Synthetic endocrine disrupting chemicals (EDCs), omnipresent in food, household, and personal care products, have been implicated in adverse trends in human reproduction, including infertility and increasing demand for assisted reproduction. Here, we study the action of 96 ubiquitous EDCs on human sperm. We show that structurally diverse EDCs activate the sperm‐specific CatSper channel and, thereby, evoke an intracellular Ca 2+ increase, a motility response, and acrosomal exocytosis. Moreover, EDCs desensitize sperm for physiological CatSper ligands and cooperate in low‐dose mixtures to elevate Ca 2+ levels in sperm. We conclude that EDCs interfere with various sperm functions and, thereby, might impair human fertilization.

A plethora of endocrine disrupting chemicals (EDCs)—omnipresent in food, household, and personal care products—interfere with various human sperm functions and thus might impair fertilization.

Introduction In mammalian sperm, CatSper represents the principal Ca2+ channel, controlling intracellular Ca2+ concentration ([Ca2+] i ) and motility 1234. Male mice lacking CatSper are infertile, because CatSper−/− sperm fail to undergo rheotaxis and hyperactivation 15. Mutations in CatSper genes have been correlated with male infertility 67. In human sperm, progesterone and prostaglandins, two hormones released into the oviduct 8, directly activate CatSper 29. Progesterone‐ and prostaglandin‐induced Ca2+ influx has been suggested to control sperm capacitation, chemotaxis, hyperactivation, and acrosomal exocytosis 101112. In fact, human CatSper serves as a polymodal chemosensor that harbors promiscuous binding sites for structurally diverse ligands: In vitro, CatSper is directly activated by hydrophobic agents like synthetic odorants that can mimic the action of female ligands 13. Moreover, CatSper is also activated by p,p′‐DDE, a metabolite of dichlorodiphenyltrichloroethane (DDT) 14. These observations indicate that EDCs in reproductive fluids might commonly interfere with human sperm function. EDCs mimic the action of hormones and affect their production or metabolism. EDCs have been linked to decreasing fertility rates in the Western world 1516, testis cancer, and widespread infertility 16171819 (see also Sharpe RM, 2012; DOI 10.1038/embor.2012.50). However, due to the lack of appropriate human models, the actions of EDCs are debated. Here, we systematically study the action on human sperm of ubiquitous EDCs, including biocides, plasticizers, components of personal care products, surfactants, pharmaceuticals, phytoestrogens, and polychlorinated biphenyls (Fig 1A, Supplementary Table S1). We show by Ca2+ fluorimetry, patch‐clamp recordings, and motility analysis that structurally diverse EDCs, at concentrations present in human body fluids, directly activate CatSper and, thereby, interfere with various sperm functions. Our findings substantiate common concerns regarding the negative impact of EDCs on male reproductive health and should be considered for future regulations toward a more restrictive use of EDCs. Figure 1.Structurally diverse EDCs directly activate CatSper in human sperm Composition of the compound library comprising 96 EDCs. Bisphenol A (BPA) does not evoke Ca2+ signals in human sperm. ΔF/F 0 (%) indicates the percentage change in fluorescence (ΔF) with respect to the mean basal fluorescence (F 0 ) before the application of buffer, progesterone (2 μM) or chemicals. 4‐Methylbenzylidene camphor (4‐MBC)‐evoked Ca2+ signals in human sperm; progesterone = 2 μM. Mean (n = 4–6) Ca2+ signal amplitudes evoked by EDCs at 0.1, 1, and 10 μM (blue), compared to signal amplitudes evoked by progesterone (2 μM, red) and buffer (black). Shaded area: buffer ± 3 SD; EDCs that evoked amplitudes > mean buffer + 3 SD were defined as ‘active’. 4‐MBC‐induced Ca2+ signals (10 μM) in the absence (buffer) and presence of the CatSper inhibitor MDL12330A (100 μM). Triclosan (TCS)‐induced Ca2+ signals (10 μM) in the absence (buffer) and presence of the CatSper inhibitor MDL12330A (100 μM). Relative inhibition of EDC (3‐30 μM)‐ and progesterone (2 μM)‐induced Ca2+ signals by MDL12330A (100 μM) [TCS, 4‐octylphenol (4‐OP), benzophenone‐3 (BP‐3), 4,4′‐DDT, n‐nonylparaben (n‐NP), 4‐MBC: n = 3; progesterone, 3‐benzylidene camphor (3‐BC), α‐zearalenol, di‐n‐butyl phthalate (DnBP), homosalate (HMS), padimate O (OD‐PABA): n = 4]. 4‐MBC (10 μM) reversibly enhanced monovalent whole‐cell CatSper currents (NaDVF + 4‐MBC) in human sperm, recorded in Na+‐based divalent‐free solution (NaDVF), in the absence of intracellular divalent ions. Voltage was stepped from 0 ± 80 mV in increments of 10 mV. HS: currents recorded in the presence of extracellular Ca2+ and Mg2+ . Current‐voltage relation of currents shown in (H). Increase in monovalent CatSper currents at −60 mV evoked by DnBP (100 μM; n = 4), 4‐MBC (10 μM; n = 9), TCS (10 μM; n = 4), and progesterone (2 μM; n = 6). Data information: All values are given as mean ± SD. Data information: All values are given as mean ± SD.

Discussion The EDC action on human CatSper could affect fertilization in several ways: Changes in [Ca2+] i control sperm navigation across the female genital tract, hyperactivation, and acrosomal exocytosis 533343536. Various physical and chemical cues provided across the oviduct assist sperm to coordinate these functions. EDCs in reproductive fluids might disturb the precisely coordinated sequence of events underlying fertilization: EDCs could evoke motility responses and acrosome reaction at the wrong time and wrong place; moreover, desensitization of sperm for female factors might hamper navigation toward the egg and penetration of its vestments. More data concerning EDC concentrations in seminal and oviductal fluids are required to strengthen and extend these conclusions. Of note, EDC action on sperm might be even more complex: Besides those EDCs that activate CatSper, other EDCs might inhibit rather than activate the channel. Like the action of progesterone and prostaglandins 9, the EDC action on CatSper also seems to be specific for humans: In mouse sperm, CatSper‐mediated Ca2+ signals were evoked by an alkaline/depolarizing medium (K8.6) 37 or the cGMP derivative 8‐Br‐cGMP 113 (Supplementary Fig S5). In contrast, α‐zearalenol, 4‐MBC, n‐NP, DnBP, and 4,4′‐DDT did not evoke Ca2+ responses (Supplementary Fig S5), demonstrating that mice are not a suitable model to study the EDC action on sperm and fertility. The no‐observed‐adverse‐effect‐level (NOAEL) standard declares safety thresholds for individual EDCs. Our finding that EDCs cooperatively elevate Ca2+ levels challenges the validity of this standard procedure. To understand the action of EDC mixtures in mechanistic terms, it needs to be studied whether EDCs act additively or even synergistically. Here, we provide a direct link between exposure to EDCs and potential adverse effects on fertilization in humans. About 800 omnipresent man‐made chemicals are suspected to interfere with the endocrine system. To this day, the majority of these potential EDCs have not been evaluated for their action in humans 1619. This deficit has been largely due to the lack of suitable models or procedures to systematically test large numbers of chemicals. Here, we introduce a medium‐throughput assay that allows the rapid test of hundreds to thousands of chemicals for their potential to interfere with human sperm function. We trust that this new tool will greatly facilitate evaluating these chemicals with respect to their threat for human reproduction.

Materials and Methods Detailed Material and Methods are provided in the Supplementary Materials and Methods. Human and mouse sperm were prepared as described 238; changes in [Ca2+] i and pH i were measured in human sperm loaded with the Ca2+ indicator Fluo‐4 and the pH i indicator BCECF, respectively, in 384‐microtiter plates in a fluorescence plate reader (Fluostar Omega, BMG Labtech, Germany) at 30°C 213. Mouse sperm were loaded with the Ca2+ indicator Cal‐520 (5 μM) (ATT Bioquest, USA) and changes in [Ca2+] i were measured in a rapid‐mixing device (SFM‐400; Biologic, France) in the stopped‐flow mode 38. Whole‐cell recordings were performed as described 213. Seals between pipette and sperm were formed at the cytoplasmic droplet or the neck region. Monovalent currents were recorded in HS solution, containing Ca2+ and Mg2+, and in Na+‐based divalent‐free (NaDVF) bath solution. The pipette (10–15 MΩ) solution contained (in mM): 130 Cs‐aspartate, 50 HEPES, 5 EGTA, and 5 CsCl adjusted to pH 7.3 with CsOH. The osmolarity of intra‐ and extracellular solutions was ~320 mOsm. For motility experiments, the flagellar beat of head‐tethered sperm was recorded under an inverted microscope at 37°C. Flagellar beat asymmetry and frequency were analyzed by MATLAB (Mathworks, Germany). Acrosomal exocytosis was assessed by PNA‐FITC staining. Data are given as mean ± standard deviation (SD); n = number of experiments.

Acknowledgements We thank N. Kotzur, B.V. Hansen, O. Nielsen, M. Simonsen, and M. Krause for their help with the evaluation and handling of sperm samples and chemicals. We thank H. Krause for preparing the manuscript. We thank C. Lingle for helpful discussions and comments on the manuscript.

Author contributions NES and TS conceived the project. CS, AM, DLE, LA, CB, KA, AR, HF, BW, MB, DW, and TS designed and performed experiments. AM and TS wrote the manuscript. All authors revised and edited the manuscript.

Funding This work was supported by the German Research Foundation (SFB645), the Danish National Advanced Technology Foundation (005‐2010‐3 and 14‐2013‐4), and the Danish Environmental Protection Agency through the ‘Centre on Endocrine Disruptors’.