Bilirubin Concentration in Follicular Fluid Is Increased in Infertile Females, Correlates with Decreased Antioxidant Levels and Increased Nitric Oxide Metabolites, and Negatively Affects Outcome Measures of In Vitro Fertilization.

In a previous study, we showed that various low-molecular-weight compounds in follicular fluid (FF) samples of control fertile females (CFF) have different concentrations compared to those found in FF of infertile females (IF), before and after their categorization into different subgroups, according to their clinical diagnosis of infertility. Using the same FF samples of this previous study, we here analyzed the FF concentrations of free and bound bilirubin and compared the results obtained in CFF, IF and the different subgroups of IF (endometriosis, EM, polycystic ovary syndrome, PCOS, age-related reduced ovarian reserve, AR-ROR, reduced ovarian reserve, ROR, genetic infertility, GI and unexplained infertility, UI). The results clearly indicated that CFF had lower values of free, bound and total bilirubin compared to the respective values measured in pooled IF. These differences were observed even when IF were categorized into EM, PCOS, AR-ROR, ROR, GI and UI, with EM and PCOS showing the highest values of free, bound and total bilirubin among the six subgroups. Using previous results of ascorbic acid, GSH and nitrite + nitrate measured in the same FF samples of the same FF donors, we found that total bilirubin in FF increased as a function of decreased values of ascorbic acid and GSH, and increased concentrations of nitrite + nitrate. The values of total bilirubin negatively correlated with the clinical parameters of fertilization procedures (number of retrieved oocytes, mature oocytes, fertilized oocytes, blastocysts, high-quality blastocysts) and with clinical pregnancies and birth rates. Bilirubin concentrations in FF were not linked to those found in serum samples of FF donors, thereby strongly suggesting that its over production was due to higher activity of heme oxygenase-1 (HO-1), the key enzyme responsible for bilirubin formation, in granulosa cells, or cumulus cells or oocytes of IF and ultimately leading to bilirubin accumulation in FF. Since increased activity of HO-1 is one of the main enzymatic intracellular mechanisms of defense towards external insults (oxidative/nitrosative stress, inflammation), and since we found correlations among bilirubin and oxidative/nitrosative stress in these FF samples, it may reasonably be supposed that bilirubin increase in FF of IF is the result of protracted exposures to the aforementioned insults evidently playing relevant roles in female infertility.

Metabolic Signature of Energy Metabolism Alterations and Excess Nitric Oxide Production in Culture Media Correlate with Low Human Embryo Quality and Unsuccessful Pregnancy.

Notwithstanding the great improvement of ART, the overall rate of successful pregnancies from implanted human embryos is definitely low. The current routine embryo quality assessment is performed only through morphological criteria, which has poor predictive capacity since only a minor percentage of those in the highest class give rise to successful pregnancy. Previous studies highlighted the potentiality of the analysis of metabolites in human embryo culture media, useful for the selection of embryos for implantation. In the present study, we analyzed in blind 66 human embryo culture media at 5 days after in vitro fertilization with the aim of quantifying compounds released by cell metabolism that were not present as normal constituents of the human embryo growth media, including purines, pyrimidines, nitrite, and nitrate. Only some purines were detectable (hypoxanthine and uric acid) in the majority of samples, while nitrite and nitrate were always detectable. When matching biochemical results with morphological evaluation, it was found that low grade embryos (n = 12) had significantly higher levels of all the compounds of interest. Moreover, when matching biochemical results according to successful (n = 17) or unsuccessful (n = 25) pregnancy, it was found that human embryos from the latter group released higher concentrations of hypoxanthine, uric acid, nitrite, and nitrate in the culture media. Additionally, those embryos that developed into successful pregnancies were all associated with the birth of healthy newborns. These results, although carried out on a relatively low number of samples, indicate that the analysis of the aforementioned compounds in the culture media of human embryos is a potentially useful tool for the selection of embryos for implantation, possibly leading to an increase in the overall rate of ART.