Oxidative stress profiles in spent culture media of day 3 and day 5 frozen-thawed embryos: a prospective cohort study

This study aimed to compare oxidative stress parameters in the culture media of frozen-thawed cleavage-stage (Day 3) and blastocyst-stage (Day 5) embryos and to evaluate their association with implantation outcomes. In this prospective cohort study, 101 infertile couples undergoing IVF-ICSI treatment were included, with 50 women receiving frozen-thawed cleavage-stage (Day 3) embryo transfers and 51 women receiving frozen-thawed blastocyst-stage (Day 5) embryo transfers. Following embryo thawing and prior to embryo transfer, spent culture media samples were collected for oxidative stress analysis. Total Antioxidant Status (TAS), Total Oxidant Status (TOS), and Oxidative Stress Index (OSI) were measured using standardized colorimetric assays. Clinical pregnancy was defined as the presence of fetal cardiac activity confirmed by ultrasound at 6–7 weeks. Live birth was defined as the delivery of a live infant after 24 weeks of gestation. Oxidative stress parameters were additionally compared between patients with and without clinical pregnancy separately within the Day 3 and Day 5 groups, and stage-adjusted logistic regression models were used to evaluate whether each biomarker was independently associated with clinical pregnancy. A total of 101 patients were included, with 50 undergoing Day 3 and 51 undergoing Day 5 transfers. Clinical pregnancy rates were significantly higher in the Day 5 group (52.9% vs. 32.0%, p = 0.033), and the live birth rate was also higher (47.0% vs. 28.0%, p = 0.036). Day 5 spent culture media had higher TAS ( p = 0.005), lower TOS ( p = 0.001), and lower OSI ( p < 0.001). However, within both developmental-stage strata, TAS, TOS, and OSI did not differ significantly between patients with and without clinical pregnancy (all p > 0.20). In stage-adjusted logistic regression, TAS (aOR per 1-SD increase: 1.30, 95% CI 0.71–2.40; p = 0.399), TOS (aOR: 1.00, 95% CI 0.64–1.55; p = 0.991), and OSI (aOR: 0.97, 95% CI 0.61–1.54; p = 0.885) were not independently associated with clinical pregnancy. Day 5 embryos were associated with a distinct oxidative stress profile characterized by higher TAS, lower TOS, and lower OSI, as well as higher clinical pregnancy and live birth rates, compared with Day 3 embryos. Nevertheless, the absence of within-stage differences and the negative stage-adjusted regression analyses indicate that TAS, TOS, and OSI primarily reflect embryo developmental stage or biological selection in this cohort rather than provide independent information on clinical pregnancy. Larger prospective studies are needed before these measures can be considered candidate non-invasive biomarkers for embryo selection.