Hyperhomocysteinemia and pregnancy outcomes in women with polycystic ovary syndrome: A case-control study

Abstract Background One of the reproductive medicine challenges is to determine the role of hyperhomocysteinemia in the pathogenesis of polycystic ovary syndrome (PCOS), especially in women with recurrent pregnancy loss (RPL). Objective Determine the correlation between hyperhomocysteinemia and pregnancy outcome in women with PCOS. Materials and Methods This case-control study involved 245 women (20-30 yr) and was conducted in Georgia, Tbilisi from 2019-2022. Of these, 175 were women with PCOS (study group) and 70 were healthy women (control group). Women with PCOS were divided into group I with RPL (n = 90), and group II with live births (n = 85). Group I was divided into subgroups A and B with and without insulin resistance. The investigation measured homocysteine (Hcy), follicle-stimulating, luteinizing, anti-Mullerian hormones, total and free testosterone were determined. To determine the ovarian volume and antral follicle count, participants also underwent an ultrasound examination. Results In women with PCOS, the average Hcy level was significantly higher than in the controls, p < 0.05. In group I, the average Hcy level was significantly higher than in group II and controls, p < 0.05. There was no significant difference in average Hcy level between group II and controls. The average Hcy level in group I, subgroup A was significantly higher than in subgroup B, p < 0.05. The average total, free testosterone levels, and homeostatic model assessment-insulin resistance levels (HOMA-IR) in group I was significantly higher than in group II and controls. HOMA-IR in group II and controls did not differ significantly. The average anti-Mullerian hormone levels in women with PCOS were significantly higher than controls, p < 0.05. No significant difference was observed in average anti-Mullerian hormone level, ovarian volume, antral follicle count, and body mass index between the comparison groups of PCOS. In group I, a positive correlation between Hcy with HOMA-IR was detected. Conclusion Serum Hcy levels are elevated in women with PCOS and RPL, which correlates with their insulin resistance status.


Introduction
Polycystic ovary syndrome (PCOS) is one of the most common endocrinopathies associated with metabolic derailments and complex disorders involving reproductive and cardiovascular systems (1,2).
In cases of PCOS, multiple individuals or combined factors lead to fertility issues (3,4). PCOS is one of the most important risk factors not only for infertility but also for spontaneous abortion.
According to various studies, the frequency range of miscarriages in PCOS have been reported to range from 25-73% (5,6).
Hyperhomocysteinemia (Hhcy) has been considered an independent risk factor for developing several diseases, including cardiovascular disease, arterial and venous thrombosis, neuropsychogenic diseases, and adverse pregnancy outcomes (11,12). It has been established that Hhcy increases the hypercoagulable state of pregnancy and the likelihood of developing thrombosis in the maternal-fetal circulatory system and, as a result, the undesirable outcome of pregnancy (11)(12)(13)(14).
In the early stages of pregnancy, including the

Materials and Methods
The case-control study involved 245 Georgian women (20-30 yr). Of which, 175 women were with • Group I-90 women, experienced RPL. RPL is defined as 2 or more clinical pregnancy losses (20).

Criteria for inclusion in the study
All the participants did not take a drug containing sex hormones 6 months before inclusion in the study.

Ethical considerations
All the participants in the study were informed in advance, the essence and goal of the study were explained, and written consent for their participation in the study was obtained. The Ethics Committee of the 'Center for Reproductive Medicine Universe' agreed to conduct the study.

Results
The mean age of women with PCOS (study group) was 26.8 ± 3.4 and the mean age of control group was 25.2 ± 3.9 yr. No statistical difference was observed between groups regarding age.
In the study group, women with PCOS (group I and group II together) average Hcy levels were significantly higher than in controls (p < 0.05).
Average Hcy level in group I was significantly higher than in group II and the control group (p < 0.05, Table I p < 0.05) and controls (HHcy: 39%; IR: 6.5%) ( Figure 1). The incidence of insulin resistance in PCOS women with live births (group II) was significantly higher than in controls, but the incidence of Hhcy in group II and control group did not differ significantly (Figure 1). HOMA-IR in group I was significantly higher than in group II and control group (p < 0.05). HOMA-IR in PCOS women with live births and controls did not differ significantly (Table I). There was no significant difference in average AMH and LH levels, ovarian volume, antral follicle count, and BMI between comparison groups of PCOS (Table I).
In group I, the average level of T, FT, FAI, SHBG, and Ferriman-Gallwey modified scale were significantly higher than in group II and controls (Table I).
In group I statistically significant positive correlation between Hcy with HOMA-IR was detected (p < 0.05) (Figure 2).
In group I, subgroup A-a statistically significant positive correlation was found between Hcy and HOMA-IR, BMI, and OV/v (p < 0.05).
In group I, subgroup B-there was only a statistically significant positive correlation between Hcy and the Ov/v (p < 0.05).
In group II and control group, there was no correlation between Hcy with HOMA-IR and hormonal and ovarian morphological characteristics. Ov/v (cm 3 ) 12.9 ± 6.5 ∧ 11 ± 5.7 □ 6.8 ± 1.5 AFC 31 ± 11.5 ∧ 29 ± 6.5 □ 8.3 ± 6.5 Data are presented as Mean ± SD, comparison of the data in group I and group II: *p < 0.05. Comparison of the data at group I and control group: ∧ p < 0.05. Comparison of the data at group II and control group:

Discussion
PCOS is a multifactorial syndrome and is associated with important reproductive disorders.
Several confounding factors involving in the pathogenesis of PCOS, individually or in combination, may contribute to developing thrombosis and lead to RPL (21). This study indicated a link between Hhcy and pregnancy outcomes in PCOS patients. Our study shows that average Hcy levels were significantly higher in PCOS patients who experienced RPL than in PCOS patients with a live birth in anamnesis and controls.
According to our results, Hhcy may be considered as one of the causes of pregnancy loss in patients with PCOS. Our findings are consistent with the studies of a few other authors (14,17,22), but some studies did not find a significant difference in Hcy levels among PCOS women with RPL and without pregnancy complications (19,23).
PCOS is commonly associated with metabolic and cardiovascular complications, and IR is the potential pathogenetic mechanism for both (22,24,25). Insulin resistance and compensatory hyperinsulinemia are one of the most important events in the pathogenesis of PCOS. Our investigation showed that the average HOMA-IR in PCOS patients with RPL was significantly higher compared with PCOS patients with live births and controls. Our study results showed that the insulin resistance index in PCOS patients with live births and controls did not differ significantly. A similar finding was confirmed by other studies (16,19,23).
Other authors, however, did not find a significant association between insulin resistance and RPL in PCOS patients (26).
When we compared the groups of PCOS women, we found that in patients with RPL the average level of T, FT, and FAI were significantly higher than in the group with live births. There were positive correlations among serum Hcy, HOMA-IR, and androgen levels in PCOS women with RPL. Few studies found similar outcomes. They described that in PCOS women, compensatory hyperinsulinemia due to insulin resistance may lead to ovarian and adrenal androgen hypersecretion. Furthermore, there is an increased level of FT due to high insulin levels suppressing hepatic production of SHBG (26).
The relationship between insulin resistance status and Hhcy in PCOS women is still a topic of debate. Some studies found a positive correlation between Hcy and insulin resistance in PCOS women, which was related to thrombosis in the fetoplacental system and may be considered as a cause of pregnancy loss (2,3,5,15,18,26). Others did not confirm this association and proposed that serum Hcy levels rise independently in PCOS women (17,27). Our research indicated a relationship between Hhcy and insulin resistance in women with PCOS and RPL. According to our results, we detected a positive correlation between Hcy and insulin resistance in PCOS women with RPL. The results may indicate the importance of measuring the Hcy levels and insulin resistance status in women with PCOS for predicting RPL.
Our study results showed that in PCOS women with RPL and insulin resistance, average Hcy levels were significantly higher than in PCOS patients with RPL without insulin resistance, but other characteristics in comparable subgroups did not differ significantly. Similar findings were confirmed by other studies (28), however, some studies reported higher levels of AMH in PCOS women with insulin resistance, compared with those with normal insulin sensitivity (9,29). In further contrast, International Journal of Reproductive BioMedicine Hyperhomocysteinemia in women with PCOS some studies found a negative correlation between AMH and HOMA-IR has also been reported (14).
A strong relationship exists between serum Hcy level and insulin resistance status in women with PCOS and RPL, which contributes to the long-term complications of PCOS. However, larger sample sizes and randomized trials are needed to establish the role of Hhcy in women with PCOS and RPL and to rule out genetic factors of Hhcy.
Screening for Hcy status and correction of Hhcy in women with PCOS will help avoid pregnancy losses by preventing thrombosis in the maternalfetal circulatory system and improving reproductive performance and pregnancy outcome.

Conclusion
Serum Hcy levels are elevated in women with PCOS and RPL, which correlates with their insulin resistance status. Screening for Hcy status and correction of Hhyc and insulin resistance in women with PCOS might improve reproductive outcomes.