cellular uptake of polystyrene nanoplastics by oocytes and their impact on subsequent embryo development: a preliminary insight using a bovine in vitro model

Abstract Study question Can polystyrene nanoplastics enter cumulus-enclosed oocytes (CEOs) and consequently alter oocyte quality and embryo development? Is this effect particle size-dependent? Summary answer After 24h in vitro exposure, polystyrene-nanoplastics entered the extracellular matrix and cytoplasm of peripheral cumulus cells, but not oocytes. Preimplantation embryo development was not altered. What is known already The ubiquitous use of plastic resulted in extreme global environmental plastic pollution. Chemical and physical processes contribute to the fragmentation of these macroplastic pollutants into heterogenous nanoplastics (NPs). Plastic intake is inevitable for humans by inhalation and ingestion of contaminated air, food (e.g. plastic ingested fish) and drinks (e.g. leakage of plastic bottles). Once imbibed, particles smaller than 150 µm can spread across the entire human body and reach the blood and lymphatic tissue. Due to the neovascularisation associated with an increased vascular permeability during follicular development, nanoparticles might reach the ovarian follicles, potentially affecting oocyte quality and embryo development. Study design, size, duration In a bottom-up approach, using a bovine in vitro oocyte maturation model, CEOs were exposed for 24h to 100µg/mL plain Nile Red-labelled polystyrene-nanoplastics (PS-NP) with average diameter of 50nm (PS-NP-50) or plain Flash Red-labelled PS-NP with average diameter of 200nm (PS-NP-200) (4 replicates using 714 CEOs). CEOs in NP-free control media (CONT) and in-vehicle control (SOLV) were included. Oocytes were in vitro fertilized (for 20h) and presumptive zygotes were cultured until 8 days post-insemination (p.i.). Participants/materials, setting, methods CEOs were fixed after IVM and examined for cumulus cell (CCs) expansion. The presence of NPs was assessed with laser-scanning Leica SP8 confocal microscope. Cleavage (48h p.i.) and blastocyst rates (8 days p.i.) were recorded together with differential staining of trophectoderm (TE, CDX2) and inner cell mass (ICM, Hoechst). The apoptotic-cell-index of the CCs and blastocysts was examined with Caspase-3 immunostaining. Images were quantified using ImageJ. Data were analysed using binary logistic regression and ANOVA. Main results and the role of chance At 24h of IVM, both PS-NP-50 and PS-NP-200 could be detected in the extracellular matrix surrounding the CCs and even within the cytoplasm of a few CCs in the outer layers of the CEOs. No NPs could be detected within the oocytes. Irrespective of the size of the NP’s, no significant differences in cumulus expansion rate or embryo cleavage rate were observed and no difference in apoptotic-cell-index of the CCs could be demonstrated due to a plastic effect (P > 0.1). However, the blastocyst/cleaved rate of PS-NP-50 (56%) was significantly higher than SOLV (36%, p < 0.05). On top, PS-NP-50 showed a lower apoptotic-cell-index (4.11±0.90) of the TE compared to CONT (7.08±0.99, p < 0.1), SOLV (9.09±1.49, p < 0.05) and PS-NP-200 (7.65±1.09, p < 0.05). There is a complete lack of univocal and sound scientific evidence of the actual amount of human plastic intake, making it difficult to determine a physiologically relevant concentration for testing possible effects. However, despite the high plastic concentration used in our model, no deteriorating effects on oocyte quality and embryo development were proven. Limitations, reasons for caution Exposure for only 24h during IVM resulted in a limited timeframe for uptake, consequently exerting its effects. However, long-term in vitro follicle culture models are not as straightforward. Limited reports give insight into the final destination of NPs inside the body, addressing the necessity for in vivo trials as confirmation. Wider implications of the findings While this study indicate that PS-NPs are not toxic for oocyte developmental competence upon direct exposure, this does not rule out the potential impact on e.g. embryo gene expression patterns and epigenetic programming. Future studies should also focus on indirect effects on the oocyte as PS-NP may alter follicular physiology. Trial registration number not applicable