOR | POR | GOR | POR | GOR | POR | GOR | || | AMHR2 rs2002555 | AA | 32.8±3.4 | 31.6±3.4 | 2.05±1.6 | 2.4±1.4 | 48.4±27.1 | 69.8±30.7 | 7.03±2.5 | 6.3±2.0 | 4.95±2.2 | 4.7±2.3 | 3 (2-4) | 12 (9.7-15) | | AG+GG | 32.4±3.3 | 32.1±3.4 | 1.86±1.2 | 2.3±1.1 | 43.4±20.4 | 68.7±18.1 | 7.87±2.9 | 6.6±2.4 | 4.87±1.8 | 5.2±2.6 | 4 (3-4) | 12 (8-16) | | | P value | 0.58 | 0.38 | 0.60 | 0.72 | 0.38 | 0.84 | 0.40 | 0.53 | 0.87 | 0.26 | 0.05 | 0.84 | | | LHCGR rs2293275 | AA | 31.9±3.1 | 31.7±3.5 | 1.78±1.2 | 2.3±1.3 | 49.5±29.1 | 70.0±28.1 | 8.26±2.0 | 6.3±2.1 | 4.72±1.9 | 4.9±2.3 | 4 (2-4) | 12 (9-16) | | AG+GG | 33.2±3.4 | 31.7±3.0 | 2.11±1.7 | 2.6±1.6 | 43.8±20.3 | 68.0±28.0 | 7.04±3.0 | 6.6±2.0 | 5.06±2.2 | 4.7±2.5 | 3 (2-4) | 11 (9-14) | | | P value | 0.11 | 0.99 | 0.35 | 0.29 | 0.33 | 0.70 | 0.06 | 0.52 | 0.49 | 0.79 | 0.71 | 0.07 | | | MTHFR rs1801131 | AA | 32.6±3.1 | 31.7±3.5 | 1.86±1.2 | 2.4±1.3 | 45.8±23.1 | 72.0±28.5 | 8.25±2.3 | 6.2±2.1 | 5.22±2.3 | 4.8±2.2 | 3 (2.5-4) | 12 (10-15) | | AC+CC | 32.7±3.5 | 31.7±2.9 | 2.06±1.6 | 2.4±1.6 | 46.7±25.8 | 60.4±24.0 | 6.98±2.8 | 7.0±2.0 | 4.65±1.8 | 5.1±2.7 | 3 (2-4) | 10 (8-15) | | | P value | 0.88 | 0.98 | 0.58 | 0.79 | 0.88 | 0.04 | 0.05 | 0.06 | 0.24 | 0.49 | 0.51 | 0.16 | | | MTHFR rs1801133 | CC | 31.4±3.5 | 31.8±3.4 | 2.51±2.0 | 2.5±1.6 | 51.4±25.4 | 67.9±25.9 | 7.91±1.9 | 6.5±2.0 | 4.80±2.4 | 5.1±2.5 | 3 (2-4) | 12 (10-16) | | CT+TT | 33.0±3.2 | 31.6±3.4 | 1.80±1.2 | 2.1±0.91 | 44.7±24.1 | 72.5±31.5 | 7.46±2.8 | 6.1±2.2 | 4.95±2.0 | 4.4±2.0 | 4 (2-4) | 12 (8-14) | | | P value | 0.08 | 0.85 | 0.11 | 0.07 | 0.32 | 0.34 | 0.54 | 0.24 | 0.79 | 0.12 | 0.29 | 0.09 | | | PGR rs10895068 | GG | 32.1±3.5 | 31.3±3.0 | 2.02±1.1 | 2.3±1.5 | 39.9±19.9 | 67.4±27.9 | 8.09±2.6 | 6.4±2.3 | 4.48±1.8 | 4.8±2.2 | 3 (2-4) | 12 (10-15) | | GA+AA | 32.9±3.2 | 31.4±3.5 | 1.94±1.6 | 2.4±1.3 | 49.9±26.1 | 70.6±28.1 | 7.27±2.6 | 6.4±2.0 | 5.16±2.1 | 4.8±2.4 | 3.5 (2-4) | 12 (9-15.25) | | | P value | 0.29 | 0.11 | 0.82 | 0.70 | 0.10 | 0.52 | 0.21 | 0.97 | 0.18 | 0.90 | 0.66 | 0.21 | | | SERPINE1 rs1799889 | 5G5G | 34.2±2.1 | 31.8±3.3 | 1.58±1.1 | 2.3±1.1 | 37.3±18.5 | 70.6±28.9 | 7.29±2.9 | 6.5±2.2 | 5.11±2.0 | 4.6±2.2 | 3 (2-4) | 12 (9-15) | | 5G4G+4G4G | 32.1±3.5 | 31.5±3.5 | 2.09±1.5 | 2.7±1.8 | 49.0±25.5 | 66.7±25.7 | 7.65±2.6 | 6.2±1.7 | 4.85±2.1 | 5.4±2.6 | 3 (2-4) | 12 (10-15) | | | P value | 0.02 | 0.59 | 0.23 | 0.14 | 0.09 | 0.43 | 0.62 | 0.42 | 0.64 | 0.06 | 0.72 | 0.72 | P values were performed based on the chi-square or t-test, and a value of <0.05 was considered statistically significant. Significant P values are shown in bold. *Total oocytes are presented as median (interquartile range) AMH anti mullerian hormone, ES estradiol, FSH follicle-stimulating hormone, LH luteinizing hormone Besides, to uncover any potentially confounding impact of multiple clinical−demographic factors (age, AMH, FSH, and LH) and polymorphic genotypes of AMHR2 (rs2002555), LHCGR (rs2293275), MTHFR (rs1801131 and rs1801133), PGR (rs10895068), and SERPINE1 (rs1799889) variations between patients and controls, an analysis based on multivariate logistic regression was performed (Table 5). When compared between patients and controls, our results demonstrated that poor response status could be correlated with ES, FSH, and LH levels as well as polymorphic genotypes of MTHFR (rs1801131 and rs1801133) and SERPINE1. However, only FSH and SERPINE1 polymorphic genotypes remain significant after correction for multiple analyses. Therefore, FSH levels might increase the risk of aberrant ovarian response when the polymorphic genotype of SERPINE1 is present. Table 5. | Factor | OR | 95% CI | P value | Pc | |---|---|---|---|---| | Age | 0.93 | 0.77–1.13 | 0.47 | - | | AMH | 1.15 | 0.85–1.56 | 0.34 | - | | ES | 1.03 | 1.00–1.05 | 0.14 | | | FSH | 0.63 | 0.47–0.83 | 0.001 | 0.01 | | LH | 2.66 | 1.78–3.99 | 0.000 | - | | AMHR2 rs2002555, AA vs. AG+ GG | 1.42 | 0.42–4.80 | 0.57 | - | | LHCGR rs2293275, AA vs. AG+ GG | 3.18 | 0.86–11.6 | 0.08 | - | | MTHFR rs1801131, AA vs. AC+ CC | 4.79 | 1.42–16.1 | 0.01 | 0.11 | | MTHFR rs1801133, CC vs. CT+ TT | 5.50 | 1.65–18.3 | 0.005 | 0.05 | | PGR rs10895068, GG vs. GA+ AA | 2.67 | 0.67–10.6 | 0.16 | - | | SERPINE1 rs1799889, 5G5G vs. 5G4G + 4G4G | 22.0 | 5.83–83.0 | 0.000 | 0.000 | OR odds ratio, CI confidence interval. OR and 95%CI were obtained from multivariate logistic regression model after adjusted for age, hormonal statement, and gene variations. AMH anti mullerian hormone, ES estradiol, FSH follicle-stimulating hormone, LH luteinizing hormone

The present study was designed to scrutinize whether gene variations of AMHR2, LHCGR, MTHFR, PGR, and SERPINE1 could influence the number of oocytes retrieved following ovarian stimulation in infertile patients. Comparison of POR patients with a low number of oocytes and their GOR group with good oocyte retrieval indicated that genotypes and alleles of AMHR2, LHCGR, MTHFR, and SERPINE1 variations were found to be more prevalent in the patients with a low number of retrieval oocytes. In contrast, alleles of PGR SNP were found to be distributed similarly between GOR and POR groups. However, despite a higher prevalence of these variations in the poor responders, it seems that these variations are not associated with the ovarian response. Considering few reports investigating the effects of these gene variations on COS, no independent replication of these studies has so far been reported. To the best of our knowledge, no data exist on a possible association between the ovarian response in terms of oocyte number and these SNPs in the Iranian population. AMH, which exerts its biological effects through the receptor AMHR2, plays an important role in ovarian folliculogenesis [18]. AMH is expressed in granulosa cells, contributing to the FSH sensitivity following stimulation in the ovary and thereby could be used as a biomarker of ovarian response to stimulation in clinical practice [19]. In vivo investigations in mice have also demonstrated diminished effects of FSH on follicle development as well as higher sensitivity to FSH stimulation [18]. Considering the significant impact of the AMH-AMHR2 expression pattern on sensitivity to FSH in the ovary and follicle development, respective gene polymorphisms are eligible to influence ovarian response. It has previously been shown that the AMHR2 rs2002555 variation, located on chromosome 12q13, is associated with altering the hormonal levels and elevated follicular sensitivity to FSH [20]. Our data in the present study indicated that POR patients carried more AMHR2 minor G-allele, but there is no association between this variation and a lower number of oocytes following FSH administration. In line with our data, other studies failed to find an association between the AMHRII rs2002555 gene polymorphism and ovarian reserve, response, or outcomes in ovarian stimulation [11, 21]. In contrast, Lazaros et al. reported an influence of AMHR2 genotypes on the ovarian response to standard gonadotropin stimulation [22]. The LHCGR, a member of the G protein-coupled receptor family, mediates the actions of LH which is essential for ovulation and therefore indispensable for fertility [23]. The importance of LHCGR in fertility has indeed been confirmed in animal models such as LHCGR knockout mice, indicating a complete loss of functional receptors resulting in infertility [24, 25]. The LHCGR gene is located on chromosome region 2p21, and several variations have so far been identified in this gene. Since LHCGR is involved in ovulation, genetic variation in this gene may have a role in reproductive function. There is a variation in the coding region of the LHCGR gene (rs2293275) which could affect receptor activity and sensitivity [26]. Our observations explored that POR women had a higher frequency of GG genotype. These findings could be in agreement with previous data demonstrating a higher sensitivity of women with A/A genotype or A-allele of LHCGR rs2293275 to the FSH stimulation [26–28]. In a recent study, however, we addressed a significant association between rs2293275 G-allele and OHSS, indicating the implication of this variation as a risk factor for developing OHSS [29]. Considering these findings, it may be noteworthy that this variation may render a dual effect on altering the sensitivity to ovarian stimulation, but more studies are needed to confirm these data. Involvement of folate deficiency and hyperhomocysteinemia in reproduction and an association between MTHFR polymorphisms and pregnancy complications and birth malformations has so far been reported [30, 31]. The MTHFR gene, located on chromosome 1p36.3, could influence the folate metabolism [32], and therefore the variations in MTHFR are associated with the circulating concentrations of folate and homocysteine. In the present study, women with few numbers of oocytes following ovarian stimulation (POR) had a higher distribution of minor C-allele in rs1801131 and T-allele in rs1801133 polymorphisms. In agreement with our findings, several observations found a higher prevalence of the MTHFR rs1801131 alleles in patients with diminished response to ovarian stimulation [12, 33]. These data, however, was not replicated in another study in COS outcomes [1]. In human reproduction, the PGR, a nuclear receptor transcription factor, is essential for ovarian follicle development and consequently female fertility due to controlling oocyte release from the ovaries [34]. Among several polymorphisms in the PGR gene, located on chromosome 11q22, that have been described, PGR rs10895068 is one of the most common studied variations. However, the influence of this polymorphism on oocyte release following ovarian stimulation has poorly been investigated. While this variation had no impact on COH outcome in IVF-treated women, its association has been reported with decreased pregnancy outcomes [10]. An association between PGR rs10895068 variation and elevated risk of implantation failure or increases transcription of the PGR gene has also been suggested [35, 36]. Based on the distribution of genotypes or alleles in our study, this polymorphism is not implicated in the COS outcome of women undergoing an IVF procedure in terms of releasing oocytes. Recently, we could show a protective effect of A-allele at the locus of PGR rs10895068 variation in developing OHSS [29]. Hence, although this polymorphism seems to be associated with preventing ovarian from overreacting to FSH stimulation, it does not influence diminished ovarian response. Collectively and in line with other studies, this variation in the PGR gene is more susceptible to IVF pregnancy outcome, rather than COH outcome [7]. Coagulation and fibrinolysis are mandatory processes in the implantation, and alteration in this balance could lead to IVF failure [37]. SERPINE1, which is expressed in the placenta and maternal plasma of pregnant women, is an essential fibrinolytic factor, contributed to thrombotic complications [38, 39]. Several studies revealed a decrease in fibrinolysis markers such as SERPINE1 in the women undergoing COS (reviewed in [40]). On the other side, higher expression levels of SERPINE1 has also been reported which is associated with fibrinolysis changes during the ovarian stimulation cycle as well as the risk of worse pregnancy outcome and consequently infertility [39, 41]. SERPINE1 gene is located at chromosome 7q22, and a common variation in the promoter region of this gene (rs1799889) is well characterized and functionally important [42]. It has been shown that SERPINE1 gene variation is associated with infertility by altering immunological and hormonal profiles [43]. Our findings explored a higher prevalence of polymorphic genotypes of SERPINE1 rs1799889 genes in the patients with POR compared to the GOR. These data indicate that the 4G allele might not influence the ovarian response to be more resistant to the stimulation. Association investigations between SERPINE1 rs1799889 variation and ovarian response to stimulation are highly limited. Although complications in pregnancies, recurrent miscarriage, and infertility have been linked with genotypes of SERPINE1 rs1799889 [44–46], Boudjenah et al. did not find any impact of SERPINE1 polymorphism on the IVF outcome [1]. The data presented in this study should be interpreted with limitations. This study is based on cohort data with a relatively limited sample size. Nevertheless, finding an association between most of the studied variations and ovarian response to the stimulation might be helpful for further investigations, possibly with larger sample size. Another limitation could be explained by the lack of analysis of the possible influence of studied polymorphisms on gene or protein expressions. Since gene variations could potentially affect the levels of gene expression or protein secretion, these levels have not been detected in our investigation. In conclusion, the findings of this study indicate that women with variations in AMHR2, LHCGR, MTHFR, and SERPINE1 genes appear to be more frequent in the poor responders to ovarian stimulation. However, despite the higher frequency, these variations are not associated with ovarian response. Nevertheless, further investigations with a larger sample size are mandatory to confirm these data before considering these effects in treatment strategies.

We would like to thank the medical and nursing staff of the Erfan, Laleh, and Taleghani hospitals in Tehran, Iran, for their helpful collaboration. Declarations Conflict of interest The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Sayyed Mohammad Hossein Ghaderian and Reza Akbarzadeh contributed equally to this work.

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