Per- and polyfluoroalkyl substances (PFAS) and the effect on apoptosis in early porcine embryo development in vitro

Abstract: The aim of this study was to evaluate the effects of PFHxS (perfluorohexane sulfonic acid) and PFOS (perfluorooctane sulfonic acid) on early embryo development parameters and apoptosis on day 6 post fertilization in porcine embryos. A second purpose was to evaluate, and establish, the TUNEL assay in our IVF laboratory. PFHxS and PFOS belong to the PFAS (per- and polyfluoroalkyl substances) group, which is a large and diverse group of man-made compounds used in e.g. textiles and fire-fighting foam. Embryos were produced in vitro using oocytes obtained from abattoir ovaries. Oocytes were matured for 45 hours, with the addition of PFHxS (40 µg/mL) and PFOS (0.1 µg/mL), respectively. Oocytes were then fertilized, and presumed zygotes were cultured. Cleavage rate was recorded 48 hours post fertilization. Developmental stages and grades, as well as number, of resulting blastocysts were recorded for each group on day 5 and 6 post fertilization, respectively. Blastocysts were then collected, fixated, and stained using a TUNEL assay to detect nuclei of apoptotic cells. Positive and negative controls were included to evaluate the TUNEL assay. Stained blastocysts were evaluated using confocal microscopy followed by computerized image analysis. Development parameters and proportion of apoptotic cells were analysed statistically. The treatments had no significant effect on cleavage rate, cleavage rate above 2 cells, developmental stage, or grade. PFOS had no effect on blastocyst rate on day 5 or 6 post fertilization. PFHxS tended to result in higher blastocyst rate on day 5 post fertilization (p=0.07) and resulted in higher blastocyst rate on day 6 post fertilization (p=0.05). Blastocysts with higher total number of nuclei contained a significantly lower proportion of apoptotic nuclei, compared with blastocysts containing lower total numbers of nuclei (p=0.01). PFHxS-treatment tended to result in larger total numbers of nuclei (p=0.062). Furthermore, blastocysts in the PFHxS group tended to contain higher proportions of apoptotic nuclei than blastocysts in the control group (p=0.084). PFOS did not have any effect on any of these parameters. This study shows that PFHxS likely interferes with some parameters of early embryo development in porcine embryos, although not all results obtained were statistically significant. The concentration of PFOS used in this study (0.1 µg/mL) had no effect on any parameter, although it has been showed to have adverse effects on reproduction in several earlier studies. More studies are needed to investigate the effects of PFAS on apoptosis and early embryo development. Furthermore, the effect of PFAS on continued embryonic and foetal development, as well as on live offspring, also needs to be investigated.