Bovine oocyte exposure to perfluorohexane sulfonate (PFHxS) induces phenotypic, transcriptomic, and DNA methylation changes in resulting embryos in vitro.

Knowledge on the effects of perfluorohexane sulfonate (PFHxS) on ovarian function is limited. In the current study, we investigated the sensitivity of oocytes to PFHxS during in vitro maturation (IVM), including consequences on embryo development at the morphological, transcriptomic, and epigenomic levels. Bovine cumulus-oocyte complexes (COCs) were exposed to PFHxS during 22 h IVM. Following fertilisation, developmental competence was recorded until day 8 of culture. Two experiments were conducted: 1) exposure of COCs to 0.01 µg mL-1 - 100 µg mL-1 PFHxS followed by confocal imaging to detect neutral lipids and nuclei, and 2) exposure of COCs to 0.1 µg mL-1 PFHxS followed by analysis of transcriptomic and DNA methylation changes in blastocysts. Decreased oocyte developmental competence was observed upon exposure to ≥ 40 µg mL-1 PFHxS and altered lipid distribution was observed in the blastocysts upon exposure to 1-10 µg mL-1 PFHxS (not observed at lower or higher concentrations). Transcriptomic data showed that genes affected by 0.1 µg mL-1 PFHxS were enriched for pathways related to increased synthesis and production of reactive oxygen species. Enrichment for peroxisome proliferator-activated receptor-γ and oestrogen pathways was also observed. Genes linked to DNA methylation changes were enriched for similar pathways. In conclusion, exposure of the bovine oocyte to PFHxS during the narrow window of IVM affected subsequent embryonic development, as reflected by morphological and molecular changes. This suggests that PFHxS interferes with the final nuclear and cytoplasmic maturation of the oocyte leading to decreased developmental competence to blastocyst stage.

Perfluorooctane sulfonate (PFOS) exposure of bovine oocytes affects early embryonic development at human-relevant levels in an in vitro model.

Perfluorooctane sulfonate (PFOS) has been added to Stockholm Convention for global phase out, but will continue to contribute to the chemical burden in humans for a long time to come due to extreme persistence in the environment. In the body, PFOS is transferred into to the ovarian follicular fluid that surrounds the maturing oocyte. In the present study, bovine cumulus oocyte complexes were exposed to PFOS during 22 h in vitro maturation. Concentrations of 2 ng g-1 (PFOS-02) representing average human exposure and 53 ng g-1 (PFOS-53) relevant to highly exposed groups were used. After exposure, developmental competence was recorded until day 8 after fertilisation. Blastocysts were fixed and either stained to evaluate blastomere number and lipid distribution using confocal microscopy or frozen and pooled for microarray-based gene expression and DNA methylation analyses. PFOS-53 delayed the first cleavage to two-cell stage and beyond at 44 h after fertilisation (p < .01). No reduction of proportion blastocysts were seen at day 8 in either of the groups, but PFOS-53 exposure resulted in delayed development into more advanced stages of blastocysts seen as both reduced developmental stage (p = .001) and reduced number of blastomeres (p = .04). Blastocysts showed an altered lipid distribution that was more pronounced after exposure to PFOS-53 (increased total lipid volume, p=.0003, lipid volume/cell p < .0001) than PFOS-02, where only decreased average lipid droplet size (p=.02) was observed. Gene expression analyses revealed pathways differently regulated in the PFOS-treated groups compared to the controls, which were related to cell death and survival through e.g., P38 mitogen-activated protein kinases and signal transducer and activator of transcription 3, which in turn activates tumour protein 53 (TP53). Transcriptomic changes were also associated with metabolic stress response, differentiation and proliferation, which could help to explain the phenotypic changes seen in the blastocysts. The gene expression changes were more pronounced after exposure to PFOS-53 compared to PFOS-02. DNA-methylation changes were associated with similar biological functions as the transcriptomic data, with the most significantly associated pathway being TP53. Collectively, these results reveal that brief PFOS exposure during oocyte maturation alters the early embryo development at concentrations relevant to humans. This study adds to the evidence that PFOS has the potential to affect female fertility.

Suspect and non-target screening of ovarian follicular fluid and serum - identification of anthropogenic chemicals and investigation of their association to fertility.

In this work, ultra-high performance liquid chromatography-high resolution (Orbitrap) mass spectrometry-based suspect and non-target screening was applied to follicular fluid (n = 161) and serum (n = 116) from women undergoing in vitro fertilization in order to identify substances that may be associated with decreased fertility. Detected features were prioritized for identification based on (i) hazard/exposure scores in a database of chemicals on the Swedish market and an in-house database on per- and polyfluoroalkyl substances (PFAS); (ii) enrichment in follicular fluid relative to serum; and (iii) association with treatment outcomes. Non-target screening detected 20 644 features in follicular fluid and 13 740 in serum. Two hundred and sixty-two features accumulated in follicular fluid (follicular fluid: serum ratio >20) and another 252 features were associated with embryo quality. Standards were used to confirm the identities of 21 compounds, including 11 PFAS. 6-Hydroxyindole was associated with lower embryo quality and 4-aminophenol was associated with higher embryo quality. Overall, we show the complexity of follicular fluid and the applicability of suspect and non-target screening for discovering both anthropogenic and endogenous substances, which may play a role in fertility in women.

Effect of an altered hormonal environment by blood plasma collected after adrenocorticotropic administration on embryo development and gene expression in porcine embryos.

Early embryonic development may be affected by adrenal hyperactivity in stressful situations which may lead to endocrine changes in the embryo environment. A sensitive period in porcine embryo development is the 4-cell stage when the embryo genome activation occurs. A mixed in vivo-in vitro system was implemented to test whether an altered milieu around this stage could affect embryo development and blastocyst quality in the porcine model. After in vitro maturation and fertilisation, presumptive zygotes were exposed for 24 h to plasma collected after ovulation from adrenocorticotropic hormone (ACTH)-treated, non-ACTH-treated sows; and, medium without plasma, supplemented with bovine serum albumin. Subsequently, embryo development and differences in gene expression were tested among treatments. Cleavage and blastocyst rates did not differ between treatments. Blastocyst quality by morphology assessment was similar when all the resulting blastocysts were included in the analysis. However, when only expanded blastocysts (and onwards) were included in the analysis, the blastocysts from the non-ACTH plasma group showed better quality score. Blastocyst quality by morphological assessment was not mirrored by the transcription levels of various important genes for embryo development whose gene expression profile did not significantly differ among groups. It is likely that the effect of the altered environment provided by plasma from ACTH-treated sows was too short to affect embryo development. Therefore, a brief exposure to an altered endocrine environment may not have harmful consequences for the embryo once fertilisation occurs.