Exposure of human sperm to progesterone (P4) activates cation channel of sperm (CatSper) channels, inducing an intracellular Ca2+ concentration ([Ca2+]i) transient followed by repetitive [Ca2+]i activity (oscillations), which are believed to be functionally important. We investigated the potential significance of store-operated Ca2+-entry in these oscillations using the inhibitor SKF96365 (30 µM; SKF). Following pre-treatment of human sperm with 3 µM P4, exposure to SKF doubled the proportion of oscillating cells (P = 0.00004). In non-pre-treated cells, SKF had an effect similar to P4, inducing a [Ca2+]i transient in >80% of cells which was followed by oscillations in ≈50% of cells. The CatSper blocker RU1968 (11 µM) inhibited the SKF-induced [Ca2+]i increase and reversibly arrested [Ca2+]i oscillations. Using whole-cell patch clamp, we observed that SKF enhanced CatSper currents by 100% within 30 s, but amplitude then decayed to levels below control over the next minute. When cells were stimulated with P4, CatSper currents were stably increased (by 200%). Application of SKF then returned current amplitude to control level or less. When sperm were prepared in medium lacking bovine serum albumin (BSA), both P4 and SKF induced a [Ca2+]i transient in >95% of cells but the ability of SKF to induce oscillations was greatly reduced (P = 0.0009). We conclude that SKF, similar to a range of small organic molecules, activates CatSper channels, but that a secondary blocking action also occurs, which was detected only during patch-clamp recording. The failure of SKF to induce oscillations when cells were prepared without BSA emphasizes that the drug does not fully mimic the actions of P4.

Background: Progesterone (P4) influences human sperm function by stimulating an elevation of intracellular calcium concentration [Ca2+]i. Assessment of the P4 response in sperm populations indicates an association between poor [Ca2+]i increase and reduced fertilisation capacity. Analysis of P4 sensitivity single donor sperm demonstrates heterogeneity in responsiveness and dynamics of [Ca2+]i increase.

Human spermatozoa interact with a complex biochemical environment in the female reproductive tract en route to the site of fertilisation. Ovarian follicular fluid contributes to this complex milieu and is known to contain steroids such as progesterone, whose effects on sperm physiology have been widely characterised. We have previously reported that progesterone stimulates intracellular calcium concentration ([Ca2+]i) signalling and acrosome reaction in human spermatozoa. To characterise the effects of the unified complete follicular fluid steroid hormone complement on human spermatozoa, a comprehensive, data-based, ‘physiological standard’ steroid hormone balance of follicular fluid (shFF) was created from individual constituents. shFF induced a rapid biphasic [Ca2+]i elevation in human spermatozoa. Using population fluorimetry, we compared [Ca2+]i signal amplitude in cells exposed to serial applications of shFF (6 steps from 10-5X up to 1X shFF) with responses to the equivalent progesterone component alone (6 steps from 135 pM - 13.5μM). Threshold for the response to shFF was right-shifted (≈10-fold) compared to progesterone alone, but the maximum response to shFF was greatly enhanced. An acrosome reaction assay was used to assess functional effects of shFF-induced sperm calcium signalling. shFF as well as progesterone-treated spermatozoa showed a significant increase in % acrosome reaction (P < 0.01). All of this evidence suggests the modulation of progesterone-mediated responses by other follicular fluid steroids.

Lay summary A human sperm must swim to the egg to fertilise it. To do this the sperm uses different types of swimming (behaviours) as they are needed. When we watch sperm swimming we see that they regularly change behaviour, sometimes repeatedly switching between two different types. Calcium ions inside cells are crucial in controlling many cell functions and in sperm they play a key role in regulating their behaviour. Here we have measured the concentration of calcium ions inside swimming human sperm. We found that in 12/35 (34%) of the cells we assessed, the concentration of calcium changed repeatedly, averaging more than one cycle of rise and fall per minute. These changes in the concentration of calcium ions occurred as the sperm switched swimming stroke, suggesting that oscillation of calcium concentration is involved in controlling the switching of sperm behaviour. Impaired sperm motility is an important cause of subfertility in men. Understanding how sperm behaviour is controlled will allow the development of treatments that can rescue the fertility of sperm with impaired motility.

Several strands of evidence indicate the presence of marked similarities between human brain and testis. Understanding these similarities and their implications has become a topic of interest among the scientific community. Indeed, an association of intelligence with some semen quality parameters has been reported and a relation between dysfunctions of the human brain and testis has also been evident. Numerous common molecular features are evident when these tissues are compared, which is reflected in the huge number of common proteins. At the functional level, human neurons and sperm share a number of characteristics, including the importance of the exocytotic process and the presence of similar receptors and signalling pathways. The common proteins are mainly involved in exocytosis, tissue development and neuron/brain-associated biological processes. With this analysis, we conclude that human brain and testis share several biochemical characteristics which, in addition to their involvement in the speciation process, could, at least in part, be responsible for the expression of a huge number of common proteins. Nonetheless, this is an underexplored topic, and the connection between these tissues needs to be clarified, which could help to understand the dysfunctions affecting brain and testis, as well as to develop improved therapeutic strategies.

Background: CatSper is a multi-subunit sperm-specific calcium channel that mediates the progesterone-induced increase in intracellular calcium. Evidence from CatSper subunit knock-out mouse models implicates it as essential for sperm fertilisation competence. Similarly, impaired or absent P4 response in patient sperm is associated with poor or loss of fertilisation at IVF. However, genetic evidence in a limited number of cases reported genomic deletions spanning a number of loci. We reported a unique case (Williams et al., 2014) of a man (patient 1) with sperm that lacked functional CatSper expression but he had no genetic errors in CatSper subunit coding regions. Subsequently, two new proteins have been proposed to be part of the mature complex (CatSper-epsilon and CatSper-zeta).

STUDY QUESTIONHow are progesterone (P4)-induced repetitive intracellular Ca2+ concentration ([Ca2+]i) signals (oscillations) in human sperm generated?SUMMARY ANSWERP4-induced [Ca2+]i oscillations are generated in the flagellum by membrane potential (Vm)-sensitive Ca2+-influx through CatSper channels.WHAT IS KNOWN ALREADYA subset of human sperm display [Ca2+]i oscillations that regulate flagellar beating and acrosome reaction. Although pharmacological manipulations indicate involvement of stored Ca2+ in these oscillations, influx of extracellular Ca2+ is also required.STUDY DESIGN, SIZE, DURATIONThis was a laboratory study that used >20 sperm donors and involved more than 100 separate experiments and analysis of more than 1000 individual cells over a period of 2 years.PARTICIPANTS/MATERIALS, SETTING, METHODSSemen donors and patients were recruited in accordance with local ethics approval from Birmingham University and Tayside ethics committees. [Ca2+]i responses and Vm of individual cells were examined by fluorescence imaging and whole-cell current clamp.MAIN RESULTS AND THE ROLE OF CHANCEP4-induced [Ca2+]i oscillations originated in the flagellum, spreading to the neck and head (latency of 1-2 s). K+-ionophore valinomycin (1 µM) was used to investigate the role of membrane potential (Vm). Direct assessment by whole-cell current-clamp confirmed that Vm in valinomycin-exposed cells was determined primarily by K+ equilibrium potential (EK) and was rapidly 'reset' upon manipulation of [K+]o. Pre-treatment of sperm with valinomycin ([K+]o = 5.4 mM) had no effect on the P4-induced [Ca2+] transient (P = 0.95; eight experiments), but application of valinomycin to P4-pretreated sperm suppressed activity in 82% of oscillating cells (n = 257; P = 5 × 10-55 compared to control) and significantly reduced both the amplitude and frequency of persisting oscillations (P = 0.0001). Upon valinomycin washout, oscillations re-started in most cells. When valinomycin was applied in saline with elevated [K+], the inhibitory effect of valinomycin was reduced and was dependent on EK (P = 10-25). Amplitude and frequency of [Ca2+]i oscillations that persisted in the presence of valinomycin showed similar sensitivity to EK (P < 0.01). The CatSper inhibitor RU1968 (4.8 and 11 µM) caused immediate and reversible arrest of activity in 36% and 96% of oscillating cells, respectively (P < 10-10). Quinidine (300 µM) which blocks the sperm K+ current (IKsper) completely, inhibited [Ca2+]i oscillations.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis was an in-vitro study and caution must be taken when extrapolating these results to in-vivo regulation of sperm.WIDER IMPLICATIONS OF THE FINDINGS[Ca2+]i oscillations in human sperm are functionally important and their absence is associated with failed fertilisation at IVF. The data reported here provide new understanding of the mechanisms that underlie the regulation and generation (or failure) of these oscillations.STUDY FUNDING/COMPETING INTEREST(S)E.T.-N. was in receipt of a postgraduate scholarship from the CAPES Foundation (Ministry of Education, Brazil). E.M-M received travel funds from the Programa de Apoyo a los Estudios de Posgrado (Maestria y Doctorado en Ciencias Bioquimicas-Universidad Autonoma de Mexico). SGB and CLRB are recipients of a Chief Scientist Office (NHS Scotland) grant TCS/17/28. The authors have no conflicts of interest.

In attempting to formulate potential WHO guidelines for the diagnosis of male infertility, the Evidence Synthesis Group noted a paucity of high‐quality data on which to base key recommendations. As a result, a number of authors suggested that key areas of research/evidence gaps should be identified, so that appropriate funding and policy actions could be undertaken to help address key questions.

Abstract Human sperm show a variety of different behaviours (types of motility) that have different functional roles. Previous reports suggest that sperm may reversibly switch between these behaviours. We have recorded and analysed the behaviour of individual human sperm (180 cells in total), each cell monitored continuously for 3–3.5 min either under control conditions or in the presence of Ca2+-mobilising stimuli. Switching between different behaviours was assessed visually (1 s bins using four behaviour categories), and was verified by fractal dimension analysis of sperm head tracks. In the absence of stimuli, ~90% of cells showed at least one behavioural transition (mean rate under control conditions = 6.4 ± 0.8 transitions.min−1). Type 1 behaviour (progressive, activated-like motility) was most common, but the majority of cells (>70%) displayed at least three behaviour types. Treatment of sperm with Ca2+-mobilising agonists had negligible effects on the rate of switching but increased the time spent in type 2 and type 3 (hyperactivation-like) behaviours (P < 2*10−8; chi-square). Treatment with 4-aminopyridine under alkaline conditions (pHo = 8.5), a highly-potent Ca2+-mobilising stimulus, was the most effective in increasing the proportion of type 3 behaviour, biasing switching away from type 1 (P < 0.005) and dramatically extending the duration of type 3 events (P < 10−16). Other stimuli, including 300 nM progesterone and 1% human follicular fluid, had qualitatively similar effects but were less potent. We conclude that human sperm observed in vitro constitutively display a range of behaviours and regulation of motility by [Ca2+]i, at the level of the single cell, is achieved not by causing cells to adopt a ‘new’ behaviour but by changing the relative contributions of those behaviours.

BACKGROUND:Intensive research on sperm ion channels has identified members of several ion channel families in both mouse and human sperm. Gene knock-out studies have unequivocally demonstrated the importance of the calcium and potassium conductances in sperm for fertility. In both species, the calcium current is carried by the highly complex cation channel of sperm (CatSper). In mouse sperm, the potassium current has been conclusively shown to be carried by a channel consisting of the pore forming subunit SLO3 and auxiliary subunit leucine-rich repeat-containing 52 (LRRC52). However, in human sperm it is controversial whether the pore forming subunit of the channel is composed of SLO3 and/or SLO1. Deciphering the role of the proton-specific Hv1 channel is more challenging as it is only expressed in human sperm. However, definitive evidence for a role in, and importance for, human fertility can only be determined through studies using clinical samples. OBJECTIVE AND RATIONALE:This review aims to provide insight into the role of sperm ion channels in human fertilization as evidenced from recent studies of sperm from infertile men. We also summarize the key discoveries from mouse ion channel knock-out models and contrast the properties of mouse and human CatSper and potassium currents. We detail the evidence for, and consequences of, defective ion channels in human sperm and discuss hypotheses to explain how defects arise and why affected sperm have impaired fertilization potential. SEARCH METHODS:Relevant studies were identified using PubMed and were limited to ion channels that have been characterized in mouse and human sperm. Additional notable examples from other species are included as appropriate. OUTCOMES:There are now well-documented fundamental differences between the properties of CatSper and potassium channel currents in mouse and human sperm. However, in both species, sperm lacking either channel cannot fertilize in vivo and CatSper-null sperm also fail to fertilize at IVF. Sperm-lacking potassium currents are capable of fertilizing at IVF, albeit at a much lower rate. However, additional complex and heterogeneous ion channel dysfunction has been reported in sperm from infertile men, the causes of which are unknown. Similarly, the nature of the functional impairment of affected patient sperm remains elusive. There are no reports of studies of Hv1 in human sperm from infertile men. WIDER IMPLICATIONS:Recent studies using sperm from infertile men have given new insight and critical evidence supporting the supposition that calcium and potassium conductances are essential for human fertility. However, it should be highlighted that many fundamental questions remain regarding the nature of molecular and functional defects in sperm with dysfunctional ion channels. The development and application of advanced technologies remains a necessity to progress basic and clinical research in this area, with the aim of providing effective screening methodologies to identify and develop treatments for affected men in order to help prevent failed ART cycles. Conversely, development of drugs that block calcium and/or potassium conductances in sperm is a plausible strategy for producing sperm-specific contraceptives.

The development of stimuli-responsive interfaces between synthetic materials and biological systems is providing the unprecedented ability to modulate biomolecular interactions for a diverse range of biotechnological and biomedical applications. Antibody-antigen binding interactions are at the heart of many biosensing platforms, but no attempts have been made yet to control antibody-antigen binding in an on-demand fashion. Herein, a molecular surface was designed and developed that utilizes an electric potential to drive a conformational change in surface bound peptide moiety, to give on-demand control over antigen-antibody interactions on sensor chips. The molecularly engineered surfaces allow for propagation of conformational changes from the molecular switching unit to a distal progesterone antigen, resulting in promotion (ON state) or inhibition (OFF state) of progesterone antibody binding. The approach presented here can be generally applicable to other antigen-antibody systems and meets the technological needs for in situ long-term assessment of biological processes and disease monitoring on-demand.

STUDY QUESTION: How do the alkaline pH, progesterone and 4-aminopyridine interact in their effects on human sperm? SUMMARY ANSWER: Behaviour of human sperm (proportion of hyperactivated cells and motility kinematics) were related directly to [Ca2+](i) irrespective of pH or the agonist applied. WHAT IS KNOWN ALREADY?: CatSper channels of human sperm, which are central to generation of sperm [Ca2+](i) signals and induction of hyperactivated motility, are activated by intracellular alkalinization and progesterone. Progesterone (P4) is much less effective than 4-aminopyridine (4-AP) (which mobilizes stored Ca2+ but also raises pHi) as an inducer of hyperactivation. STUDY DESIGN, SIZE, DURATION: This was a laboratory study, spanning similar to 18 months that used 15 sperm donors and involved more than 100 separate experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors and patients were recruited in accordance with local ethics approval (ERN_ 12-0570R). [Ca2+](i) responses of suspended cell populations were examined by fluorimetric recording and motility parameters assessed by computer-assisted sperm analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Increasing pH(o) from 7.4 to 8.5 raised pHi (from 6.9 to 7.2) and significantly increased both [Ca2+](i) and the proportion of hyperactivated cells. Stimulation of cells with P4 (1 nM-20 mu M) induced a biphasic (transient and plateau) increase in [Ca2+](i). The [Ca2+](i) increase was of similar amplitude and dose-dependency at pH(o) = 7.4 and pH(o) = 8.5. 4-aminopyridine (0.2-5 mM) induced a biphasic [Ca2+](i) increase that was dose-dependent across the entire range tested and was strongly enhanced at pH 8.5. Motility was assessed 300 s post-stimulation, during the plateau phase of the progesterone and 4-AP-induced [Ca2+](i) responses. Progesterone had only a small effect on hyperactivated motility even at the highest dose used (20 mu M; < 5% increase in the proportion of cells classified as hyperactivated) which was insensitive to pH(o). 4-Aminopyridine potently stimulated hyperactivated motility, this effect being dose-dependent and greatly enhanced at pH(o) = 8.5. The relationship between [Ca2+](i) (fluorescence of fluo4) and proportion of hyperactivated cells, irrespective of pH(o), agonist or dose, was fitted by a single curve (second order polynomial; R-2 = 0.96). Similar analysis of curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) showed a linear relationship to [Ca2+](i) (R-2 > 0.9). LIMITATIONS, REASONS FOR CAUTION: This was an in-vitro study and caution must be taken when extrapolating these results to in vivo regulation of sperm. Though controls indicate that saturation of fluo4 did not affect the findings, at the highest doses of progesterone the true amplitude of the [Ca2+](i) transient may not have been reported by the dye. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that (i) activation of human sperm CatSper by progesterone (and presumably other ligands that act similarly) and consequent acquisition of hyperactivated motility is not significantly enhanced by intracellular alkalinization; (ii) VCL, ALH and hyperactivation are directly related to [Ca2+](i), irrespective of the mechanism by which Ca2+ is mobilized, and the ability of stimuli to induce prolonged [Ca2+](i) elevation (as occurs upon mobilization of stored Ca2+) determines the observed effect on cell behaviour.

BACKGROUND AND PURPOSE Sperm from many species share the sperm-specific Ca2+ channel CatSper that controls the intracellular Ca2+ concentration and, thereby, the swimming behaviour. A growing body of evidence suggests that the mechanisms controlling the activity of CatSper and its role during fertilization differ among species. A lack of suitable pharmacological tools has hampered the elucidation of the function of CatSper. Known inhibitors of CatSper exhibit considerable side effects and also inhibit Slo3, the principal K+ channel of mammalian sperm. The compound RU1968 was reported to suppress Ca2+ signaling in human sperm by an unknown mechanism. Here, we examined the action of RU1968 on CatSper in sperm from humans, mice, and sea urchins. EXPERIMENTAL APPROACH We resynthesized RU1968 and studied its action on sperm from humans, mice, and the sea urchin Arbacia punctulata by Ca2+ fluorimetry, single-cell Ca2+ imaging, electrophysiology, opto-chemistry, and motility analysis. KEY RESULTS RU1968 inhibited CatSper in sperm from invertebrates and mammals. The compound lacked toxic side effects in human sperm, did not affect mouse Slo3, and inhibited human Slo3 with about 15-fold lower potency than CatSper. Moreover, in human sperm, RU1968 mimicked CatSper dysfunction and suppressed motility responses evoked by progesterone, an oviductal steroid known to activate CatSper. Finally, RU1968 abolished CatSper-mediated chemotactic navigation in sea urchin sperm. CONCLUSION AND IMPLICATIONS We propose RU1968 as a novel tool to elucidate the function of CatSper channels in sperm across species.

19 Study question: How do the alkaline pH, progesterone and 4-aminopyridine interact in their effects 20 on human sperm? 21 Summary answer: Behaviour of human sperm (proportion of hyperactivated cells and motility 22 kinematics) were related directly to [Ca 2+ ]i irrespective of pH or the agonist applied. 23 What is known already? CatSper channels of human sperm, which are central to generation of 24 sperm [Ca 2+ ]i signals and induction of hyperactivated motility, are activated by intracellular 25 alkalinisation and progesterone. Progesterone (P4) is much less effective than 4-aminopyridine (4-AP) 26 (which mobilises stored Ca 2+ but also raises pHi) as an inducer of hyperactivation. 27 Study design, size, duration: This was a laboratory study, spanning approximately 18 months 28 that used 15 sperm donors and involved more than 100 separate experiments. 29 Participants/materials, setting, methods: Semen donors and patients were recruited in 30 accordance with local ethics approval (ERN_12-0570R). [Ca2+]i responses of suspended cell 31 populations were examined by fluorimetric recording and motility parameters assessed by computer32 assisted sperm analysis (CASA). 33 Main results and the role of chance: Increasing pHo from 7.4 to 8.5 raised pHi (from 6.9 to 7.2) 34 and significantly increased both [Ca 2+ ]i and the proportion of hyperactivated cells. Stimulation of cells 35 with P4 (1 nM-20 M) induced a biphasic (transient and plateau) increase in [Ca 2+ ]i. The [Ca 2+ ]i 36 increase was of similar amplitude and dose-dependency at pHo=7.4 and pHo= 8.5. 4-aminopyridine 37 (0.2-5 mM) induced a biphasic [Ca 2+ ]i increase that was dose-dependent across the entire range tested 38 and was strongly enhanced at pH 8.5. Motility was assessed 300 s post-stimulation, during the plateau 39 phase of the progesterone and 4-AP-induced [Ca 2+ ]i responses. Progesterone had only a small effect 40 on hyperactivated motility even at the highest dose used (20 M; <5% increase in the proportion of 41 cells classified as hyperactivated) which was insensitive to pHo. 4-aminopyridine potently stimulated 42 hyperactivated motility, this effect being dose-dependent and greatly enhanced at pHo=8.5. The 43 relationship between [Ca 2+ ]i (fluorescence of fluo4) and proportion of hyperactivated cells, 44 irrespective of pHo, agonist or dose, was fitted by a single curve (2 nd order polynomial; R 2 =0.96). 45 Similar analysis of curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) 46 showed a linear relationship to [Ca 2+ ]i (R 2 >0.9). 47 Limitations, reasons for caution: This was an in-vitro study and caution must be taken when 48 extrapolating these results to in vivo regulation of sperm. Though controls indicate that saturation of 49 fluo4 did not affect the findings, at the highest doses of progesterone the true amplitude of the [Ca 2+ ]i 50 transient may not have been reported by the dye. 51 Wider implications of the findings: These findings indicate that (i) activation of human sperm 52 CatSper by progesterone (and presumably other ligands that act similarly) and consequent acquisition 53 of hyperactivated motility is not significantly enhanced by intracellular alkalinisation; (ii) VCL, ALH 54 and hyperactivation are directly related to [Ca 2+ ]i, irrespective of the mechanism by which Ca 2+ is 55 mobilised, and the ability of stimuli to induce prolonged [Ca 2+ ]i elevation (as occurs upon 56 mobilisation of stored Ca 2+ ) determines the observed effect on cell behaviour. 57 Study funding/competing interest(s): CA was supported by the Nigerian government (Tertiary 58 Education Trust (TET) Fund). The authors have no conflicts of interest. 59

STUDY QUESTION: Does a man (patient 1) with a previously described deficiency in principle cation channel of sperm (CatSper) function have a mutation in the CatSper-epsilon (CATSPERE) and/or CatSper-zeta (CATSPERZ) gene? SUMMARY ANSWER: Patient 1 has a homozygous in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE. WHAT IS KNOWN ALREADY: CatSper is the principal calcium channel of mammalian spermatozoa. Spermatozoa from patient 1 had a specific loss of CatSper function and were unable to fertilize at IVF. Loss of CatSper function could not be attributed to genetic abnormalities in coding regions of seven CatSper subunits. Two additional subunits (CatSper-epsilon (CATPSERE) and CatSper-zeta (CATSPERZ)) were recently identified, and are now proposed to contribute to the formation of the mature channel complex. STUDY DESIGN, SIZE, DURATION: This was a basic medical research study analysing genomic data from a single patient (patient 1) for defects in CATSPERE and CATSPERZ. PARTICIPANTS/MATERIALS, SETTING, METHODS: The original exome sequencing data for patient 1 were analysed for mutations in CATSPERE and CATSPERZ. Sanger sequencing was conducted to confirm the presence of a rare variant. MAIN RESULTS AND THE ROLE OF CHANCE: Patient 1 is homozygous for an in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE that is predicted to be highly deleterious. LIMITATIONS, REASONS FOR CAUTION: The nature of the molecular deficit caused by the rs761237686 variant and whether it is exclusively responsible for the loss of CatSper function remain to be elucidated. WIDER IMPLICATIONS OF THE FINDINGS: Population genetics are available for a significant number of predicted deleterious variants of CatSper subunits. The consequence of homozygous and compound heterozygous forms on sperm fertilization potential could be significant. Selective targeting of CatSper subunit expression maybe a feasible strategy for the development of novel contraceptives.

STUDY QUESTION: What are the characteristics of progesterone-induced (CatSper-mediated) single cell [Ca2+](i) signals in spermatozoa from sub-fertile men and how do they relate to fertilizing ability? SUMMARY ANSWER: Single cell analysis of progesterone-induced (CatSper-mediated) [Ca2+](i) showed that reduced progesterone-sensitivity is a common feature of sperm from sub-fertile patients and is correlated with fertilization rate. WHAT IS KNOWN ALREADY: Stimulation with progesterone is a widely used method for assessing [Ca2+](i) mobilization by activation of CatSper in human spermatozoa. Although data are limited, sperm population studies have indicated an association of poor [Ca2+](i) response to progesterone with reduced fertilization ability. STUDY DESIGN, SIZE, DURATION: This was a cohort study using semen samples from 21 donors and 101 patients attending the assisted conception unit at Ninewells Hospital Dundee who were undergoing ART treatment. Patients were recruited from January 2016 to June 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors and patients were recruited in accordance with local ethics approval (13/ES/0091) from the East of Scotland Research Ethics Service (EoSRES) REC1. [Ca2+](i) responses were examined by single cell imaging and motility parameters assessed by computer-assisted sperm analysis (CASA). MAIN RESULTS AND THE ROLE OF CHANCE: For analysis, patient samples were divided into three groups IVF(+ve) (successful fertilization; 62 samples), IVF-FF (failed fertilization; eight samples) and ICSI (21 samples). A further 10 IVF samples showed large, spontaneous [Ca2+](i) oscillations and responses to progesterone could not be analysed. All patient samples loaded with the [Ca2+](i)-indicator fluo4 responded to progesterone stimulation with a biphasic increase in fluorescence (transient followed by plateau) which resembled that seen in progesterone-stimulated donor samples. The mean normalized response (progesterone-induced increase in fluorescence normalized to resting level) was significantly smaller in IVF-FF and ICSI patient groups than in donors. All samples were further analysed by plotting, for each cell, the relationship between resting fluorescence intensity and the progesterone-induced fluorescence increment. In donor samples these plots overlaid closely and had a gradient of approximate to 2 and plots for most IVF(+ve) samples closely resembled the donor distribution. However, in a subset (approximate to 10%) of IVF(+ve) samples, 3/8 IVF-FF samples and one-third of ICSI samples the gradient of the plot was significantly lower, indicating that the response to progesterone of the cells in these samples was abnormally small. Examination of the relationship between gradient (regression coefficient of the plot) in IVF samples and fertilization rate showed a positive correlation. In IVF-FF and ICSI groups, the proportion of cells in which a response to progesterone could be detected was significantly lower than in donors and IVF (+ve) patients. Approximately 20% of cells in donor, IVF(+ve) and ICSI samples generated [Ca2+](i) oscil lations when challenged with progesterone but in IVF-FF samples only approximate to 10% of cells generated oscillations and there was a significantly greater proportion of samples where no oscillations were observed. Levels of hyperactivated motility were lower in IVF(+ve) and IVF-FF groups compared to controls, IVF-FF also having lower levels than IVF (+ve). LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study and caution must be taken when extrapolating these results in vivo. WIDER IMPLICATIONS OF THE FINDINGS: This study reveals important details of impaired [Ca2+](i) signalling in sperm from sub-fertile men that cannot be detected in population studies. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by a MRC project grant (MR/M012492/1; MR/K013343/1). Additional funding was provided by Chief Scientist Office/NHS research Scotland.

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STUDY QUESTION : Does progesterone in human follicular fl uid (hFF) activate CatSper and do other components of hFF modulate this effect and/or contribute separately to hFF-induced Ca 2 + signaling? SUMMARY ANSWER : hFF potently stimulates CatSper and increases 2 + ] i , primarily due to high concentrations of progesterone, however, other components of hFF contribute to [Ca 2 + ] i signaling, including modulation of CatSper channel activity and inhibition of [Ca 2 + ] i oscillations. Ca 2 + channel in spermatozoa, progesterone-sensitive and essential for fertility. Both hFF and progesterone, which is present in hFF, in fl uence sperm function and increase their 2 + ] i WIDER IMPLICATIONS OF THE FINDINGS : This study directly demonstrates that hFF activates CatSper and establishes that the bio-logically important effects of hFF re fl ect, at least in part, action on this channel, primarily via progesterone. However, these experiments also demonstrate that other components of hFF both contribute to the [Ca 2 + ] i signal and modulate the activation of CatSper. Simple in vitro experiments performed out of the context of the complex in vivo environment need to be interpreted with caution. STUDY FUNDING / COMPETING INTEREST ( ): Funding was provided by MRC (MR/K013343/1, MR/012492/1) (S.G.B., S.J.P., C.L.R.B.) and University of Abertay (sabbatical for S.G.B.). Additional funding was provided by TENOVUS SCOTLAND (S.M.D.S.), Chief Scientist Of fi ce/NHS Research Scotland (S.M.D.S). C.L.R.B. is EIC of MHR and Chair of the WHO ESG on Diagnosis of Male infertility. The remaining authors have no conlicts of interest.

STUDY QUESTION Are significant abnormalities in outward (K+) conductance and resting membrane potential (Vm) present in the spermatozoa of patients undertaking IVF and ICSI and if so, what is their functional effect on fertilization success? SUMMARY ANSWER Negligible outward conductance (≈5% of patients) or an enhanced inward conductance (≈4% of patients), both of which caused depolarization of Vm, were associated with a low rate of fertilization following IVF. WHAT IS KNOWN ALREADY Sperm-specific potassium channel knockout mice are infertile with defects in sperm function, suggesting that these channels are essential for fertility. These observations suggest that malfunction of K+ channels in human spermatozoa might contribute significantly to the occurrence of subfertility in men. However, remarkably little is known of the nature of K+ channels in human spermatozoa or the incidence and functional consequences of K+ channel defects. STUDY DESIGN, SIZE AND DURATION Spermatozoa were obtained from healthy volunteer research donors and subfertile IVF and ICSI patients attending a hospital assisted reproductive techniques clinic between May 2013 and December 2015. In total, 40 IVF patients, 41 ICSI patients and 26 normozoospermic donors took part in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS Samples were examined using electrophysiology (whole-cell patch clamping). Where abnormal electrophysiological characteristics were identified, spermatozoa were further examined for Ca2+ influx induced by progesterone and penetration into viscous media if sufficient sample was available. Full exome sequencing was performed to specifically evaluate potassium calcium-activated channel subfamily M α 1 (KCNMA1), potassium calcium-activated channel subfamily U member 1 (KCNU1) and leucine-rich repeat containing 52 (LRRC52) genes and others associated with K+ signalling. In IVF patients, comparison with fertilization rates was done to assess the functional significance of the electrophysiological abnormalities. MAIN RESULTS AND THE ROLE OF CHANCE Patch clamp electrophysiology was used to assess outward (K+) conductance and resting membrane potential (Vm) and signalling/motility assays were used to assess functional characteristics of sperm from IVF and ICSI patient samples. The mean Vm and outward membrane conductance in sperm from IVF and ICSI patients were not significantly different from those of control (donor) sperm prepared under the same conditions, but variation between individuals was significantly greater (P< 0.02) with a large number of outliers (>25%). In particular, in ≈10% of patients (7/81), we observed either a negligible outward conductance (4 patients) or an enhanced inward current (3 patients), both of which caused depolarization of Vm. Analysis of clinical data from the IVF patients showed significant association of depolarized Vm (≥0 mV) with low fertilization rate (P= 0.012). Spermatozoa with electrophysiological abnormities (conductance and Vm) responded normally to progesterone with elevation of [Ca2+]i and penetration of viscous medium, indicating retention of cation channel of sperm (CatSper) channel function. LIMITATIONS, REASONS FOR CAUTION For practical, technical, ethical and logistical reasons, we could not obtain sufficient additional semen samples from men with conductance abnormalities to establish the cause of the conductance defects. Full exome sequencing was only available in two men with conductance defects. WIDER IMPLICATIONS OF THE FINDINGS These data add significantly to the understanding of the role of ion channels in human sperm function and its impact on male fertility. Impaired potassium channel conductance (Gm) and/or Vm regulation is both common and complex in human spermatozoa and importantly is associated with impaired fertilization capacity when the Vm of cells is completely depolarized. STUDY FUNDING/COMPETING INTEREST(S) The majority of the data were obtained using funding from MRC project grants (#MR/K013343/1, MR/012492/1). Additional funding was provided by NHS Tayside, TENOVUS, Chief Scientist Office NRS Fellowship and University of Abertay. The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER Not applicable.