Epsilon Archive for Student Projects

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.