Non-target screening of per- and polyfluoroalkyl substances in follicular fluid and their blood-follicle transfer

Per- and polyfluoroalkyl substances (PFAS) have received global attention as an important group of industrial additives. They had been widely used in various commercial and industrial products resulted in ubiquitous distribution in the environment and frequent exposure to wildlife and humans. During recent decades, efforts have been made globally to eliminate legacy PFAS, such as perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS), due to their persistence, bioaccumulation, and toxicity. Other than the well-known PFOA and PFOS, an increasing list of emerging PFAS has been applied, but received insufficient investigation on their sources, environmental distributions and health risks. Thus, identification and characterization of emerging PFAS become critical to PFAS research. Follicular fluid is an important microenvironment for oocyte development and has an important impact on reproductive health. PFAS may penetrate the blood-follicular fluid barrier and enter the follicular fluid and result in potential adverse effects on reproductive health. In the present study, we screened for emerging PFAS in follicular fluid based on non-target analysis with liquid chromatography-high-resolution mass spectrometer. Follicular fluid samples from 99 Chinese women seeking in vitro fertilization-embryo transfer treatment were analysed. The workflow for non-target screening of PFAS mainly includes chromatographic peak picking, blank feature filtering, automatic PFAS annotation, confidence level annotation and manual review. The PFAS and confidence level annotations were mainly based on the following evidences: Exact precursor ion masses, characteristic fragment ion masses, homologous series, retention time, mass defect values. After applying the workflow, a total of 26 PFAS were identified in pooled follicular fluid samples, among which 17 were PFAS with reference standards and nine were emerging PFAS including two chlorinated chlorinated polyfluoroalkyl ether sulfonic acids (Cl-PFESAs), and seven hydrosubstituted polyfluoroalkyl carboxylic acids (H-PFCAs). The two novel Cl-PFESAs, 4:2 Cl-PFESAs and 5:2 Cl-PFESA had detection frequencies higher than 90%, and their semi-quantification concentrations were comparable to those of 8:2 Cl-PFESA. Four H-PFCAs (H-PFHpA, H-PFOA, H-PFNA and H-PFUdA) had detection frequencies higher than 50%, and they all exhibited low semi-quantification concentrations. There is limited information on the sources and commercial use of these emerging PFASs. We speculate that the two Cl-PFESAs and H-PFCAs may originate from the transformation of Cl-PFESA congeners and PFCAs, in the environment or in organisms, respectively. However, these need to be confirmed by further research. Blood-follicular fluid transfer efficiency (BFTE) of these emerging PFAS was also determined. The median BFTE of 4:2 Cl-PFESAs and 5:2 Cl-PFESA were 0.86 and 0.92, respectively, which were close to the BFTE of 6:2 Cl-PFESA (0.92), indicating substantial trend in crossing the blood-follicle barrier. The BFTE of H-PFCAs (1.01 for H-PFHpA, 0.96 for H-PFOA, 1.02 for H-PFNA, 0.76 for H-PFUdA and 0.56 for H-PFDoA) exhibited a significant decreasing trend along with the increase of carbon chains, which was consistent with the pattern of BFTE of PFCAs of the same population. Our data lay a solid ground for further screening of emerging PFAS and exploration of their potential impact on reproductive health.