Effect of perfluorononanoic acid (PFNA) on bovine early embryo development in vitro

Sammanfattning: Perfluoroalkyl and polyfluoroalkyl substances (PFASs) is a large family of highly fluorinated chemicals, used in different commercial products (e.g. in clothing, shampoos, kitchenware and fire-fighting foams) utilizing their different properties, and have been found in wildlife, humans and in the environment. According to several temporal trend studies, PFNA (perfluorononanoic acid) seems to increase in concentration in the general population and in the environment. PFNA is closely related to the more well-known PFOA (perfluorooctanoic acid), a substance with known reproductive toxicity, but studies regarding PFNAs full toxicological potential effects, especially developmental toxicity, are limited. The aim of this study was to examine the early effects of PFNA exposure on oocytes during in vitro maturation. Using an in vitro model, a controlled environment is established where individual chemicals can be tested without the usage of laboratory animals. Regarding oocyte maturation and initial embryo development, the bovine model is a better model for humans, compared to the murine model. In this study a bovine in vitro embryo production model was used. Abattoir-derived bovine ovaries were used to collect cumulus oocyte complexes (n = 440). Half of these were matured in vitro under PFNA exposure (0.1 µg/ml) followed by in vitro fertilization and culture, as for the control group. The used concentration was based on a previous study where PFNA was measured in follicle fluid of women (0.4 (0.2-2.1) ng/ml). A margin of safety and the short time of exposure in this study are the reasons for the raised concentration compared to the measured values in the follicle fluid. Oocyte and embryo development were assessed regarding cumulus expansion, cleavage rate 44h post fertilization and day 7 and 8 blastocyst development and morphology. Day 8 blastocysts were stained with Mitotracker Orange (visualizes active mitochondria) and fixed in paraformaldehyde, followed by additional staining with nuclear stain DRAQ5 and lipid stain LipidTOX. For analysis of the embryos a confocal laser scanning microscope was used. The embryos were evaluated concerning number of nuclei, distribution of mitochondria, lipid droplet morphology and lipid pixel intensity. Statistical analyses were performed by linear mixed models and generalized linear mixed models, with replicate as random factor and observations on day 7 and 8 as repeated measures. A total of 440 oocytes from 8 batches were included to in vitro maturation, resulting in 88 blastocysts. No significant difference between treated and control group was seen regarding cumulus expansion, cleavage rate, blastocyst development day 7 and 8, quality grade of blastocysts, stage of blastocysts, number of nuclei, mitochondrial distribution scoring or mean lipid pixel intensity. However, there was a significant difference in lipid droplet distribution score between treated and control group (p = 0.048), suggesting an increase in blastocyst lipid content due to the PFNA exposure. It is difficult to know exactly what effect this change in lipid expression has on further embryo development and more studies are needed to investigate this.