Association of serum, follicular fluid, and embryo culture media homocysteine levels with embryo quality and pregnancy rates in assisted reproductive techniques: A prospective observational study.

Infertility is defined as the inability to conceive after one year of regular unprotected sexual intercourse and affects approximately 15% of couples of reproductive age worldwide. [ 1 ] Assisted reproductive technology (ART) is currently one of the fastest-growing fields in reproductive medicine, with its clinical application increasing steadily over recent decades. ART accounts for more than 1% of all live births in the United States and up to 3% in several European countries. [ 2 ] Homocysteine (Hcy) is a sulfur-containing, nonessential amino acid generated during methionine metabolism and plays an important role in maintaining cellular homeostasis. [ 3 ] The detrimental effects of hyperhomocysteinemia are primarily mediated through 2 mechanisms. First, free radicals generated during Hcy oxidation exert toxic effects on the vascular endothelium. Second, Hcy disrupts the coagulation cascade, impairing the antithrombotic properties of the endothelium and thereby increasing the risk of thrombosis. [ 4 ] The primary causes of hyperhomocysteinemia include inadequate intake of folate, cobalamin, pyridoxine, and methionine, as well as genetic variations affecting Hcy metabolism. [ 5 ] This condition adversely affects multiple aspects of reproductive health, including abnormalities in sperm morphology, count, and motility, as well as fetal congenital malformations, miscarriage, gestational hypertension, gestational diabetes mellitus, and low birth weight. [ 6 - 8 ] . Previous studies have demonstrated that hyperhomocysteinemia is associated with reduced pregnancy and implantation rates and an increased risk of miscarriage in infertile couples undergoing fertility treatment. [ 9 ] Follicular fluid (FF), which consists of plasma exudate and locally synthesized bioactive components, reflects the metabolic status of the oocyte and its surrounding granulosa cells. Because FF can be readily obtained during in vitro fertilization (IVF) procedures, it provides a valuable and minimally invasive biological sample for investigating the intrafollicular microenvironment. Previous studies have shown that elevated Hcy concentrations in FF are associated with impaired embryo quality and reduced fertility in women undergoing ART. [ 10 , 11 ] However, evidence regarding the association between Hcy concentrations in embryo culture media and embryo quality or pregnancy outcomes remains limited. In recent years, increasing attention has been directed toward metabolic profiling during embryonic development and the analysis of embryo culture media. Therefore, this study aimed to investigate the correlations among Hcy concentrations in serum, FF, and embryo culture media and to evaluate their associations with embryo quality and pregnancy outcomes in women undergoing controlled ovarian hyperstimulation for assisted reproduction.

Conceptualization: Xuxin Zhan, Jing Zhao, Hongli Zhu. Data curation: Xuxin Zhan, Jinxia Xie. Formal analysis: Xuxin Zhan, Xiaoting Wang. Investigation: Jinxia Xie, Hongli Zhu. Methodology: Xuxin Zhan, Jing Zhao. Software: Xiaoting Wang. Supervision: Jing Zhao, Hongli Zhu. Writing – original draft: Xuxin Zhan, Hongli Zhu. Writing – review & editing: Xuxin Zhan, Jing Zhao, Hongli Zhu.

This prospective study included 66 women who underwent IVF/ICSI treatment between November 2022 and November 2023 at Xi’an Gynecology and Obstetrics Hospital, Xi’an People’s Hospital (Xi’an Fourth Hospital), Xi’an, China. The study was approved by the Institutional Review Board of Xi’an People’s Hospital (Approval No. 20233027), and written informed consent was obtained from all participants before enrollment. Eligible participants were women aged < 42 years with a basal follicle-stimulating hormone (FSH) level < 10 mIU/mL who were undergoing their first IVF/ICSI cycle and had regular menstrual cycles. Women with polycystic ovary syndrome (PCOS), endometriosis, hydrosalpinx, endocrine disorders, diabetes mellitus, a history of smoking, or severe male-factor infertility (including anejaculation, azoospermia, or sperm morphology < 1%) were excluded. Demographic and clinical characteristics were obtained from the medical records. Blood samples were collected during the early follicular phase (day 2 of the menstrual cycle) and on the day of oocyte pick-up (OPU). Serum concentrations of FSH, luteinizing hormone (LH), and anti-Müllerian hormone were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Thermo Fisher Scientific) according to the manufacturer’s instructions. Serum homocysteine (Hcy) concentrations on the day of OPU were determined using an enzyme cycling assay with a commercially available kit (Beijing Leadman Biochemistry Co., Ltd., Beijing, China) according to the manufacturer’s instructions. Patients underwent tailored ovulation protocols based on age, anti-Müllerian hormone level, body weight, basal FSH level, and the number of antral follicles. Serum hormone levels and follicular development were dynamically monitored, with gonadotropin (Gn) doses adjusted accordingly. Ovulation was triggered with 10,000 IU of human chorionic gonadotropin (produced by Serono, Switzerland) once the largest follicle reached a diameter of at least 18 mm and 2 follicles were >16 mm in size. OPU was performed 36 hours after human chorionic gonadotropin administration under transvaginal ultrasound guidance. Follicles were punctured individually, and 2 mL or more of FF was collected, specifically targeting those containing oocytes. Additionally, the levels of Hcy were measured. Great care was taken to ensure that only uncontaminated samples were included. Following a 4-hour oocyte culture period, either IVF or intracytoplasmic sperm injection (ICSI) was performed based on semen quality. Fertilization was confirmed by the presence of 2 pronuclei and 2 polar bodies approximately 18 hours after insemination or microinjection. The dividing embryo’s morphology was assessed and graded using the ESHRE-Istanbul Consensus Workshop Grading System, focusing on morphological appearance and fragmentation. [ 12 ] After separating embryos into droplets, samples of the embryo culture medium were collected, and enzyme cycling was used to determine Hcy levels in the samples. Embryo transfer (ET) was performed 3 to 5 days after retrieval. Luteal phase supplementation with 90 mg/d progesterone sustained-release vaginal gel (Crinone®, Watford, Hertfordshire, UK) administered intravaginally was started on the evening following oocyte pick-up and continued for 14 days thereafter. An ultrasound examination 5 weeks after embryo transfer was used to confirm clinical pregnancy by the presence of a gestational sac. Statistical analyses were performed using R version 4.3.1 (R Foundation for Statistical Computing, Vienna, Austria). Missing data were handled using multiple imputation by chained equations (MICE), and the estimates from the imputed datasets were pooled according to Rubin’s rules. Continuous variables were assessed for normality using the Shapiro–Wilk test. Normally distributed variables are presented as the mean ± standard deviation and were compared using the independent-samples t -test or 1-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple comparisons when appropriate. Non-normally distributed variables are presented as the median (interquartile range [IQR]) and were compared using the Mann–Whitney U test or the Kruskal–Wallis test, followed by Dunn’s post hoc test with Bonferroni correction for multiple comparisons when appropriate. Categorical variables are expressed as frequencies (percentages) and were compared using the χ 2 test or Fisher’s exact test, as appropriate. Spearman’s rank correlation analysis was performed to evaluate the associations among continuous variables. Multiple linear regression and multivariable logistic regression models with sequential adjustment for potential confounding factors were constructed to investigate the associations between Hcy concentrations (μmol/L) and embryological as well as clinical pregnancy outcomes. A 2-sided P  < .05 was considered statistically significant.

A total of 66 women undergoing IVF/ICSI treatment were enrolled in this prospective observational study. All enrolled patients completed embryo culture and were included in the analysis of embryological outcomes based on embryo culture medium Hcy levels. Among these patients, 35 women subsequently underwent embryo transfer and were included in the analysis of clinical pregnancy outcomes. Of these, 17 patients achieved clinical pregnancy, whereas 18 patients did not. The patient selection process and analysis populations are presented in Figure 1 . Study flow diagram illustrating patient enrollment, embryo outcome analysis, and clinical pregnancy outcome analysis. FSH = follicle-stimulating hormone, IVF/ICSI = in vitro fertilization/intracytoplasmic sperm injection, PCOS = polycystic ovary syndrome. The baseline characteristics of the 66 enrolled patients were compared according to embryo culture medium Hcy levels. Patients were categorized into 3 groups based on percentile distribution: low Hcy group (<25th percentile, 75th percentile, >3.34 μmol/L). The baseline demographic characteristics, ovarian reserve parameters, infertility-related factors, and ovarian stimulation protocols are summarized in Table 1 . No significant differences were observed among the 3 groups in most baseline characteristics (all P  > .05). A significantly lower total Gn dose was observed in the high embryo culture medium Hcy group compared with the low and intermediate embryo culture medium Hcy groups ( P  < .001). In addition, baseline FSH levels were significantly different among the 3 groups ( P  = .032). Baseline characteristics of female patients categorized by embryo culture medium Hcy levels (n = 66). Follicular output rate = preovulatory follicle count (PFC)/AFC. a1*a: Statistical differences among groups were assessed using the Kruskal–Wallis test followed by Dunn’s post hoc test with Bonferroni correction. Different superscript letters indicate significant differences between groups ( P  < .05). b1*b: Statistical differences among groups were assessed using the Kruskal–Wallis test followed by Dunn’s post hoc test with Bonferroni correction. Different superscript letters indicate significant differences between groups ( P  < .05). AFC = antral follicle count, AMH = anti-Mullerian hormone, BMI = body mass index, FORT = follicular output rate, FSH = follicle-stimulating hormone, Gn = gonadotropin, Hcy = homocysteine, LH = luteinizing hormone, PFC = preovulatory follicle count. Embryological outcomes were compared among patients with different embryo culture medium Hcy levels. A significant difference was observed in blastocyst formation rate among the 3 groups ( P  = .036). Post hoc pairwise comparisons showed that the blastocyst formation rate was significantly higher in the intermediate Hcy group (2.19–3.34 μmol/L) than in the high Hcy group (>3.34 μmol/L). However, no significant differences were observed in embryo quality rate, number of oocytes retrieved, fertilization rate, normal fertilization rate, cleavage rate, or normal cleavage rate among the 3 groups (all P  > .05; Table 2 ). Embryological characteristics according to embryo culture medium Hcy levels in female patients (n = 66). a1*a: Statistical differences among groups were assessed using the Kruskal–Wallis test followed by Dunn’s post hoc test with Bonferroni correction. Different superscript letters indicate significant differences between groups ( P  < .05). Hcy = homocysteine. In the multivariable linear regression analysis, embryo culture medium Hcy levels were evaluated in relation to blastocyst formation rate (Table 3 ). Compared with the low Hcy group (3.34 μmol/L) was not significantly associated with blastocyst formation rate in the unadjusted model (β = −6.78, 95% CI: −23.46 to 9.91, P  = .429). After adjustment for female age, body mass index, and total Gn dose, the high Hcy group showed a significant negative association with blastocyst formation rate (β = −20.53, 95% CI: −38.07 to −2.98, P  = .025). This association remained significant after further adjustment for baseline FSH (β = −21.17, 95% CI: −38.58 to −3.76, P  = .020). Multivariable linear regression analysis of the association between embryo culture medium Hcy levels and blastocyst formation rate (n = 66). Model 1: unadjusted model; Model 2: adjusted for female age, BMI, and total Gn dose; Model 3: further adjusted for baseline FSH. BMI = body mass index, CI = confidence interval, FSH = follicle-stimulating hormone, Gn = gonadotropin, Hcy = homocysteine. Spearman correlation analysis was performed to evaluate the relationships among Hcy levels in serum, FF, and embryo culture medium (Fig. 2 ). The Shapiro–Wilk test indicated that serum Hcy and FF Hcy levels showed skewed distributions, whereas embryo culture medium Hcy levels followed an approximately normal distribution. A weak positive correlation was observed between serum Hcy and FF Hcy levels (rs = 0.302, P  = .014). However, no significant correlations were identified between serum Hcy and embryo culture medium Hcy levels (rs = −0.151, P  = .224) or between FF Hcy and embryo culture medium Hcy levels (rs = −0.093, P  = .456). Correlation matrix of Hcy levels in serum, follicular fluid, and embryo culture medium. Hcy = homocysteine. Among the 66 enrolled patients, 35 women underwent embryo transfer and were included in the analysis of clinical pregnancy outcomes. According to pregnancy status, patients were divided into the clinical pregnancy group (n = 17) and non-pregnancy group (n = 18). The baseline characteristics and embryological parameters between the 2 groups were compared. No significant differences were observed in demographic characteristics, ovarian reserve parameters, infertility-related factors, stimulation characteristics, or embryological outcomes between the 2 groups (all P  > .05; Table S1 , Supplemental Digital Content 1). Multiple logistic regression analyses were performed to evaluate the associations between Hcy levels in serum, FF, and embryo culture medium and pregnancy outcomes (Table 4 ). In the unadjusted model (model 1), serum Hcy levels were not significantly associated with pregnancy outcomes. Compared with the lowest tertile, neither the middle tertile nor the highest tertile of serum Hcy showed significant differences ( P  > .05). For FF Hcy, patients in the middle tertile exhibited a significantly higher likelihood of achieving pregnancy compared with those in the lowest tertile (OR = 5.33, 95% CI: 1.02–27.76, P  = .047). This association remained significant after adjustment for female age and body mass index (OR = 8.59, 95% CI: 1.31–56.20, P  = .025). However, the highest tertile of FF Hcy was not significantly associated with pregnancy outcomes. Regarding embryo culture medium Hcy levels, compared with the reference group (2.19–3.34 μmol/L), patients with Hcy levels > 3.34 μmol/L had a significantly increased probability of pregnancy in both the unadjusted model (OR = 14.00, 95% CI: 1.37–142.89, P  = .026) and the adjusted model (OR = 17.56, 95% CI: 1.57–196.21, P  = .020). Associations between HCY levels in serum, follicular fluid, and embryo culture medium and pregnancy outcomes: multivariable logistic regression analysis (n = 35). Model 1: unadjusted model; Model 2: adjusted for female age, BMI. BMI = body mass index, CI = confidence interval, Hcy = homocysteine.

This prospective study comprehensively investigated the associations of Hcy levels in serum, FF, and embryo culture medium with reproductive outcomes in women undergoing IVF/ICSI. We observed a positive correlation between serum and FF Hcy levels, whereas embryo culture medium Hcy was not significantly correlated with either serum or FF Hcy. Regarding embryological outcomes, higher embryo culture medium Hcy levels were associated with a reduced blastocyst formation rate after adjustment for potential confounding factors. In terms of clinical pregnancy outcomes, serum Hcy showed no significant association, whereas Hcy levels in FF and embryo culture medium were associated with pregnancy outcomes in multivariable analyses. However, these findings should be interpreted cautiously due to the limited sample size and wide confidence intervals. In this study, we investigated the distribution and correlation patterns of Hcy levels across 3 biological compartments, including serum, FF, and embryo culture medium, during assisted reproduction. Consistent with previous findings [ 13 ] showing positive correlations between serum and FF levels of ovarian follicular microenvironment (OCM)-related metabolites, including vitamin B12, folate (FA), and Hcy, our study also observed a weak positive correlation between serum Hcy and FF Hcy levels (rs = 0.302, P  = .014). This association may be attributed to the fact that FF is partially derived from plasma exudation, allowing Hcy from the peripheral circulation to enter the follicular microenvironment through the blood-follicle barrier and influence intrafollicular Hcy levels. [ 13 ] However, the weak correlation indicates that FF Hcy is not solely determined by serum Hcy levels, and local metabolic processes may also contribute to its regulation. [ 14 , 15 ] In contrast, embryo culture medium Hcy levels were not significantly correlated with either serum or FF Hcy levels, suggesting that embryo culture medium Hcy may reflect metabolic characteristics distinct from those of the maternal circulation and follicular environment. According to the baseline characteristics in our study, most demographic characteristics and ovarian function parameters were comparable among patients with different embryo culture medium Hcy levels; however, significant differences were observed in the total gonadotropin dose and basal FSH levels. These findings suggest that variations in ovarian stimulation response and baseline ovarian reserve status may influence early embryonic metabolic characteristics and subsequently affect Hcy levels in embryo culture media. Overall, Hcy exhibits source-specific distribution patterns across different reproductive microenvironments, and Hcy levels from different biological compartments may provide complementary biological information. [ 16 ] Previous studies have primarily focused on the relationships between serum and FF Hcy levels and reproductive outcomes in IVF/ICSI cycles. However, the association between Hcy levels in human embryo culture media and embryonic developmental potential remains poorly understood. Recently, metabolomic analysis of embryo culture media has emerged as a promising noninvasive approach for embryo evaluation, as metabolite profiles in culture media may reflect embryo metabolic activity and provide complementary information to conventional morphological assessment. Gardner et al [ 17 ] reported that embryos developing into blastocysts exhibited distinct glucose and pyruvate metabolic profiles compared with those that failed to reach the blastocyst stage. Seli et al [ 18 ] further suggested that combining metabolic viability scores with morphological assessment could improve the evaluation of embryonic developmental potential. In the present study, higher embryo culture medium Hcy levels were significantly associated with a lower blastocyst formation rate after adjustment for potential confounders, suggesting that culture medium Hcy may reflect metabolic alterations during embryonic development and provide additional information for evaluating embryonic developmental potential. Although the underlying mechanisms remain unclear, Hcy, as an intermediate metabolite in the methionine cycle and 1-carbon metabolism pathway, may influence embryonic development through alterations in DNA methylation, epigenetic regulation, oxidative stress, and metabolic homeostasis. [ 19 ] Given the limited evidence currently available and the relatively small sample size of this study, further large-scale multicenter studies are required to validate these findings and clarify the biological mechanisms underlying the association between embryo culture medium Hcy and embryonic developmental potential. Since Hcy levels in different biological compartments may reflect distinct metabolic environments, their associations with pregnancy outcomes may vary. In the present study, serum Hcy levels were not significantly associated with clinical pregnancy outcomes after multivariable adjustment. Previous studies [ 20 ] have demonstrated that women with PCOS generally exhibit elevated serum Hcy levels, and such metabolic abnormalities may be associated with impaired reproductive outcomes. Asanidze et al [ 21 ] reported that women with PCOS and recurrent pregnancy loss exhibited higher serum Hcy concentrations than those with previous live births and healthy controls, suggesting that elevated Hcy levels may contribute to adverse pregnancy outcomes in this specific metabolic population. However, these findings may be influenced by metabolic disturbances associated with PCOS. Since patients with PCOS were excluded from our study, the potential confounding effect of this metabolic disorder was minimized. Consistent with our findings, Liu et al [ 22 ] reported no significant association between serum Hcy levels and IVF/ICSI pregnancy outcomes in couples with unexplained infertility. In addition, Hcy levels in FF and embryo culture medium may provide additional information regarding assisted reproductive outcomes. Previous studies have suggested [ 13 ] that FF Hcy reflects the local 1-carbon metabolic status of the follicular microenvironment and is involved in oocyte maturation, maintenance of follicular function, and early embryonic development. However, the associations between FF Hcy, embryo quality, and pregnancy outcomes remain inconsistent across studies. Some studies have suggested that elevated Hcy levels may impair oocyte quality and embryonic development by inducing oxidative stress, inflammatory responses, and aberrant DNA methylation, [ 23 - 25 ] whereas others have found no significant association between FF Hcy and embryo quality, [ 26 ] indicating that the role of Hcy in reproduction is likely regulated by complex metabolic processes. In the present study, FF Hcy was also associated with clinical pregnancy outcomes. Compared with the lowest tertile, women with intermediate FF Hcy levels exhibited a higher likelihood of achieving clinical pregnancy, whereas no similar association was observed in the highest tertile. These findings suggest that embryonic development may require an optimal level of Hcy and that maintaining Hcy within an appropriate range in the follicular microenvironment may help prevent its potentially detrimental effects. Furthermore, embryo culture medium Hcy was also associated with both clinical pregnancy outcomes and embryological outcomes, suggesting that Hcy in embryo culture medium may reflect metabolic alterations during embryonic development. Nevertheless, given the limited evidence currently available and the wide confidence intervals observed in our analyses, these findings should be interpreted with caution and require validation in larger, well-designed prospective studies. This study has several limitations. First, it was conducted at a single center with a relatively limited sample size. In particular, the number of clinical pregnancy events was relatively small, which may have reduced the statistical power and contributed to the wide confidence intervals observed for some effect estimates. Second, Hcy levels were measured at a single time point, precluding the assessment of dynamic changes in Hcy throughout the assisted reproduction process and their potential relationships with pregnancy outcomes. Third, although several potential confounding factors were adjusted for, residual confounding from unmeasured metabolic or lifestyle factors cannot be completely excluded. Future large-scale, multicenter prospective studies are warranted to validate these findings and further clarify the potential role and underlying mechanisms of Hcy from different biological compartments in assisted reproduction.

To the best of our knowledge, this is the first study to simultaneously evaluate the correlations of Hcy levels among 3 biological compartments – serum, FF, and embryo culture medium – and to explore their potential relationships with embryological and clinical pregnancy outcomes. Our findings suggest that Hcy derived from different biological compartments may reflect distinct metabolic characteristics during assisted reproduction. In particular, embryo culture medium Hcy was associated with embryological outcomes, whereas both FF and embryo culture medium Hcy may provide additional information regarding clinical pregnancy outcomes.

The authors would like to thank the clinicians and laboratory staff at the Center for the Reproductive Medicine Centre of Xi’an People’s Hospital (Xi’an No. 4 Hospital) for their contribution.