CYP2A6 polymorphisms as predictors of IVF efficacy: the role of local estrogen metabolism under hyperestrogenic conditions

Introduction

Despite the impressive progress of assisted reproductive technologies (ART) over recent decades, the effectiveness of in vitro fertilization (IVF) programs remains limited. According to the European Society of Human Reproduction and Embryology, the rate of clinical pregnancy per single embryo transfer attempt does not exceed 35% in leading reproductive centers [1]. The search for prognostic biomarkers that allow personalization of endometrial preparation protocols and hormonal support of the luteal phase remains a priority direction of fundamental and clinical research in this field.

In recent years, increasing attention of researchers has been drawn to the cytochrome P450 (CYP450) system — a superfamily of phase I metabolism enzymes of xenobiotics and endogenous compounds. Traditionally, these enzymes were considered primarily in the context of hepatic drug metabolism; however, convincing evidence has accumulated regarding their extrahepatic expression and participation in the regulation of steroid homeostasis. In particular, the CYP2A6 enzyme is of particular interest in the context of reproductive physiology. Immunohistochemical studies have demonstrated that CYP2A6 is expressed in tissues sensitive to sex steroids, including the ovaries, uterus, mammary gland, testes, and adrenal glands [2]. Moreover, a functional estrogen-responsive element (ERE) has been identified in the promoter of the CYP2A6 gene, and direct binding of the ERα receptor to this element has been confirmed [2]. The level of CYP2A6 protein in glandular endometrial cells is significantly higher in the proliferative phase of the menstrual cycle, which correlates with local estrogen secretion and indicates the biological role of this enzyme in estrogen-sensitive tissues [2]. Induction of CYP2A6 is observed during pregnancy, suggesting a specific role of hormonal regulation of CYP2A6 in the gestational period [3, 4]. It is known that constitutive CYP2A6 activity is higher in women than in men, and the use of oral estrogen-containing contraceptives or hormone replacement therapy further increases enzymatic activity [5, 6]. At the same time, differences in CYP2A6 activity between men and women in the postmenopausal period are absent, which confirms the determining role of estrogens in regulating the expression of this gene [5]. Progesterone can also modulate CYP2A6 expression, although to a lesser extent than estrogen [3]. Thus, CYP2A6 is a hormone-sensitive gene whose expression is regulated by key steroids involved in preparing the endometrium for implantation and maintaining pregnancy.

The CYP2A6 gene is localized on the long arm of chromosome 19 at locus 13.2 as part of a gene cluster approximately 350 kb in length, which also includes the CYP2A7, CYP2A13, CYP2B, and CYP2F subfamilies [7, 8]. The CYP2A6 gene is characterized by an exceptionally high level of genetic polymorphism: to date, more than 40 allelic variants have been characterized [9]. The heritability of CYP2A6-mediated metabolism is estimated at 60–80% [9]. Using next-generation sequencing methods, seven high-frequency non-coding single nucleotide polymorphisms (SNPs) have been identified that significantly contribute to the phenotypic variability of enzyme activity, including rs57837628, rs56113850, and rs8192733 [10].

Most studies on the role of CYP2A6 in reproductive medicine have focused on its involvement in the metabolism of letrozole — an aromatase inhibitor widely used for ovulation induction in patients with polycystic ovary syndrome (PCOS) [11]. However, the endogenous role of CYP2A6 in reproductive physiology remains virtually unexplored in the context of IVF outcomes, especially in patients with tubal-peritoneal infertility, where the main treatment method is IVF rather than pharmacological ovulation induction. Given the above, it can be assumed that genetic variants of CYP2A6 affect endometrial receptivity, implantation efficiency, and IVF program outcomes.

Objective

To evaluate the association of CYP2A6 gene polymorphisms with the outcomes of IVF programs in women with tubal-peritoneal infertility.

Materials and methods

Design and participants. The work was carried out as a prospective pilot study approved by the Local Ethics Committee of Ogarev Mordovia State University (Protocol No. 116 dated 12.05.2023). During the period from May 2023 to January 2025, screening of 270 patients with a verified diagnosis of tubal-peritoneal infertility (ICD-10 code: N97.1) was conducted at the ART department of the perinatal center of the Mordovian Republican Clinical Hospital. All participants signed voluntary informed consent to participate in the study and to process personal data. The age range was 25–35 years (31.4 ± 3.7 years); all women belonged to the Caucasian population.

IVF protocol. Patients underwent standardized IVF cycles using their own oocytes. Controlled ovarian stimulation (COS) was performed according to a short protocol with gonadotropin-releasing hormone antagonists (ganirelix or cetrorelix at a daily dose of 0.25 mg subcutaneously) in combination with recombinant follicle-stimulating hormone (follitropin alfa) at an initial dose of 100–150 IU/day with subsequent individual adjustment. Final oocyte maturation was initiated by subcutaneous administration of 250 μg of choriogonadotropin alfa. All therapeutic measures complied with current National Clinical Guidelines for the diagnosis and treatment of female infertility using ART.

Sample formation and selection criteria. From the total number of examined patients, 96 patients were selected for final analysis in accordance with predetermined criteria. Inclusion in the study required: preserved ovarian reserve (assessed by anti-Müllerian hormone level and antral follicle count); absence of organic endometrial pathology according to transvaginal ultrasound and hysteroscopy; normal karyotype in both spouses; transfer of good-quality embryos. Exclusion criteria included: male factor infertility; presence of extragenital or genital pathology that is an absolute contraindication to IVF; smoking; refusal to continue participation at any stage.

The primary endpoint of effectiveness was the onset of clinical pregnancy, confirmed by ultrasound visualization of the fetal sac and fetal heartbeat at 21 days after transfer. Based on the results of the IVF cycle, two comparable groups were formed: the first group (n=48) included women with pregnancy after the first attempt, and the second group (n=48) included patients who did not achieve pregnancy, with three or more unsuccessful IVF cycles in their history. The groups did not differ statistically significantly in age, anti-Müllerian hormone level, or number of antral follicles.

Genotyping. Venous blood in a volume of 6 ml was collected into vacuum tubes with EDTA anticoagulant. Samples were stored at -20°C (short-term storage) and at -70°C (long-term storage). Genomic DNA extraction was performed using magnetic sorbent kits manufactured by Genotek (Russia) on an Allsheng Auto-Pure 96 automatic station (China). Genotyping was performed by genome-wide scanning on Infinium Global Screening Array-24 v3.0 biochips (Illumina, USA) using an iScan instrument in strict accordance with the Infinium HTS Assay Guide regulations [12] at the Genotek laboratory. The following SNPs were studied: rs1809810, rs7259706, rs7260629, rs8192733, rs12459249, rs28399433, rs56113850, rs57837628, rs113288603. All procedures related to DNA isolation, genotyping, and storage of genetic material were carried out exclusively in the Russian Federation without data transfer outside the country, which fully complies with the norms of Federal Law No. 86-FZ "On State Regulation in the Field of Genetic Engineering Activities".

Statistical analysis. Data analysis was performed using Statistica 12.5 (StatSoft, Inc.). For quantitative variables, the normality of distribution was checked using the Shapiro-Wilk test; all such characteristics are presented as arithmetic mean and standard deviation (M±SD). Intergroup differences for quantitative indicators were assessed using Student's t-test. To compare genotype and allele frequencies, Pearson's χ² test was used; when the expected frequency in a cell was less than 5, Fisher's exact test was used. The correspondence of genotype distribution to Hardy-Weinberg equilibrium was checked in the comparison group using the χ² test. The strength of association between polymorphic variants and IVF effectiveness was assessed through odds ratio (OR) with 95% confidence interval (CI). The critical significance level was set at 0.05. Given the limited sample size (n=96), to confirm the reliability of the identified associations, a permutation analysis was performed using the Monte-Carlo procedure (10,000 random permutations of phenotypic labels while preserving the genotypic structure). The result was considered statistically significant at an empirical value of p-perm <0.05.

Results

Statistically significant differences between the groups were found for two polymorphisms: rs8192733 and rs57837628 (p <0.05) (Table 1). For the remaining five polymorphisms, no statistically significant differences were found between the groups (p >0.05 in all cases) (Table 1). Rs1809810 should be considered as requiring confirmation in expanded samples.

Table 1. Distribution of CYP2A6 gene genotypes in women with tubal-peritoneal infertility

Notes: ¹ – OR and 95% CI were not calculated due to the absence of the T/A genotype in the pregnancy group. ² – p corresponds to Fisher's exact test. ³ – p corresponds to p-remt <0.05 (Monte-Carlo permutation test, 10,000 permutations).

Study limitations

This study has a number of limitations that should be considered when interpreting the results. First, the relatively small sample size (n=96) limits statistical power. To minimize the risk of false-positive findings, permutation analysis (10,000 permutations) was used. The stability of the associations of polymorphic variants rs8192733 and rs57837628 under this procedure, as well as the high level of odds ratio (OR >2.3), suggest that the identified associations are not artifacts of the small sample and can be considered reliable, despite the pilot status of the study. Second, the absence of direct measurements of CYP2A6 activity in endometrial tissues or follicular fluid does not allow establishing a direct link between genotype and enzymatic activity in target tissues. Third, the study was conducted on patients of a single ethnic group (Caucasian population), which limits the extrapolation of results to other populations, given the known interethnic differences in the distribution of CYP2A6 alleles.

Discussion

In this study, a comprehensive analysis of the associations of polymorphic variants of the CYP2A6 gene with IVF program outcomes in patients with tubal-peritoneal infertility was conducted for the first time. Statistically significant associations were established for two polymorphic loci — rs8192733 and rs57837628. The obtained results allow us to formulate the hypothesis that genetically determined variability in CYP2A6 activity modulates local hormonal homeostasis in the endometrium and ovaries, thereby affecting endometrial receptivity and implantation success. A key element of the proposed hypothesis is the proven estrogen-dependent regulation of CYP2A6 expression [2, 3]. Genetic polymorphisms that alter enzyme activity may likely affect the metabolism of endogenous steroids in target tissues, thereby creating a different hormonal background for embryo implantation, especially under conditions of hyperestrogenism.

In IVF programs, controlled ovarian stimulation naturally leads to the development of hyperestrogenism — estradiol levels on the day of trigger administration for final oocyte maturation often exceed 2000–3000 pg/mL, which is several times higher than physiological peak concentrations. It is known that supraphysiological levels of estradiol induced by COS create a suboptimal preimplantation microenvironment and reduce endometrial receptivity [12]. At the molecular level, high doses of estradiol significantly affect the expression of key genes involved in the formation of endometrial receptivity, altering not only the timing of the "implantation window" but also its molecular composition, which potentially worsens implantation success [13].

The rs8192733 polymorphism is located in the 3'-untranslated region (3'-UTR) of the CYP2A6 gene and belongs to regulatory variants that affect its expression. In the study by Tanner et al., rs8192733 was identified as one of seven high-frequency non-coding SNPs that significantly contribute to the phenotypic variability of CYP2A6 [10]. Additionally, rs8192733 is associated with increased nicotine metabolism rates [14], confirming its functional significance.

According to Tanner et al., the C/C and C/C genotypes of rs8192733 are associated with an increased rate of CYP2A6 substrate metabolism [10]. In our study, the C/C genotype is associated with pregnancy onset (OR = 6.922; p <0.001), while the G/G genotype is associated with unfavorable IVF outcomes (OR = 0.053; p <0.001). Under conditions of hyperestrogenism induced by COS, it can be assumed that increased CYP2A6 activity (C/C genotype) contributes to more effective local inactivation of excess estrogens in the endometrium, preventing the negative effect of supraphysiological estradiol concentrations on receptivity and the "implantation window" [13]. In contrast, reduced enzyme activity (G/G genotype) may lead to slowed estrogen metabolism, their excessive accumulation in endometrial tissue, and impaired implantation.

The rs57837628 polymorphism also belongs to non-coding regulatory variants, identified as an important participant in CYP2A6 phenotypes and associated with variability in enzyme activity [10].

In our study, an association of the rs57837628 G/G genotype with a 2.4-fold chance of clinical pregnancy onset was identified (OR = 2.369; p = 0.010), while the A/G genotype demonstrated an unfavorable effect (OR = 0.231; p <0.001).

Interpretation of these results requires consideration of the complex regulatory architecture of the CYP2A6 locus. Since rs57837628 is a non-coding regulatory variant, its effect is likely mediated by changes in gene expression levels rather than protein structure. The homozygous G/G genotype may provide a high level of CYP2A6 expression sufficient to maintain physiological steroid metabolism under hyperestrogenic conditions. The heterozygous A/G genotype appears to be associated with lower expression, which may contribute to delayed estrogen clearance, creating unfavorable conditions for implantation. However, the functional effect of rs57837628 has not been definitively established in the literature, and the proposed interpretation requires experimental confirmation.

Unlike rs8192733 and rs57837628, the association of rs1809810 with IVF outcome was not confirmed by permutation test due to the absence of heterozygotes in group 1; this locus requires study in expanded samples.

The obtained results complement existing knowledge, demonstrating that genetic variability of CYP2A6 may have clinical significance not only in the context of xenobiotic metabolism but also in the regulation of reproductive processes. In contrast to the work of Kareem et al. [11], where no effect of CYP2A6 polymorphism on letrozole efficacy in PCOS was found, our study is the first to show an association of regulatory CYP2A6 SNPs with IVF outcomes in patients without PCOS. This emphasizes that the role of CYP2A6 may differ depending on the therapy context and type of infertility.

Conclusion

Thus, the results of this study indicate that polymorphic variants of the CYP2A6 gene — rs8192733 and rs57837628 — are associated with IVF program outcomes in patients with tubal-peritoneal infertility. Within the framework of the proposed hypothesis about the modulation of local hormonal homeostasis through genetically determined variability in CYP2A6 activity, it can be concluded that excessively low activity (G/G rs8192733 and, probably, A/G rs57837628) under hyperestrogenism conditions may lead to slowed estrogen metabolism, their excessive accumulation, and disruption of the "implantation window." High CYP2A6 activity (C/C rs8192733 and G/G rs57837628) promotes accelerated local inactivation of excess estrogens in the endometrium, preventing the negative effect of supraphysiological estradiol concentrations on receptivity and maintaining optimal conditions for implantation.

The obtained results require further experimental and clinical verification; however, already now the obtained data open new prospects for personalized prediction of ART effectiveness and the development of individual approaches to hormonal support of IVF cycles.