Ovarian follicular fluid levels of phthalates and benzophenones in relation to fertility outcomes.

BACKGROUND: Many endocrine disrupting chemicals (EDCs), for instance phthalates and benzophenones, are associated with adverse fertility outcomes and semen quality parameters. OBJECTIVE: To evaluate if concentrations of selected phthalate metabolites and benzophenones measured in follicular fluid are associated with fertility outcomes (i.e., reproductive hormones, antral follicle count, detected heartbeat at gestational week 7, and live birth) and, in a supplementary study, if measured concentrations of chemicals in follicular fluid can exert biological effects on human spermatozoa. METHODS: Overall, 111 couples from a fertility clinic in Denmark contributed with 155 follicular fluid samples. Concentrations of 43 metabolites from 19 phthalates and phthalate substitutes and six benzophenones were measured in follicular fluid using liquid chromatography-tandem mass spectrometry. Multiple linear and logistic regression with an applied generalized estimating equation model allowing more than one measurement per woman assessed the association between follicular EDC levels and fertility outcomes. The assessment of biological effects of individual and mixtures of EDCs on human spermatozoa was conducted through a human sperm cell based Ca2+-fluorimetric assay. RESULTS: Benzophenone-3 (BP-3) and seven metabolites of five phthalates were detectable in follicular fluid. Women with metabolites of dibutyl phthalate isomers in the highest tertiles had lower antral follicle count (MiBP: ß = -5.35 [95 % CI: -9.06; -2.00], MnBP: ß = -5.25 [95 % CI: -9.00; -2.00]) and lower odds for detecting a heartbeat at gestational week 7 (MiBP: OR = 0.35 [95 % CI: 0.14; 0.91], MnBP: OR = 0.39 [95 % CI: 0.13; 1.15]). Mixtures of the measured concentrations of BP-3 and the seven phthalate metabolites induced a small significant increase in the intracellular calcium ion concentration in human spermatozoa from healthy donors (n = 3). DISCUSSION: Phthalate metabolites and BP-3 were detectable in follicular fluid and high concentrations of some phthalate metabolites were linked with lower chance of successful fertility treatment outcomes. Chemical mixture concentrations in follicular fluid induced a calcium response in human spermatozoa highlighting possible biological effects at physiologically relevant concentrations.

Presence of the vitamin D inactivating enzyme CYP24A1 in human sperm and prediction of the success of intrauterine insemination: A prospective study.

Male fertility is routinely evaluated by semen analysis, although semen quality variables such as; sperm count, motility and morphology have low predictive value for spontaneous pregnancies and fertility treatment outcomes. Vitamin D has been suggested to be beneficial for male reproduction. The vitamin D receptor and the vitamin D inactivating enzyme CYP24A1 are co-expressed in high quality sperm. Presence of CYP24A1 at the annulus of human sperm can distinguish between sperm from healthy and infertile men with high specificity and is positively correlated with semen quality. The high expression level in the testis of FAM57B2, which is activated by 24,25OH2D3, indicates an uncharacterized biological role for CYP24A1 in male reproduction. Moreover, activated vitamin D has been shown to induce sperm motility and promote fertilization in vitro. Here, we prospectively investigated whether the fraction of CYP24A1 positive sperm was a better predictor of clinical pregnancy than semen analysis by including 240 fertility treatments (169 couples) from a single fertility centre in Denmark. ROC-curve based analysis showed that the percentage of sperm expressing CYP24A1 was a better predictor of successful pregnancy outcome after intrauterine inseminations (IUI) than both sperm concentration and motility (p < 0.05). Interestingly, samples with CYP24A1 staining >67% of the sperm increased the likelihood of achieving pregnancy 4-fold after IUI compared with samples having fewer sperm with detectable CYP24A1 (p < 0.05). Neither CYP24A1 nor any of the other assessed semen quality variables were predictive for the treatment outcome of the more invasive assisted reproductive techniques (IVF and ICSI). In conclusion, our results provide proof of principle for a CYP24A1-based sperm test to improve fertility outcome for infertile patients referred for IUI and supports a role for vitamin D metabolites during fertilization.