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Male contraceptive options and infertility treatments are limited. To help overcome these limitations we established a small molecule library as a toolbox for assay development and screening campaigns using human spermatozoa. We have profiled all compounds in our automated high-throughput screening platform and provide the dataset, which allows direct side-by-side comparisons of compound effects and assays using live human spermatozoa.

Despite recent advances in male reproductive health research, there remain many elements of male infertility where our understanding is incomplete. Consequently, diagnostic tools and treatments for men with sperm dysfunction, other than medically assisted reproduction, are limited. On the other hand, the gaps in our knowledge of the mechanisms which underpin sperm function have hampered the development of male non-hormonal contraceptives. The study of mature spermatozoa is inherently difficult. They are a unique and highly specialised cell type which does not actively transcribe or translate proteins and cannot be cultured for long periods of time or matured in vitro. One large-scale approach to both increasing the understanding of sperm function and the discovery and development of compounds that can modulate sperm function is to directly observe responses to compounds with phenotypic screening techniques. These target agnostic approaches can be developed into high-throughput screening platforms with the potential to drastically increase advances in the field. Here, we discuss the rationale and development of high-throughput phenotypic screening platforms for mature human spermatozoa and the multiple potential applications these present, as well as the current limitations and leaps in our understanding and the capabilities needed to overcome them. Further development and use of these technologies could lead to the identification of compounds which positively or negatively affect sperm cell motility or function or novel platforms for toxicology or environmental chemical testing among other applications. Ultimately, each of these potential applications is also likely to increase the understanding within the field of sperm biology.

There is a need for non-hormonal contraceptives. One area that needs further investigation is the development of male contraceptives. Comparatively little is understood about potential drug targets in men to achieve a reversible contraceptive effect. In this article, we review the need for male contraceptives and some thoughts around the characteristics of a male contraceptive and the potential development pathway. We then discuss different potential approaches to discovering male contraceptives and then highlight potential targets that have been discussed in the literature.

STUDY QUESTION Can a high-throughput screening (HTS) platform facilitate male fertility drug discovery? SUMMARY ANSWER An HTS platform identified a large number of compounds that enhanced sperm motility. WHAT IS KNOWN ALREADY Several efforts to find small molecules modulating sperm function have been performed but none have used high-throughput technology. STUDY DESIGN, SIZE, DURATION Healthy donor semen samples were used and samples were pooled (3–5 donors per pool). Primary screening was performed singly; dose–response screening was performed in duplicate (using independent donor pools). PARTICIPANTS/MATERIALS, SETTING, METHODS Spermatozoa isolated from healthy donors were prepared by density gradient centrifugation and incubated in 384-well plates with compounds (6.25 μM) to identify those compounds with enhancing effects on motility. Approximately 17 000 compounds from the libraries, ReFRAME, Prestwick, Tocris, LOPAC, CLOUD and MMV Pathogen Box, were screened. Dose–response experiments of screening hits were performed to confirm the enhancing effect on sperm motility. Experiments were performed in a university setting. MAIN RESULTS AND THE ROLE OF CHANCE From our primary single concentration screening, 105 compounds elicited an enhancing effect on sperm motility compared to dimethylsulphoxide-treated wells. Confirmed enhancing compounds were grouped based on their annotated targets/target classes. A major target class, phosphodiesterase inhibitors, were identified, in particular PDE10A inhibitors as well as number of compounds not previously known to enhance human sperm motility, such as those related to GABA signalling. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Although this approach provides data about the activity of the compound, it is only a starting point. For example, further substantive experiments are necessary to provide a more comprehensive picture of each compound’s activity, the effect on the kinetics of the cell populations and subpopulations, and their potential mechanisms of action. Compounds have been tested with prepared donor spermatozoa, incubated under non-capacitating conditions, and only incubated with compounds for a relatively short period of time. Therefore, the effect of compounds under different conditions, for example in whole semen, for longer incubation times, or using samples from patient groups, may be different and require further study. All experiments were performed in vitro. WIDER IMPLICATIONS OF THE FINDINGS This phenotypic screening assay identified a large number of compounds that increased sperm motility. In addition to furthering our understanding of human sperm function, for example identifying new avenues for discovery, we highlight potential compounds as promising start-point for a medicinal chemistry programme for potential enhancement of male fertility. Moreover, with disclosure of the results of screening, we present a substantial resource to inform further work in the field. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Bill and Melinda Gates Foundation, Scottish Funding Council and Scottish Universities Life Science Alliance. C.L.R.B. is Editor for RBMO. C.L.R.B. receives funding from Chief Scientists Office (Scotland), ESHRE and Genus PLC, consulting fees from Exscientia and lecture fees from Cooper Surgical and Ferring. S.M.d.S. is an Associate Editor of Human Reproduction, and an Associate Editor of Reproduction and Fertility. S.M.d.S. receives funding from Cooper Surgical and British Dietetic Society. No other authors declared a COI.

Aberrant gene expression during gametogenesis is one of the factors underlying infertility, which affects roughly 15% of couples worldwide.Deleted-in-Azoospermia-Like (DAZL),a member of theDAZ-genefamily, encodes an mRNA-specific regulator of translation which is essential for gametogenesis in both sexes. In this study we show that DAZL controls gene expression in oocytes by regulating the length of the mRNA poly(A) tail, a major determinant of temporal and amplitudinal gene regulation in germ cells, in which gene expression is regulated entirely post-transcriptionally. We show that DAZL does not induce polyadenylation but that binding of DAZL efficiently inhibits mRNA deadenylation induced by oocyte maturation. We reveal that this activity depends on DAZL-mediated recruitment of poly(A)-binding protein, PABP, to the mRNA. Although DAZL also activates mRNA translation via PABP recruitment, mechanistic analysis revealed that neither translation nor translational activation are required for DAZL to stabilise the poly(A) tail, suggesting two mutually independent posttranscriptional roles for the DAZL-PABP complex. We show that recruited PABP must maintain its ability to bind RNA, leading to a model in which DAZL recruits PABP and/or stabilises PABP binding to poly(A) thereby preventing access of deadenylases. These results indicate that the role of DAZL in regulating germ-cell mRNA fate is more complex than previously thought and inform on the poorly understood links between mRNA translation and deadenylation, showing that they can be mechanistically separable.

There is an urgent need to develop new methods for male contraception, however a major barrier to drug discovery has been the lack of validated targets and the absence of an effective high-throughput phenotypic screening system. To address this deficit, we developed a fully-automated robotic screening platform that provided quantitative evaluation of compound activity against two key attributes of human sperm function: motility and acrosome reaction. In order to accelerate contraceptive development, we screened the comprehensive collection of 12,000 molecules that make up the ReFRAME repurposing library, comprising nearly all the small molecules that have been approved or have undergone clinical development, or have significant preclinical profiling. We identified several compounds that potently inhibit motility representing either novel drug candidates or routes to target identification. This platform will now allow for major drug discovery programmes that address the critical gap in the contraceptive portfolio as well as uncover novel human sperm biology.

Masculinization of the external genitalia in humans is dependent on formation of 5α-dihydrotestosterone (DHT) through both the canonical androgenic pathway and an alternative (backdoor) pathway. The fetal testes are essential for canonical androgen production but little is known about the synthesis of backdoor androgens despite their known critical role in masculinization. In this study, we have measured plasma and tissue levels of endogenous steroids in second trimester human male fetuses using multi-dimensional and high-resolution mass-spectrometry. Results show that androsterone is the principal backdoor androgen in the fetal circulation and that DHT is undetectable (

Background Human fetal adrenal glands are highly active and, with the placenta, regulate circulating progesterone, estrogen and corticosteroids in the fetus. At birth the adrenals are essential for neonate salt retention through secretion of aldosterone, while adequate glucocorticoids are required to prevent adrenal insufficiency. The objective of this study was to carry out the first comprehensive analysis of adrenal steroid levels and steroidogenic enzyme expression in normal second trimester human fetuses. Methods This was an observational study of steroids, messenger RNA transcripts and proteins in adrenals from up to 109 second trimester fetuses (11 weeks to 21 weeks) at the Universities of Aberdeen and Glasgow. The study design was balanced to show effects of maternal smoking. Results Concentrations of 19 intra-adrenal steroids were quantified using liquid chromatography and mass spectrometry. Pregnenolone was the most abundant steroid while levels of 17α-hydroxyprogesterone, dehydroepiandrosterone sulphate (DHEAS) and progesterone were also high. Cortisol was present in all adrenals, but aldosterone was undetected and Δ4 androgens were low/undetected. CYP17A1, CYP21A2 and CYP11A1 were all highly expressed and the proteins localized to the adrenal fetal zone. There was low-level expression of HSD3B and CYP11B2, with HSD3B located mainly in the definitive zone. Maternal smoking altered fetal plasma adrenocorticotropic hormone (ACTH) (P = 0.052) and intra-adrenal progesterone, 17α-hydroxyprogesterone and 16α-hydroxyprogesterone, but not plasma or intra-adrenal cortisol, or intra-adrenal DHEAS. Fetal adrenal GATA6 and NR5A1 were increased by maternal smoking. Conclusions The human fetal adrenal gland produces cortisol but very low levels of Δ4 androgens and no detectable aldosterone throughout the second trimester. The presence of cortisol in fetal adrenals suggests that adrenal regulation of circulating fetal ACTH remains a factor in development of congenital adrenal hyperplasia during the second trimester, while a relative lack of aldosterone explains the salt-wasting disorders frequently seen in extreme pre-term neonates. Finally, maternal smoking may alter fetal adrenal sensitivity to ACTH, which could have knock-on effects on post-natal health. Electronic supplementary material The online version of this article (10.1186/s12916-018-1009-7) contains supplementary material, which is available to authorized users.

Masculinization of the external genitalia in humans is dependent on formation of 5α-dihydrotestosterone (DHT) through both the canonical androgenic pathway and an alternative (backdoor) pathway. The fetal testes are essential for canonical androgen production, but little is known about the synthesis of backdoor androgens, despite their known critical role in masculinization. In this study, we have measured plasma and tissue levels of endogenous steroids in second trimester human fetuses using multidimensional and high-resolution mass spectrometry. Results show that androsterone is the principal backdoor androgen in the male fetal circulation and that DHT is undetectable (, Fetal human masculinisation depends on testosterone production by the testes and an alternative “backdoor” androgen. This study shows that this androgen is likely to be androsterone, which is sexually dimorphic in the fetus but does not come from the testes; instead, synthesis probably depends on placental substrates., Author summary The human penis starts to develop before birth from a structure called the genital tubercle. This process is dependent on the secretion of testosterone from the fetal testes and subsequent conversion of testosterone into dihydrotestosterone (DHT) by enzymes in the genital tubercle. Recently, an alternative "backdoor" route to the formation of DHT, which does not require testosterone, has also been shown to be essential for normal development of the human penis. In this study we provide evidence indicating that androsterone is the major backdoor androgen involved in human masculinization and that it is produced in nongonadal tissues. Steroid hormone levels were measured in the plasma of second trimester human fetuses, and testosterone and androsterone were the only androgens with higher levels in males than in females. Analysis of tissue steroid levels showed that plasma androsterone did not primarily originate from the testes but, instead, was probably formed in other tissues via metabolism of placental progesterone. These data indicate, therefore, that masculinization of the human fetus depends on steroid hormone secretion from both the testes and the placenta, and would explain why placental dysfunction is associated with disorders of sex development.

Most common male reproductive disorders are linked to lower testosterone exposure in fetal life, although the factors responsible for suppressing fetal testosterone remain largely unknown. Protracted use of acetaminophen during pregnancy is associated with increased risk of cryptorchidism in sons, but effects on fetal testosterone production have not been demonstrated. We used a validated xenograft model to expose human fetal testes to clinically relevant doses and regimens of acetaminophen. Exposure to a therapeutic dose of acetaminophen for 7 days significantly reduced plasma testosterone (45% reduction; P = 0.025) and seminal vesicle weight (a biomarker of androgen exposure; 18% reduction; P = 0.005) in castrate host mice bearing human fetal testis xenografts, whereas acetaminophen exposure for just 1 day did not alter either parameter. Plasma acetaminophen concentrations (at 1 hour after the final dose) in exposed host mice were substantially below those reported in humans after a therapeutic oral dose. Subsequent in utero exposure studies in rats indicated that the acetaminophen-induced reduction in testosterone likely results from reduced expression of key steroidogenic enzymes (Cyp11a1, Cyp17a1). Our results suggest that protracted use of acetaminophen (1 week) may suppress fetal testosterone production, which could have adverse consequences. Further studies are required to establish the dose-response and treatment-duration relationships to delineate the maximum dose and treatment period without this adverse effect.