Advances in assisted reproductive technologies (ART) have significantly improved treatment outcomes for various forms of infertility. However, despite the standardization of approaches, the high variability in individual patient response to controlled ovarian stimulation (COS) remains one of the most acute and unresolved challenges in clinical reproductive medicine [1, 2]. Existing standard protocols, based predominantly on clinical-laboratory and ultrasound parameters, do not always allow for highly accurate prediction and optimization of in vitro fertilization (IVF) outcomes.

Accumulating fundamental and clinical data indicate that the genetic component is a key predictor of interindividual differences in response to gonadotropin therapy [2-4]. Genetic factors account for 20 to 40% of the variance in individual ovarian reactivity to exogenous regulatory signals, which directly affects folliculogenesis processes and the overall effectiveness of IVF protocols.

COS is a fundamental component of ART, aimed at inducing multifollicular growth and obtaining a therapeutic pool of mature oocytes. Most modern COS protocols are based on exogenous administration of recombinant or urinary follicle-stimulating hormone (FSH) followed by trigger administration of human chorionic gonadotropin (hCG) to initiate final oocyte maturation. The integration of pharmacogenetic principles into the management of infertile women opens fundamentally new opportunities for treatment personalization [1-3, 5]. Thus, the use of individualized FSH dosages calculated based on the patient's genetic profile demonstrates superiority over empirical regimens, significantly increasing cumulative pregnancy and live birth rates [6].

According to systematic review data, up to one-third of interindividual differences in ART effectiveness are attributable to polymorphisms of genes encoding gonadotropin and sex steroid receptors [5]. Single nucleotide polymorphisms (SNPs) can modify the expression level, affinity, and signaling cascade of target receptors, as well as hormonal metabolic pathways, thereby predetermining tissue sensitivity to pharmacotherapy [2]. In particular, polymorphisms of gonadotropin receptor genes (FSHR, LHCGR) modulate granulosa cell response to gonadotropin administration. On the other hand, genetic variability of sex steroid receptors (ESR1, ESR2, PGR) critically affects endometrial receptivity and the follicular microenvironment in response to fluctuations in estrogen and progesterone levels, determining the success of embryo implantation and pregnancy maintenance.

Despite the obvious pathogenetic significance, the results of existing studies on isolated polymorphisms (e.g., the most extensively studied Asn680Ser variant of the FSHR gene or the PvuII polymorphism of the ESR1 gene) remain highly contradictory and heterogeneous, ranging from complete absence of effect to diametrically opposite clinical associations [6-9]. Such dissociation of results dictates the need for comprehensive assessment of gene-genotypic profiles in clinically homogeneous patient groups.

To conduct a comprehensive assessment of the possible association of polymorphic variants of gonadotropin receptor genes (FSHR: rs6165, rs6166, rs1394205; LHCGR: rs2293275, rs4073366, rs4953616, rs7579411) and sex steroid hormone receptor genes (ESR1: rs2234693, rs3020394, rs3798577, rs9340799; ESR2: rs4986938; PGR: rs1042838) with clinical pregnancy rates in IVF programs among women with tubal-peritoneal infertility, to identify potentially significant predictors for subsequent personalization of COS protocols.

This prospective pilot study, approved by the Local Ethics Committee of the National Research Mordovia State University (Protocol No. 116 dated 12.05.2023), included 270 patients with confirmed tubal-peritoneal infertility (ICD-10: N97.1) who were treated at the ART Department of the Perinatal Center of the Mordovian Republican Clinical Hospital during the period May 2023 – January 2025. All participants provided written informed consent. Age range was 25-35 years (M±SD: 31.4±3.7 years), with Caucasian ethnicity.

All patients underwent a standardized IVF program using their own oocytes. Ovarian stimulation was performed using a short protocol with gonadotropin-releasing hormone antagonists (ganirelix or cetrorelix 0.25 mg/day subcutaneously) and recombinant FSH (follitropin alfa at a starting dose of 150-225 IU/day subcutaneously with subsequent dose adjustment); choriogonadotropin alfa 250 μg subcutaneously served as the trigger for final oocyte maturation. Patient management was conducted in accordance with current National Clinical Guidelines for the treatment of female infertility and the use of ART.

The final sample included 96 patients selected according to pre-established criteria. Inclusion criteria: age 25-35 years; documented tubal-peritoneal infertility (ICD-10 code: N97.1); normal ovarian reserve; absence of endometrial pathology according to ultrasound and hysteroscopy; normal karyotype in both partners; transfer of good-quality embryos. Exclusion criteria: male factor infertility; BMI >30 kg/m²; extragenital or genital pathology constituting an absolute contraindication to IVF; refusal to participate at any stage of the study.

The primary endpoint was the onset of clinical pregnancy, verified echographically on day 21 after embryo transfer. Based on IVF cycle outcome, two groups were formed: Group 1 (n=48) — patients with pregnancy on the first attempt; Group 2 (n=48) — patients without pregnancy and with three or more unsuccessful IVF cycles in their history. The groups were comparable in terms of age, body mass index, anti-Müllerian hormone levels, and antral follicle count.

Venous blood (6 mL) was collected on an empty stomach into EDTA vacuum tubes and stored at -20 °C (short-term) and -70 °C (long-term). Genomic DNA was extracted using magnetic sorbent reagents (Genotek, Russia) on an Allsheng Auto-Pure 96 automated platform (China).

Genotyping of 13 SNPs, selected based on a systematic literature review as markers with predictive significance for IVF outcomes, was performed using genome-wide typing on Infinium Global Screening Array-24 v3.0 microarrays (Illumina, USA) with an iScan scanner according to the Infinium HTS Assay protocol [10]: FSHR — rs6166, rs6165, rs1394205; LHCGR — rs2293275, rs4073366, rs4953616, rs7579411; ESR1 — rs2234693, rs3020394, rs3798577, rs9340799; ESR2 — rs4986938; PGR — rs1042838. All stages of genetic analysis, including DNA extraction, genotyping, and biomaterial storage, were conducted exclusively within the territory of the Russian Federation. No cross-border transfer of genetic data occurred, which fully complies with the requirements of Federal Law No. 86-FZ.

Statistical analysis was performed using Statistica 12.5 (StatSoft). The conformity of quantitative variables to the normal distribution was tested using the Shapiro-Wilk test; data are presented as M±SD. Student's t-test was used for intergroup comparisons of quantitative traits. Genotype and allele frequencies were compared using Pearson's χ² test; Fisher's exact test was applied when the expected number of observations in a cell was less than 5. Conformity of observed genotype frequencies to Hardy-Weinberg equilibrium (HWE) was assessed in the comparison group using the χ² method. The strength of associations between polymorphic variants and IVF outcomes was expressed as odds ratios (OR) with 95% confidence intervals (95% CI). The threshold for statistical significance was set at p<0.05.

Given the limited sample size (n=96), additional verification of identified associations was performed using permutation testing (Monte Carlo procedure, 10,000 phenotype label permutations with fixed genotype structure). A result was considered statistically significant at an empirical significance level of p-perm <0.05.

The results of the comparative analysis of genotype frequency distribution for 13 SNPs in gonadotropin receptor genes (FSHR, LHCGR) and sex steroid receptor genes (ESR1, ESR2, PGR) between patients with favorable IVF outcomes (Group 1) and women with unsuccessful outcomes (Group 2) are presented in Table 1. The genotype frequency distribution of the studied polymorphisms conformed to Hardy-Weinberg equilibrium (p>0.05). Statistically significant intergroup differences were verified for two loci of the ESR1 gene: rs2234693 and rs3020394.

Analysis of rs2234693 genotype distribution revealed a negative prognostic value of the heterozygous C/T variant. The frequency of this genotype in the unfavorable outcome group was 26 cases versus 16 in the successful implantation group, confirming a significant association with IVF program ineffectiveness (p=0.024; OR=0.593; 95% CI 0.170-0.890).

The rs3020394 locus demonstrated high predictive significance. The minor homozygous G/G genotype was completely absent in the pregnancy group but was recorded in 8 patients in Group 2, indicating its potential negative role (Table 1). Conversely, the A/A genotype was significantly more frequent in Group 1, and carriers of this genotype had a 2.5-fold higher chance of favorable IVF outcomes compared to carriers of other genotypes (p=0.025).

For other ESR1 polymorphisms — rs3798577 and rs9340799 — no statistically reliable association with clinical outcomes was found (p>0.05).

None of the tested polymorphic variants of gonadotropin receptors FSHR and LHCGR showed a statistically significant association with clinical pregnancy rates (p>0.05). The genotype frequency distribution of the rs6165 and rs6166 variants of the FSHR gene was virtually identical in women with successful and unsuccessful IVF attempts (Table 1). The promoter polymorphism rs1394205 demonstrated a weak trend toward accumulation of the C/C genotype in Group 2 (27 cases vs. 19), but the differences did not reach statistical significance (p=0.102).

The frequencies of the studied LHCGR loci (rs2293275, rs4073366, rs4953616) showed no significant intergroup deviations. At the rs7579411 locus, the C/C genotype was significantly more frequent in patients with pregnancy, but permutation testing did not confirm the significance of the association with IVF outcome (Table 1).

Investigation of genetic variability in ESR2 and PGR isoforms did not reveal prognostic markers among the analyzed sites. It should be noted that the minor homozygous A/A variant, recorded in 2 patients in Group 1 and absent in the implantation failure group (p=0.247), cannot serve as an isolated criterion for ART cycle effectiveness.

GenePolymorphismGenotypesGroup 1 (n=48)Group 2 (n=48)χ²OR95% CIpFSHRrs6165C/C43—1.3640.288-6.4480.695²  C/T26260——1.0  T/T8190.0440.9160.402-2.0840.834 rs6166C/C43—1.3030.275-6.1690.738²  C/T26240.0371.0830.482-2.4370.847  T/T18190.1420.8530.372-1.9520.706 rs1394205C/C19272.6710.5100.226-1.1480.102  C/T27192.6711.9620.871-4.4220.102  T/T220——1.0LHCGRrs2293275C/C13150.2020.8170.338-1.9740.653  C/T23230——1.0  T/T12100.2361.2670.487-3.2920.627 rs4073366C/C31340.4290.7110.318-1.7720.513  C/G13120.8161.1140.448-2.7740.054  G/G42—2.0910.364-11.9960.399² rs4953616C/C240.7110.4780.083-2.7740.054²  C/T13170.7760.6770.284-1.6150.378  T/T33271.61.7110.742-3.9450.206 rs7579411C/C19104.0022.4901.007-6.1570.046  C/T24302.0640.5520.245-1.2450.151  T/T680.3340.7140.228-2.2420.564ESR1rs2234693C/C18103.2272.280.918-5.6600.072  C/T16275.0970.5930.170-0.8900.024³  T/T14110.4871.2730.554-3.4640.485 rs3020394A/A28175.0612.5531.120-5.8200.025³  A/G20230.3790.7760.347-1.7390.539  G/G08————¹ rs3798577C/C44—0.9050.211-3.8710.893²  C/T26210.3751.30.561-3.0110.540  T/T16170.3040.7840.330-1.8620.582 rs9340799A/A19170.1781.1950.522-2.7330.673  G/A860.3341.40.446-4.3930.563  G/G21250.6680.7160.320-1.5980.414ESR2rs4986938C/C17242.0860.5480.242-1.2430.149  C/T27202.0431.80.802-4.0420.153  T/T440——0.153PGRrs1042838C/C8121.0110.60.22-1.6330.315  C/A38321.8991.90.758-4.7650.168  A/A20————¹

Notes:¹ — not calculated due to absence of cases in one group;² — p-value corresponds to Fisher's exact test;³ — p-value corresponds to p-perm <0.05.

This study identified a statistically significant association between the heterozygous C/T genotype of the rs2234693 (PvuII) polymorphism of the ESR1 gene and unfavorable IVF outcomes (p=0.024; OR=0.593; 95% CI 0.170-0.890), indicating an increased risk of implantation failure and ART cycle ineffectiveness in this patient category.

The molecular-genetic mechanism of this phenomenon is closely related to the localization of the polymorphism and its functional effect on gene transcriptional activity. The rs2234693 single nucleotide substitution is located in the first intron of the ESR1 gene. According to the literature, this region is involved in alternative splicing processes and modulates the final mRNA level of the estrogen receptor alpha (ERα) [11, 12]. The substitution of cytosine with thymine (T allele) disrupts the key regulatory interaction with the transcription factor B-myb, blocking the enhancer effect, which leads to a significant reduction in ESR1 gene expression and a deficiency of functional ERα receptors [13]. It can be hypothesized that in heterozygous carriers (C/T genotype), due to the presence of the minor T allele, the stoichiometric ratio and density of ERα receptors on the endometrial cell surface are disrupted. Even in the context of adequate or high (under COS conditions) levels of exogenous and endogenous estrogens, endometrial tissue is unable to develop an adequate biological response due to receptor deficiency. Thus, a phenotype of relative "estrogen resistance" is formed.

Furthermore, adequate ERα expression during the proliferative phase of the menstrual cycle is critically important for complete stromal and epithelial proliferation of the uterus, induction of progesterone receptors, and subsequent secretory transformation [14]. Reduced functional activity of ERα in C/T genotype carriers leads to disruption of synchronization of endometrial maturation processes. This shifts or blocks the formation of the so-called "implantation window," making the endometrium functionally immature and non-receptive at the time of high-quality embryo transfer, which accounts for the high rate of failures.

In addition to the uterine factor, ERα is expressed in ovarian granulosa cells [15]. Suboptimal receptor expression levels in heterozygotes may disrupt local estrogen-dependent signaling during COS, potentially affecting oocyte quality despite their visually normal morphological maturation.

An important result of the analysis was the identification of a protective association of the A/A genotype (p=0.025; OR=2.553; 95% CI 1.120-5.820) and the negative prognostic value of the homozygous G/G genotype of the rs3020394 polymorphism of the ESR1 gene. In the specialized reproductive medicine literature, this marker is mentioned less frequently than the classical PvuII (rs2234693) or XbaI polymorphisms. Nevertheless, its role is considered in the context of altering gene regulatory activity and systemic estrogen effects [16]. The SNP at the rs3020394 locus may exert regulatory influence on ESR1 mRNA stability or alter the affinity of promoter regions to key nuclear transcription factors due to its localization in modified histone sites in enhancer regions and DNase-hypersensitive sites.

Thus, the most likely explanation is that rs3020394, possessing regulatory potential, may alter ERα expression or function. This, in turn, modifies estrogen-dependent processes critically important for endometrial receptivity and successful trophoblast invasion. Under COS conditions, when plasma estradiol levels exceed physiological values many times over, adequate tissue response depends not on hormone concentration but on receptor density and reactivity. In A/A genotype carriers, sufficient ERα density promotes endometrial secretory transformation under progesterone action during the luteal phase, leading to synchronization of molecular-cellular processes including pinopode maturation, integrin and cytokine expression, forming the "implantation window" and making the endometrium functionally receptive to a quality embryo.

The fact that the G/G genotype of the rs3020394 polymorphism of the ESR1 gene occurred exclusively in the group with repeated IVF failures (8 cases vs. 0) supports the hypothesis that this polymorphism may serve as an important "hidden" marker of endometrial receptivity defects, which exacerbates infertility risks in the presence of tubal-peritoneal factor. Pharmacogenetic determination of this locus before entering an ART protocol allows identification of a very high-risk group for recurrent implantation failure and requires the development of individualized pathogenetic endometrial preparation regimens.

It is worth noting that similar associations have previously been described for other ESR1 gene polymorphisms. For example, in studies on the Korean population, combinations of ESR1 genotypes with other polymorphisms were associated with a reduced risk of recurrent implantation failure [7]. This confirms that ESR1 genetic variability in general is an important factor influencing IVF program success.

The study established that polymorphisms of gonadotropin receptor genes (FSHR: rs6165, rs6166, rs1394205; LHCGR: rs2293275, rs4073366, rs4953616, rs7579411), as well as steroid hormone receptor genes (ESR2: rs4986938 and PGR: rs1042838), did not demonstrate a statistically significant association with clinical pregnancy rates in patients with tubal-peritoneal infertility (p>0.05).

Thus, isolated polymorphisms of the FSHR, LHCGR, ESR2, and PGR genes cannot be used as independent prognostic markers of ART clinical effectiveness in patients with tubal-peritoneal infertility. Their isolated influence on reproductive outcomes is likely successfully counterbalanced by pharmacological ovarian effects and personalization of starting dosages of exogenous gonadotropins during controlled ovarian stimulation, as well as standard hormonal support of the luteal phase. This underscores the unique predictive significance of the rs2234693 and rs3020394 loci of the ESR1 gene, polymorphisms of which, in contrast, are associated with persistent tissue resistance not overcome by standard therapeutic hormone dosages.

Two methodological limitations must be considered when interpreting the results. Multiple comparisons (13 SNPs) in a limited sample size potentially increase the probability of false-positive findings; this risk was controlled via permutation analysis (10,000 permutations), and only associations confirmed by the p-perm criterion are included in the present work. Furthermore, the targeted selection of patients with tubal-peritoneal infertility, preserved ovarian reserve, and Caucasian ethnicity limits the generalizability of the conclusions to other nosological forms of female infertility, different population groups, and patients with diminished ovarian reserve.

This prospective pilot study demonstrated the high scientific and practical significance of integrating pharmacogenetic methods into the management algorithms for patients with tubal-peritoneal infertility in ART programs. Comprehensive analysis of 13 SNPs allowed the identification of potential genetic predictors of IVF outcome. It was established that the A/A genotype of rs3020394 of the ESR1 gene is associated with clinical pregnancy achievement, while the G/G genotype of rs3020394 and the C/T genotype of rs2234693 of the ESR1 gene are associated with unfavorable outcomes and likely play an important role in the pathogenesis of recurrent implantation failure.

Identification of unfavorable allelic variants of the ESR1 gene allows prediction of the risk of recurrent implantation failure even before the patient enters an ART protocol in somatically healthy women with normal ovarian reserve. For patients carrying the C/T rs2234693 and G/G rs3020394 genotypes of the ESR1 gene, it is advisable to recommend extended pre-gravid preparation aimed at overcoming tissue estrogen resistance, as well as consideration of embryo transfer in modified cryoprotocols or against the background of individually selected hormone replacement therapy regimens.