Bisphenol A (BPA), a plasticizer and endocrine disruptor, has been substituted by bisphenol S (BPS), a structural analogue that had already shown adverse effects on granulosa cell steroidogenesis. The objective of this study was to assess the effect of chronic exposure to BPS, a possible endocrine disruptor, on steroid hormones in the ovary, oviduct and plasma using the ewe as a model. Given the interaction between steroidogenesis and the metabolic status, the BPS effect was tested according to two diet groups. Eighty adult ewes were allotted to restricted (R) and well-fed (WF) groups, that were further subdivided into two subgroups. Ewes were exposed to 50 µg BPS/kg/day in their diet (R50 and WF50 groups) or were unexposed controls (R0 and WF0 groups). After at least 3 months of BPS exposure, preovulatory follicular fluid, oviduct fluid and plasma were collected and steroid hormones were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). A deleterious effect of restricted diet on the volume of oviduct fluid and numbers of pre-ovulatory follicles was observed. Exposure to BPS impaired estradiol concentrations in both follicular and oviduct fluids of well-fed ewes and progesterone, estradiol and estrone concentrations in plasma of restricted ewes. In addition, a significant interaction between metabolic status and BPS exposure was observed for seven steroids, including estradiol. In conclusion, BPS acts in ewes as an endocrine disruptor with differential actions according to metabolic status.

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STUDY QUESTION Do plastic laboratory consumables and cell culture media used in ART contain bisphenols? SUMMARY ANSWER The majority of human embryo culture media assessed contained bisphenol S close to the nanomolar concentration range, while no release of bisphenols by plastic consumables was detected under routine conditions. WHAT IS KNOWN ALREADY The deleterious effect of the endocrine disruptor bisphenol A (BPA) on female fertility raised concerns regarding ART outcome. BPA was detected neither in media nor in the majority of plastic consumables used in ART; however, it might have already been replaced by its structural analogs, including bisphenol S (BPS). STUDY DESIGN, SIZE, DURATION Seventeen plastic consumables and 18 cell culture and ART media were assessed for the presence of bisphenols. PARTICIPANTS/MATERIALS, SETTING, METHODS Ten different bisphenols (bisphenol A, S, AF, AP, B, C, E, F, P and Z) were measured using an isotopic dilution according to an on-line solid phase extraction/liquid chromatography/mass spectrometry method. MAIN RESULTS AND THE ROLE OF CHANCE While the plastic consumables did not release bisphenols under routine conditions, 16 of the 18 cell culture and ART media assessed contained BPS. Six media exhibited BPS concentrations higher than 1 nM and reached up to 6.7 nM (1693 ng/l). LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Further studies are required to investigate a greater number of ART media to identify less potentially harmful ones, in terms of bisphenol content. WIDER IMPLICATIONS OF THE FINDINGS As BPS has already been reported to impair oocyte quality at nanomolar concentrations, its presence in ART media, at a similar concentration range, could contribute to a decrease in the ART success rate. Thus far, there has been no regulation of these compounds in the ART context. STUDY FUNDING/COMPETING INTERESTS This study was financially supported by the ‘Centre-Val de Loire’ Region (Bemol project, APR IR 2017), INRAE, BRGM, the French National Research Agency (project ANR-18-CE34-0011-01 MAMBO) and the BioMedicine Agency (Project 18AMP006 FertiPhenol). The authors declare that they have no conflict of interest that could be perceived as prejudicing the impartiality of the reported research.

Background Ovarian granulosa cells (GC) are essential for the development and maturation of a proper oocyte. GC are sensitive to endocrine disruptors, including bisphenol A (BPA) and its analogue bisphenol S (BPS), plasticisers present in everyday consumer products. BPA exhibits greater binding affinity for the membrane oestrogen receptor (GPER) than for the nuclear oestrogen receptors (ERα and ERβ). Here, we analysed the effects of BPA and BPS on the steroidogenesis of ovine GC in vitro, as well as their early mechanisms of action, the ovine being a relevant model to study human reproductive impairment. Disruption of GC steroidogenesis might alter oocyte quality and consequently fertility rate. In addition, we compared the effects of a specific GPER agonist (G-1) and antagonist (G-15) to those of BPA and BPS. Ewe GC were cultured with BPA or BPS (10 or 50 µM) or G-1 (1 µM) and/or G-15 (10 µM) for 48 h to study steroidogenesis. Results Both BPA and BPS (10 µM) altered the secretion of progesterone, however, only BPS (10 µM) affected oestradiol secretion. RNA-seq was performed on GC after 1 h of culture with BPA or BPS (50 µM) or G-1 (10 µM), followed by real-time PCR analyses of differentially expressed genes after 12, 24 and 48 h of culture. The absence of induced GPER target genes showed that BPA and BPS did not activate GPER in GC after 1 h of treatment. These molecules exhibited mainly independent early mechanisms of action. Gene ontology analysis showed that after 1 h of treatment, BPA mainly disrupted the expression of the genes involved in metabolism and transcription, while BPS had a smaller effect and impaired cellular communications. BPA had a transient effect on the expression of CHAC1 (NOTCH signalling and oxidative balance), JUN (linked to MAPK pathway), NR4A1 (oestradiol secretion inhibition), ARRDC4 (endocytose of GPCR) and KLF10 (cell growth, differentiation and apoptosis), while expression changes were maintained over time for the genes LSMEM1 (linked to MAPK pathway), TXNIP (oxidative stress) and LIF (cell cycle regulation) after 12 and 48 h, respectively. Conclusion In conclusion, although they exhibited similar effects, BPA and BPS impaired different molecular pathways in GC in vitro. New investigations will be necessary to follow the temporal changes of these genes over time, as well as the biological processes involved.

Bisphenol S (BPS) affects terminal folliculogenesis by impairing steroidogenesis in granulosa cells from different species. Nevertheless, limited data are available on its effects during basal folliculogenesis. In this study, we evaluate in vitro the effects of a long-term BPS exposure on a model of basal follicular development in a mono-ovulatory species. We cultured ovine preantral follicles (180–240 μm, n = 168) with BPS (0.1 μM (possible human exposure dose) or 10 μM (high dose)) and monitored antrum appearance and follicular survival and growth for 15 days. We measured hormonal secretions (oestradiol (at day 13 [D13]), progesterone and anti-Müllerian hormone [D15]) and expression of key follicular development and redox status genes (D15) in medium and whole follicles, respectively. BPS (0.1 µM) decreased oestradiol secretion compared with the control (−48.8%, p < 0.001), without significantly impairing antrum appearance, follicular survival and growth, anti-Müllerian hormone and progesterone secretion and target gene expression. Thus, BPS could also impair oestradiol secretion during basal folliculogenesis as it is the case during terminal folliculogenesis. It questions the use of BPS as a safe BPA substitute in the human environment. More studies are required to elucidate mechanisms of action of BPS and its effects throughout basal follicular development.

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Study question Do plastic laboratory consumables and cell culture media used in human ART contain bisphenols? Summary answer Human embryo development media contained bisphenols close to the nanomolar concentration range while no release of bisphenols by plastic consumables was detected under routine conditions. What is known already The deleterious effect of the endocrine disruptor bisphenol A (BPA) on female fertility raised concerns regarding ART outcome. BPA was detected neither in media nor in the majority of plastic consumables used in ART, however it might have already been replaced by its structural analogs, including bisphenol S (BPS). Study design, size, duration Seventeen plastic consumables and 18 cell culture and ART media were assessed for the presence of bisphenols. Participants/materials, setting, methods Ten different bisphenols (bisphenol A, S, AF, AP, B, C, E, F, P, and Z) were measured using an isotopic dilution according to an on-line solid phase extraction / liquid chromatography/mass spectrometry method. Main results and the role of chance While all the plastic consumables of this study did contain bisphenols, none of them did release bisphenols under routine conditions. Moreover, 16 of the 18 cell culture and ART media assessed contained bisphenols, including 8 among the 10 media used in human ART. Five human ART media exhibited bisphenol concentrations higher than 0.8 nM and reached up to 3.2 nM (799 ng/L). Limitations, reasons for caution Further studies are required to investigate a greater number of ART media to identify less potentially harmful ones, in terms of bisphenol content. Wider implications of the findings: As BPS has already been reported to impair oocyte quality at nanomolar concentrations, its presence in ART media, at a similar concentration range, could contribute to a decrease in the ART success rate. Thus far, there has been no regulation of these compounds in the ART context. Trial registration number Not applicable

Study question Do plastic laboratory consumables and cell culture media used in human ART contain bisphenols? Summary answer Human embryo development media contained bisphenols close to the nanomolar concentration range while no release of bisphenols by plastic consumables was detected under routine conditions. What is known already The deleterious effect of the endocrine disruptor bisphenol A (BPA) on female fertility raised concerns regarding ART outcome. BPA was detected neither in media nor in the majority of plastic consumables used in ART, however it might have already been replaced by its structural analogs, including bisphenol S (BPS). Study design, size, duration Seventeen plastic consumables and 18 cell culture and ART media were assessed for the presence of bisphenols. Participants/materials, setting, methods Ten different bisphenols (bisphenol A, S, AF, AP, B, C, E, F, P, and Z) were measured using an isotopic dilution according to an on-line solid phase extraction / liquid chromatography/mass spectrometry method. Main results and the role of chance While all the plastic consumables of this study did contain bisphenols, none of them did release bisphenols under routine conditions. Moreover, 16 of the 18 cell culture and ART media assessed contained bisphenols, including 8 among the 10 media used in human ART. Five human ART media exhibited bisphenol concentrations higher than 0.8 nM and reached up to 3.2 nM (799 ng/L). Limitations, reasons for caution Further studies are required to investigate a greater number of ART media to identify less potentially harmful ones, in terms of bisphenol content. Wider implications of the findings As BPS has already been reported to impair oocyte quality at nanomolar concentrations, its presence in ART media, at a similar concentration range, could contribute to a decrease in the ART success rate. Thus far, there has been no regulation of these compounds in the ART context. Trial registration number Not applicable