Perfluorooctanoic acid alters progesterone activity in human endometrial cells and induces reproductive alterations in young women.

Female fecundity is finely regulated by hormonal signaling, representing a potential target for endocrine-disrupting chemicals. Among the chemicals of most concern are the perfluoroalkyl substances (PFAS), widely used in consumer goods, that are associated with adverse effects on reproductive health. In this context, the endometrium clearly represents an important fertility determining factor. The aim of this study was to investigate PFAS interference on hormonal endometrial regulation. This study was performed within a screening protocol to evaluate reproductive health in high schools. We studied a cohort of 146 exposed females aged 18-21 from the Veneto region in Italy, one of the four areas worldwide heavily polluted with PFAS, and 1080 non-exposed controls. In experiments on Ishikawa cells included UV-Vis spectroscopy, microarray analysis and qPCR. We report a significant dysregulation of the genetic cascade leading to embryo implantation and endometrial receptivity. The most differentially-expressed genes upon PFOA coincubation were ITGB8, KLF5, WNT11, SULT1E1, ALPPL2 and G0S2 (all p < 0.01). By qPCR, we confirmed an antagonistic effect of PFOA on all these genes, which was reversed at higher progesterone levels. Molecular interference of PFOA on progesterone was confirmed by an increase in the intensity of absorption spectra at 250 nm in a dose-dependent manner, but not in the presence of ß-estradiol. Age at menarche (+164 days, p = 0.006) and the frequency of girls with irregular periods (29.5% vs 21.5%, p = 0.022) were significantly higher in the exposed group. Our results are indicative of endocrine-disrupting activity of PFAS on progesterone-mediated endometrial function.

Perfluoro-octanoic acid impairs sperm motility through the alteration of plasma membrane.

CONTEXT: Perfluoroalkyl-substances (PFAS) are chemical additives considered harmful for humans. We recently showed that accumulation of perfluoro-octanoic acid (PFOA) in human semen of exposed subjects was associated with altered motility parameters of sperm cells, suggesting direct toxicity. OBJECTIVES: To determine whether direct exposure of human spermatozoa to PFOA was associated to impairment of cell function. PATIENTS AND METHODS: Spermatozoa isolated from semen samples of ten normozoospermic healthy donors were exposed up to 2 h to PFOA, at concentrations from 0.1 to 10 ng/mL. Viability and motility parameters were evaluated by Sperm Class Analyser. Cell respiratory function was assessed by both mitochondrial probe JC-1 and respiratory control ratio (RCR) determination. Sperm accumulation of PFOA was quantified by liquid chromatography-mass spectrometry. Expression of organic ion-transporters OATP1 and SLCO1B2 was assessed by immunofluorescence and respective role in PFOA accumulation was evaluated by either blockade with probenecid or membrane scavenging through ß-cyclodextrin (ß-CD). Plasma membrane fluidity and electrochemical potential (ΔΨp) were evaluated, respectively, with Merocyanine-540 and Di-3-ANEPPDHQ fluorescent probes. RESULTS: Compared to untreated controls, a threefold increase of the percentage of non-motile sperms was observed after 2 h of exposure to PFOA regardless of the concentration of PFOA, whilst RCR was significantly reduced. Only scavenging with ß-CD was effective in reducing PFOA accumulation, suggesting membrane involvement. Altered membrane fluidity, reduced ΔΨp and sperm motility loss associated with exposure to PFOA were reverted by ß-CD treatment. CONCLUSION: PFOA alters human sperm motility through plasma-membrane disruption, an effect recovered by incubation with ß-CD.

E2F1 copy number variations contribute to spermatogenic impairment and cryptorchidism by increasing susceptibility to heat stress.

BACKGROUND: Copy number variations (CNVs) play an important role in the onset of several diseases, and recently research focused on the relationship between these structural variants and diseases of the reproductive tract, including male infertility and cryptorchidism. OBJECTIVES: To evaluate the contribution of copy number variations of E2F1 gene to idiopathic male infertility and the factors influencing expression of this gene. MATERIALS AND METHODS: We performed a retrospective study on 540 subjects recruited from September 2014 to February 2015. TaqMan CNV assay was used to analyze E2F1 CNV. Real-time PCR was used to assess E2F1 and HSP70 expression level in heat stressed and transfected cells with three E2F1 copies. RESULTS: We found a significant difference in the frequency of altered E2F1 copies in patients (12/343, 3.5%) compared with controls (0/197) (p = 0.005). Six patients with E2F1 CNV had history of cryptorchidism, but the prevalence between men with idiopathic infertility (6/243, 2.5%) and infertile men with history of cryptorchidism (6/100, 6.0%) was not statistically different (p = 0.1). E2F1 expression increased under heat stress conditions, especially in cells carrying more copies of gene and this was associated with increased expression of HSP70. DISCUSSION: Our data suggest that an abnormal E2F1 expression caused by multiple copies of E2F1 gene predisposes to the onset of infertility and that the risk further increases if subjects with altered E2F1 copies have stressful conditions, such as heat stress or history of cryptorchidism. CONCLUSION: This study shows a link between E2F1 CNV and male infertility, suggesting that the increased risk of spermatogenic impairment associated with higher E2F1 copies might be due to higher susceptibility to stressful conditions.

Impaired sperm function in infertile men relies on the membrane sterol pattern.

Membrane cholesterol removal appears a key step for the gain of fertility potential during sperm maturation. However, the membrane sterol pattern in sperm cells from infertile patients, with impaired sperm parameters, has been poorly investigated. To elucidate a causative link between sperm membrane composition in male fertility, here we have investigated the levels of cholesterol and its oxidized derivatives 7ß-hydroxycholesterol and 7-keto-cholesterol in sixteen infertile patients with oligo-asthenozoospermia and 16 normozoospermic (N) fertile subjects. Furthermore, ten of 16 N fertile subjects agreed to receive a defined testicular thermal challenge by adhering to a programme of sauna sessions for 1 month. Semen samples were obtained from each of the participants, and sperm parameters were assessed according to the World Health Organization criteria. Sperm levels of cholesterol, 7ß-hydroxycholesterol and 7-keto-cholesterol were quantified by ultra-pressure liquid chromatography mass spectrometry. The results showed that oligo-asthenozoospermia patients had a huge amount of cholesterol content compared with fertile subjects (12.40 ± 6.05 µg/106 cells vs. 0.45 ± 0.28 µg/106 cells, p < 0.001, N and oligo-asthenozoospermia, respectively). Also, oxidized derivatives were significantly higher in oligo-asthenozoospermia patients (7ß-hydroxycholesterol: 1.96 ± 1.03 ng/106 cells vs. 0.075 ± 0.05 ng/106 cells, p < 0.001 and 7-keto-cholesterol: 1.11 ± 0.72 ng/106 cells vs. 0.005 ± 0.003 ng/106 cells, p < 0.001). Moreover, sauna exposure, in parallel with a progressive worsening of sperm motility parameters, was associated with a reversible increase in sperm cholesterol after the third and fourth week of treatment, whilst 7ß-hydroxycholesterol and 7-keto-cholesterol levels showed an earlier enhancement starting from the second week. Our data show for the first time in humans a strong difference in the cholesterol and its oxidized derivatives of infertile and fertile subjects. These findings suggest a strict biochemical link relating testis function, sperm membrane status and male fertility potential.