Features of the profile of serum autoantibodies to reproductive hormones and steroidogenic enzymes in patients with endometriosis-related infertility and infertile patients without endometriosis

Female infertility is a serious issue in modern reproductive medicine, with a significant increase in the number of patients diagnosed annually. In 2023, more than 65,000 women in Russia received this diagnosis for the first time, bringing the total number of women with infertility to over 250,000 [1]. Assisted reproductive technologies (ART) are widely used to support the reproductive functions of these patients. According to the ART Registry Report from the Russian Association of Human Reproduction, the total number of ART cycles performed in Russia in 2022 surpassed 170,000, averaging approximately 1,200 ART cycles per 1 million population [2], which was slightly higher than the 1,100 cycles per 1 million population recorded in 2019 [3].

Epidemiological and clinical studies have shown a consistent association between infertility and endometriosis. Available data indicate that the risk of infertility in patients with endometriosis is 2-4 times higher than that in the general population [4]. Conversely, the likelihood of having endometriosis among patients with infertility is approximately 50%. Endometriosis-associated infertility has been identified in over half of women who have undergone laparoscopic intervention for infertility [5].

Endometriosis is a multifactorial disease that directly and indirectly affects fertility, frequently affecting the ovaries and impairing their function [6]. Recent systematic reviews suggest that patients with endometriomas have a significantly reduced number of antral follicles [7] as well as lower levels of anti-Müllerian hormone (AMH), which indicates ovarian reserve, compared to healthy women and patients with non-endometrioid benign ovarian cysts [8]. Several studies have also indicated that endometriosis can negatively affect the quality of oocytes, embryos, and outcomes in in vitro fertilization (IVF) cycles [6].

Despite the recognized clinical link between endometriosis and infertility, the exact mechanisms underlying infertility development are not fully understood. It is important to note that endometriosis is associated with complex disorders of the immune system, characterized by changes in systemic and local immunity as well as functional disorders of effector and antigen-presenting cells [9]. Endometriosis is often accompanied by polyclonal activation of B lymphocytes, increased production of autoantibodies and cytokines, cell-mediated abnormalities, concomitant autoimmune diseases, and effects of immunomodulators [10, 11]. In this context, endometriosis has recently been considered an autoimmune disease.

A systematic analysis of studies investigating the impact of five types of autoantibodies, namely antithyroid, antiphospholipid, antinuclear, antibodies affecting the reproductive system, and antibodies associated with celiac disease, showed that most autoantibodies significantly affect the frequency of miscarriages, whereas their impact on clinical pregnancy and live birth rates varies depending on the type of autoantibody [12].

Anti-ovarian antibodies (AOA), including antibodies targeting multiple ovarian proteins and histological structures, have been found in 30% of patients with premature ovarian insufficiency (POI) and 26% of patients undergoing IVF [13]. According to the authors, testing for AOA before entering an IVF cycle is important for predicting reproductive outcomes and prescribing immunosuppressive therapy. Such therapy can enhance the effectiveness of the IVF program and reduce both physician labor costs and patients’ material expenses.

Studies investigating autoimmune mechanisms in the development of endometriosis, POI, and failures in assisted reproductive technology (ART) programs are ongoing. Recent research has demonstrated the diagnostic and pathogenetic significance of autoantibodies to endometrial antigens as well as steroid and gonadotropic hormones in endometriosis [14]. In cases of POI, a high diagnostic value has been revealed for autoantibodies targeting key steroidogenic enzymes of cytochrome P450 – specifically aromatase (CYP19A1), the enzyme that cleaves the side chain of cholesterol (CYP11A1), and 21-hydroxylase (CYP21A2) – which catalyze certain stages of steroid hormone biosynthesis in the ovaries, adrenal cortex, and other tissues [15].

Of particular interest is the study of the profile of autoantibodies to critical hormones of the reproductive system, including those produced in the ovaries. Anti-Müllerian hormone (AMH), produced by granulosa cells of preantral and antral follicles, has high prognostic significance in assessing ovarian reserve, affects the hypothalamic-pituitary-gonadal axis, and acts as a neuroendocrine regulator of fertility [16, 17].

In light of the above, the aim of this study was to conduct a comparative analysis of the profile of autoantibodies to hormones of the reproductive system and steroidogenic enzymes in patients with endometriosis-associated infertility and those without endometriosis.

Materials and methods

The study included 88 patients with infertility who were referred to the V.I. Kulakov NMRC for OG&P of the Ministry of Health of Russia for reproductive function implementation using ART programs. Group 1 consisted of patients with an established diagnosis of extragenital endometriosis (n=44) and group 2 included patients without endometriosis (n=44). The diagnosis of endometriosis was based on ultrasound and magnetic resonance imaging data and was confirmed by laparoscopic surgery and histological examination in 27/44 (61.4%) patients.

The inclusion criteria were reproductive age (18-45 years), signed informed consent to participate in the study, and treatment with ART. The exclusion criteria included refusal to participate; contraindications to ART; donor programs; surrogacy programs; morbid obesity (body mass index ≥40.0 kg/m²); HIV infection; and severe somatic, autoimmune, or oncological diseases.

All patients were examined in accordance with the current clinical guidelines "Female Infertility" and the Order of the Ministry of Health of Russia dated July 31, 2020, N 803n, concerning the procedure for using assisted reproductive technologies as well as contraindications and restrictions on their use. Blood samples for serum autoantibody determination were collected from women in the follicular phase on the 2nd to 5th day of the menstrual cycle, before entering the ART program. Immunological investigations included the determination of serum autoantibodies to steroid hormones (estradiol and progesterone), follicle-stimulating hormone (FSH), AMH, and steroidogenic enzymes (CYP19A1, CYP11A1, CYP21A2). These were assessed using modifications of the indirect solid-phase enzyme-linked immunosorbent assay (ELISA) developed in the laboratory of clinical immunology at V.I. Kulakov NMRC for OG&P of the Ministry of Health of Russia, and described previously [14, 15].

For ELISA, we used microplates with a high adsorption capacity for polystyrene, estradiol-BSA conjugates (Sigma-Aldrich, USA), and progesterone-BSA (OOO HEMA, Russia), along with purified FSH from the human pituitary gland (Sigma-Aldrich, USA) and recombinant human cytochrome P450 enzymes (CYP19A1, CYP11A1, CYP21A2) from Cloud-Clone Corp. (USA), recombinant human AMH (OOO Hitest, Russia), and mouse monoclonal antibodies against human IgM and IgG, labeled with horseradish peroxidase. Additionally, buffer, substrate-chromogenic solutions, and stop reagent for ELISA were obtained from OOO HEMA, Russia. Before the study, blood serum was diluted 1:100. The optical density (OD) was measured at a wavelength of 450 nm using an Infinite F50 microplate photometer (TECAN, Austria). An ELISA result was considered positive if the mean OD of the test sample exceeded the mean OD of the negative controls by more than two standard deviations (2δ).

Statistical analysis

Statistical analysis was performed using Microsoft Office Excel 2016 (Microsoft Corp., USA) and MedCalc v. 12 (MedCalc Software Ltd., Belgium). The distribution of continuous variables was tested for normality using the Kolmogorov–Smirnov test and Shapiro–Wilk W-test. Non-normally distributed continuous variables were presented as median (Me) and range of minimum to maximum (min; max) or interquartile range (Q25; Q75). Data between groups were compared using the non-parametric Mann–Whitney U test. Qualitative data are presented as counts (n) and percentages (%), with the chi-squared test (χ²) used to assess differences between groups. The relationships between variables were evaluated by calculating Spearman’s correlation coefficient (r). Differences were considered statistically significant at p<0.05.

Results

Patients with infertility were divided into two groups: group 1 included 44 patients with an established diagnosis of extragenital endometriosis (EGE), while group 2 consisted of 44 patients without endometriosis. The groups were comparable in terms of age, body mass index, age at menarche, and menstrual cycle duration. There were no significant differences in the number of pregnancies, medical abortions, spontaneous miscarriages, frequency of fibroids, endometrial polyps, chronic endometritis, sexually transmitted infections (STIs), number of myomectomies, frequency of primary and secondary infertility, duration of infertility, number of IVF attempts, somatic and endocrinological morbidities, or hormone levels (Table 1). Both groups had a high frequency of uterine fibroids (>20%), endometrial polyps (>20%), thyroid diseases (>20%), and history of IVF programs (38.6% in each group).

Group 1 consisted of patients with stage 2–3 EGE, including ovarian (24/44, 54.5%) and pelvic peritoneal (35/44, 79.5%) endometriosis. Additionally, 27/44 (61.4%) patients had previously undergone surgery for EGE, including 23/44 (52.3%) who had undergone ovarian resection. Moreover, group 1 had longer menstruation, significantly more frequent adenomyosis (29.5%) and gastrointestinal (GI) diseases (15.9%), and a higher incidence of ovarian resection (52.3%). In contrast, group 2 had a higher incidence of ectopic pregnancy (22.7%) and tubectomy (34.1%) than those with a high incidence of STIs (22.7%). Notably, a combination of tubectomy and secondary infertility was observed in the of 13/44 (29.5%) cases. All women in both groups had a regular menstrual cycle, with the exception of one woman with oligomenorrhea in group 2.

When analyzing hormonal status, the majority of patients in group 1 (36/44 (81.8%)) and group 2 (42/44 (95.5%)) had AMH levels corresponding to normal ovarian reserve. A decrease in AMH levels < 1.2 ng/ml was detected in 8/44 (18.2%) patients in group 1 and in 2/44 (4.5%) patients in group 2, significantly more often in group 1 than in group 2 (p=0.04). The FSH levels were within the normal range in all patients. Using the developed IFA modifications, a wide range of autoimmune antibodies was detected in infertile patients, including antibodies to steroid hormones, steroidogenic enzymes, FSH, and AMH (Fig. 1). Autoantibodies of classes M and G were detected in both groups 1 and 2 with high frequency: antibodies to AMH in 13/44 (29.5%) and 19/44 (43.2%) patients, antibodies to estradiol in 15/44 (34.1%) and 9/44 (20.5%) patients, antibodies to progesterone in 14/44 (31.8%) and 8/44 (18.2%) patients, antibodies to FSH in 15/44 (34.1%) and 16/44 (36.4%) patients, and antibodies to steroidogenic enzymes in 14/44 (31.8%) and 21/44 (47.7%) patients. The frequency of antibody detection did not differ significantly between the two groups. However, in patients in group 2, antibodies to steroidogenic enzymes and AMH were detected significantly more often than antibodies to estradiol (20.5%; p=0.02) and progesterone (18.2%; p=0.01).

Antibodies (M, G) to AMH were detected in 13/44 (29.5%) patients with endometriosis and in 19/44 (43.2%) patients without endometriosis (p=0.18). Among the seropositive patients in group 1, 10/44 (76.9%) had primary infertility and 3/44 (23.1%) had secondary infertility (p=0.01). In group 2, 9/44 (47.4%) patients had primary infertility and 10/44 (52.6%) had secondary infertility (p=0.75). In most seropositive patients, infertility was observed for a long time (more than 2 years): in group 1, 11/13 (84.6%) and in group 2, 17/19 (89.5%). In group 1, endometrioid cysts were diagnosed in 6/13 (46.2%) seropositive patients, adenomyosis in 6/13 (46.2%), and ovarian resection was performed in 6/13 (46.2%). In group 2, ectopic pregnancies were noted in 7/19 (36.8%) patients, and tubectomy was performed in 8/19 (42.1%) patients. Unsuccessful IVF attempts occurred in group 1 in 4/13 (30.8%) seropositive patients in group 2 in 7/19 (36.8%) patients (p=0.55).

A comparison of the detection rates of classes M and G serum autoantibodies in patients in the study groups is presented in Table 2. In group 1, high frequencies of IgG antibodies were found against estradiol (29.5% of patients), progesterone (29.5%), aromatase (CYP19A1) (27.3%), and FSH (20.5%). IgG antibodies to estradiol were detected more frequently in group 1 than in group 2 (11.4%; p=0.04). In group 2, the most frequently detected IgG antibodies were against FSH (34.1%) and AMH (34.1%), with IgG antibodies to AMH being more prevalent in group 2 than in group 1 (13.6%; p=0.03). Combinations of antibodies to several steroidogenic enzymes were found in 9/44 (20.5%) patients in group 1 and in 11/44 (25%) patients in group 2 (p=0.79).

Of the 10 women with reduced AMH levels, 9 had IgG antibodies to FSH, AMH, steroid hormones, and steroidogenic enzymes: to FSH in 4, to AMH in 4, to CYP19A1 in 3, to CYP21A2 in 2, to PG in 3, and to estradiol in 1. Table 3 presents the median levels of serum autoantibodies of classes M and G to steroidogenic enzymes and hormones in the study groups of patients with infertility. The comparison showed that group 1 had higher levels of IgG antibodies to estradiol and IgM antibodies to progesterone than group 2, whereas group 2 exhibited higher levels of IgG antibodies to AMH and IgM antibodies to the CYP11A1 enzyme, and a tendency toward an increase in IgM antibodies to the CYP21A2 enzyme compared to group 1. In group 2, among the 15 patients who were seropositive for IgG antibodies to AMH, 10 (66.7%) had secondary infertility and 9 (60%) had undergone tubectomy in the past.

Figure 2 shows the individual profiles of serum antibodies in patients with and without endometriosis. In five patients with endometriosis, elevated levels of IgM and IgG antibodies to FSH, estradiol, progesterone, and CYP19A1 were frequently observed, and in some cases, IgM antibodies to AMH were also present. In contrast, patients without endometriosis frequently exhibited elevated levels of antibodies to AMH, FSH, and CYP19A1.

A strong or moderate direct correlation was found between the levels of serum autoantibodies to steroidogenic enzymes and hormones in patients of the studied groups (Fig. 3). In group 1, patients with endometriosis showed a strong direct correlation (r>0.7) between the levels of IgM antibodies to estradiol and CYP11A1, as well as between IgM antibodies to AMH and IgM antibodies to estradiol, CYP11A1, and CYP21A2. Additionally, there was a direct correlation between IgM antibodies to progesterone and FSH, and between IgG antibodies to CYP21A2 and CYP19A1. In group 2, a direct correlation (r>0.7) was found between the levels of IgM antibodies to estradiol and CYP11A1, IgM antibodies to progesterone and FSH, and IgM and IgG antibodies to CYP19A1 and CYP11A1. IgM antibodies to estradiol and AMH positively correlated with antibodies to steroidogenic enzymes (r from 0.52 to 0.67).

Discussion

This study was a comparative analysis of serum autoantibody profiles in patients with both primary and secondary infertility with and without endometriosis. The diagnosis of endometriosis was based on ultrasound and magnetic resonance imaging data and confirmed by laparoscopic surgery in 61.4% of cases. Additionally, more than half of the patients in group 1 had endometrioid ovarian cysts and had previously undergone ovarian resection. Among the patients in group 2, the most common infertility factor was tuboperitoneal, with tubectomy due to ectopic pregnancy or pelvic inflammatory disease performed in 34.1% of the women, and 15.9% of these patients had a history of ovarian resection.

The study groups were comparable in terms of basic demographic and clinical-anamnestic data, including the number of patients with primary and secondary infertility, duration of infertility, frequency and number of IVF programs performed, and endocrinological morbidities. Notably, most women in both groups had regular menstrual cycles. However, in group 1, patients with endometriosis presented with a longer duration of menstruation, and diagnoses of adenomyosis and gastrointestinal diseases were more frequent. Furthermore, cases of ovarian resection were more prevalent than those in group 2. In contrast, group 2 had a higher incidence of ectopic pregnancy and tubectomy, with most tubectomy cases observed in patients with secondary infertility and a significant prevalence of STIs. Despite these differences, the average FSH, estradiol, and AMH levels did not differ between the groups. However, a decrease in AMH levels below the normal range, indicating reduced ovarian reserve, was detected more frequently in group 1 than in group 2.

A study of the serum autoantibody profile revealed a wide range of autoantibodies to steroid hormones, FSH, AMH, and steroidogenic enzymes in infertile patients, highlighting differences in the autoantibody profile between patients with and without endometriosis. In patients with endometriosis, a high frequency of total antibodies (IgM and IgG) with different specificities was found, targeting both steroid hormones and FSH, AMH, and steroidogenic enzymes; however, the frequency of their detection did not differ significantly. In contrast, in patients without endometriosis, total antibodies to AMH, steroidogenic enzymes, and FSH were commonly detected, with antibodies to AMH and steroidogenic enzymes being significantly more prevalent than antibodies to estradiol and progesterone. Notably, this study marks the first detection of IgM and IgG autoantibodies to AMH in patients with infertility. Antibodies to AMH were found in both patients with endometriosis and those without endometriosis, regardless of whether they had long-term (>2 years) primary or secondary infertility.

Among the seropositive patients in group 1, those with primary infertility predominated; nearly half had a history of endometrioid cysts, adenomyosis, and past ovarian resections. Conversely, in group 2, more than half of the seropositive patients experienced secondary infertility, with a history of ectopic pregnancies and tubectomies. Over 30% of the patients in both groups had previously unsuccessful IVF attempts. In group 1, IgG antibodies to estradiol, progesterone, CYP19A1, and FSH were detected at a high frequency (greater than 20%), whereas in group 2, IgG antibodies to FSH, AMH, and CYP19A1 were prevalent. Furthermore, IgG antibodies to estradiol and IgM antibodies to FSH were found more frequently in patients with endometriosis, whereas IgG antibodies to AMH were more common in patients without endometriosis.

Group 1 demonstrated higher median levels of IgG antibodies to estradiol and IgM antibodies to progesterone, whereas group 2 exhibited elevated levels of IgG antibodies to AMH and IgM antibodies to CYP11A1 and CYP21A2. Distinct individual serum antibody profiles were observed in patients with infertility. In the individual profiles of endometriosis patients, there was an increase in IgM and IgG antibody levels targeting FSH, estradiol, progesterone, and CYP19A1. Some patients also exhibit elevated levels of IgM antibodies to AMH. Conversely, patients without endometriosis often show increased antibody levels of AMH, FSH, and CYP19A1, with less frequent increases observed for progesterone and estradiol.

These findings align with results from a previous study demonstrating a diverse range of autoantibodies in patients with endometriosis, including antibodies to steroids and gonadotropic hormones, which vary across different forms of endometriosis. Higher levels of autoantibodies to steroid hormones were associated with endometrioid ovarian cysts [18]. Antibodies to estradiol and progesterone were found to be significantly more frequent in endometriomas than in deep endometriosis or in women without endometriosis. It has been hypothesized that these antibodies may play a role in the pathophysiology of endometriosis, particularly by contributing to the development of endometrial resistance to progesterone and serving as diagnostic markers for the condition [14].

In addition, the present study demonstrated a high frequency of detection of antibodies to key steroidogenic enzymes (CYP11A1, CYP19A1, and CYP21A2) in patients with endometriosis. These enzymes catalyze reactions in the steroid hormone biosynthesis pathway in the ovaries, placenta, adipose tissue, and adrenal glands, with a particular emphasis on antibodies to aromatase, which facilitates the formation of estrone and estradiol in the ovaries and adipose tissue. The increased production of autoantibodies to steroid hormones, FSH, and steroidogenic enzymes is driven by excess estrogen biosynthesis in the ovaries, ectopic endometrioid foci, and peripheral adipose tissue, as well as by increased progesterone synthesis in endometriosis foci [19].

A strong direct correlation was observed in group 1 between the levels of IgM antibodies to estradiol, AMH, and the steroidogenic enzymes CYP11A1 and CYP21A2, between IgM antibodies to progesterone and FSH, and between IgG antibodies to CYP21A2 and CYP19A1. In group 2, a direct correlation was identified between the levels of IgM antibodies to estradiol and CYP11A1, IgM antibodies to progesterone and FSH, and M and G antibodies to CYP19A1 and CYP11A1. Furthermore, IgM antibodies to estradiol and AMH positively correlated with antibodies to steroidogenic enzymes.

The observed direct correlation between serum antibodies and steroid hormones, steroidogenic enzymes, FSH, and AMH may be attributed to the polyclonal activation of B lymphocytes, which is characteristic of endometriosis, along with the functional interactions between hormones and enzymes. FSH stimulates folliculogenesis and secretion of steroid hormones, gonadal peptides, and growth factors in the ovaries. In particular, FSH has a positive stimulatory effect on the expression of AMH [20] and serves as the main inducer of aromatase activity in granulosa cells [21].

It is important to note that in group 2, among patients with infertility who did not have endometriosis, the leading cause of infertility was tubal-peritoneal factor, antibodies to FSH and AMH predominated, unlike in endometriosis, where antibodies to steroid hormones were more common. Additionally, antibodies to steroidogenic enzymes were also detected. According to a recent study, autoantibodies to steroidogenic enzymes (CYP11A1, CYP19A1, CYP21A2) are risk factors for POI and exhibit high diagnostic value for this pathology [15]. Furthermore, the likelihood of developing POI in seropositive patients was significantly higher, by 26.8, 14.7, and 13.6, respectively, than that in seronegative patients. Levels of these autoantibodies correlate with menstrual cycle disorders and a reduction in the number of antral follicles in the ovaries. Recent studies have shown that AMH can regulate gonadotropic cells of the anterior pituitary gland and various types of hypothalamic cells through receptors located outside the gonads. This regulation effectively controls the secretion of gonadotropin-releasing hormone by hypothalamic neurons, thereby promoting gonadotropin secretion. This suggests that AMH may function as a neuroendocrine regulator of fertility [16]. Moreover, recent research in a mouse model has demonstrated that AMH participates in the periodic remodeling of gonadotropin-releasing hormone endings of neurons, tanycytes, and neurohemal junctions at the level of the median eminence of the hypothalamus, a process that is impaired in polycystic ovary syndrome [22]. It is presumed that autoantibodies can disrupt the biological function of this hormone, contributing to impaired fertility.

Interestingly, increased production of autoantibodies to hormones and steroidogenic enzymes was observed in the majority of patients with a regular menstrual cycle and normal ovarian reserve (81.8% in group 1 and 95.5% in group 2) at the time of the study. However, since autoantibodies can appear long before the onset of an autoimmune disease, it can be hypothesized that over time, as they reach a high level, these antibodies may disrupt steroidogenesis and the menstrual cycle, decrease ovarian reserve, and ultimately lead to the development of an autoimmune form of premature ovarian insufficiency. This hypothesis is supported by the presence of high levels of antibodies to hormones and steroidogenic enzymes in patients with reduced ovarian reserve in this study, as well as the previously shown high probability of developing premature ovarian insufficiency in seropositive women [15].

Conclusion

Patients with infertility have a diverse profile of serum autoantibodies, including antibodies to steroid hormones, FSH, AMH, and steroidogenic enzymes. In patients with endometriosis-associated infertility, IgG antibodies to estradiol, progesterone, and CYP19A1 predominate. In contrast, IgG antibodies to FSH and AMH predominate in patients without endometriosis. Patients with reduced ovarian reserve also have antibodies to hormones and steroidogenic enzymes, which are risk factors for developing the autoimmune form of POI. Evaluation of the autoantibody profile prior to initiation of ART will allow optimization of the ART protocol through a personalized approach to therapy prescription and patient management. Further prospective studies are needed to evaluate the impact of autoantibodies on embryonic and clinical outcomes in ART programs and to identify effective immunosuppressive and immunotherapies.