Sperm Toolbox-A selection of small molecules to study human spermatozoa.

Male contraceptive options and infertility treatments are limited, and almost all innovation has been limited to updates to medically assisted reproduction protocols and methods. To accelerate the development of drugs that can either improve or inhibit fertility, we established a small molecule library as a toolbox for assay development and screening campaigns using human spermatozoa. We have profiled all compounds in the Sperm Toolbox in several automated high-throughput assays that measure stimulation or inhibition of sperm motility or the acrosome reaction. We have assayed motility under non-capacitating and capacitating conditions to distinguish between pathways operating under these different physiological states. We also assayed cell viability to ensure any effects on sperm function are specific. A key advantage of our studies is that all compounds are assayed together in the same experimental conditions, which allows quantitative comparisons of their effects in complementary functional assays. We have combined the resulting datasets to generate fingerprints of the Sperm Toolbox compounds on sperm function. The data are included in an on-line R-based app for convenient querying.

Fertility preservation provision in the NHS: a national assessment of care policies.

Fertility preservation has gained momentum in recent years. As cancer survival rates improve, late effects of loss of gonadal function have increased the need to consider fertility preservation. NICE recommends offering cryopreservation of gametes or embryos to patients undergoing gonadotoxic therapy, highlighting that this should be extrapolated to those with non-malignant conditions that pose a risk to fertility. We investigated whether variation in fertility preservation provision exists across the United Kingdom, with a view to identifying equitable models of provision. In England, cryopreservation of gametes and embryos is funded for all patients undergoing treatment for cancer, but eligibility criteria and duration of storage funding vary widely. In Scotland, a national policy is applied, with health boards equitably providing funding for cryopreservation of gametes, embryos, and ovarian and testicular tissue for those undergoing treatment for benign and malignant conditions which impair fertility, including gender incongruence. In Wales and Northern Ireland, cryopreservation of gametes and embryos is funded for those undergoing treatment likely to make them infertile, but ovarian tissue cryopreservation is not funded. Funding criteria for fertility preservation in England, Wales, and Northern Ireland deviates from NICE guidance. Standardization of fertility preservation policies is needed to provide equity of access for patients.

Sperm cryopreservation for impaired spermatogenesis.

Sperm cryopreservation for men with severely impaired spermatogenesis is one of the commonest reasons for short-term sperm storage, usually in advance of fertility treatment. Cryopreservation is generally very effective, although not all spermatozoa survive the process of freezing and thawing. This review considers various aspects of freezing sperm, including an overview of methods, appropriate use of cryoprotectants and practical considerations, as well as oxidative stress and mechanisms of cell cryodamage.

Reproductive axis ageing and fertility in men.

Compared to women, increasing male age is not accompanied by such marked changes in reproductive function but changes certainly do happen. These include alterations to the hypothalamo-pituitary-testicular axis, with resultant implications for testosterone production and bioavailability as well as spermatogenesis. There is a decline in sexual function as men age, with a dramatic increase in the prevalence of erectile dysfunction after the age of 40, which is a marker for both clinically evident as well as covert coronary artery disease. Despite a quantitative decline in spermatogenesis and reduced fecundability, the male potential for fertility persists throughout adult life, however there are also increasingly recognised alterations in sperm quality and function with significant implications for offspring health. These changes are relevant to both natural and medically assisted conception.

Elevated and Sustained Intracellular Calcium Signalling Is Necessary for Efficacious Induction of the Human Sperm Acrosome Reaction.

Progesterone and prostaglandin E1 are postulated to trigger the human sperm acrosome reaction (AR). However, their reported efficacy is very variable which likely, in part, reflects the plethora of experimental conditions and methodologies used to detect this physiologically relevant event. The purpose of this study was to develop an assay for the robust induction and objective measurement of the complete AR. Sperm from healthy volunteers or patients undertaking IVF were treated with a variety of ligands (progesterone, prostaglandin E1 or NH4Cl, alone or in combinations). AR, motility and intracellular calcium measurements were measured using flow cytometry, computer-assisted sperm analysis (CASA) and fluorimetry, respectively. The AR was significantly increased by the simultaneous application of progesterone, prostaglandin E1 and NH4Cl, following an elevated and sustained intracellular calcium concentration. However, we observed notable inter- and intra-donor sample heterogeneity of the AR induction. When studying the patient samples, we found no relationship between the IVF fertilization rate and the AR. We conclude that progesterone and prostaglandin E1 alone do not significantly increase the percentage of live acrosome-reacted sperm. This assay has utility for drug discovery and sperm toxicology studies but is not predictive for IVF success.

Male infertility: what on earth is going on? Pilot international questionnaire study regarding clinical evaluation and fertility treatment for men.

Infertility is a time-consuming and exhaustive process, which disproportionally affects women. Although concerns have been raised about deficiencies in clinical evaluation of infertile men, there is currently little published data documenting this. A SurveyMonkey questionnaire was therefore created to capture current clinical practice of fertility specialists working in IVF clinics. Responses were collected May - July 2021. 112 clinicians completed the pilot survey with respondents from Europe (n=49; 43.8%), Africa (n=39, 34.8%), North America (n=6; 5.4%), Asia (n=16; 14.3%), South America (n=1; 0.9%) and Australasia (n=1;0.9%). 41% fertility specialists (45/110) reported taking only a brief medical history and 24% reported that they never routinely examined infertile male patients. 54% fertility specialists also reported issues getting men to undertake diagnostic semen analysis. Treatment for male infertility spanned Assisted Reproductive Technology (ART), with themes of individualised medicine influencing treatment recommendations. 48.2% clinicians reported using empirical medical therapy (EMT) for unexplained male infertility. Notably, 3.6% respondents recommended testosterone treatment, despite likely negative impact on spermatogenesis. However, high levels of opportunistic general health advice were reported, including discussion of life exposures thought to be important for male reproductive health. This study adds novel evidence and highlights current deficiencies in clinical practice relating to male infertility. Evaluation of the infertile male using simple medical tools (detailed history taking and clinical examination) has the potential to identify treatable or reversible conditions and should be an immediate focus for education and improvement in Reproductive Medicine. Investment in research and development is much needed in the field of andrology, to develop effective non-ART treatment options for male infertility.

Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology.

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

Compounds enhancing human sperm motility identified using a high-throughput phenotypic screening platform.

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.

IVF or ICSI for fertility preservation?

In IVF, eggs and sperm are added together for fertilisation to occur whereas ICSI involves injecting a single sperm into each egg. ICSI is very effective where sperm count or swimming is poor (male infertility) but is slightly riskier than IVF in terms of health problems in children, although these risks are small. However, the risk of no eggs fertilising is higher for IVF compared to ICSI and couples undertaking fertility preservation, for example, before cancer treatment, usually only have time for one attempt. Using fertility preservation treatment cycle data reported to Human Fertilisation and Embryology Authority (HFEA), this study shows that ICSI results in higher number of fertilised eggs and embryos for storage or treatment compared to IVF. However, 19% of eggs are not used in ICSI treatment, so IVF appears to be better overall. Clinics should choose IVF or ICSI for fertility preservation depending on sperm characteristics rather than using ICSI for all.

Artificial Egg Activation Using Calcium Ionophore.

Artificial oocyte activation, most commonly using calcium ionophore, is a treatment add-on utilized to avoid recurrence of abnormally low or total failed fertilization following in vitro fertilization/intracytoplasmic sperm injection. It aims to modify defective physiological processes, specifically calcium-mediated cell signaling that are critical to events required for fertilization. Routine application of artificial oocyte activation is neither required nor recommended; however, it represents an invaluable intervention for a subgroup of patients affected by sperm-related oocyte activation deficiency.

Assisted reproductive technology, justice and autonomy in an era of COVID-19.

Fertility services were significantly curtailed or suspended as an initial response to the coronavirus (COVID-19) pandemic earlier this year, following guidance from European Society for Human Reproduction and Embryology (ESHRE) and the American Society for Reproductive Medicine (ASRM) as well as a General Direction (GD0014) issued by the Human Fertilisation and Embryo Authority (HFEA). It is difficult to argue with triage of medical care and resources in the face of anticipated overwhelming demand, but this situation resulted in considerable distress, as shown by a change.org petition opposing ASRM recommendations, which has gathered over 21,000 signatures to date. Although halting assisted reproductive technology (ART) as the pandemic unfolded was ethical because public health goals superseded individual patient autonomy, the fertility sector now faces a greater challenge balancing ethical considerations in an era characterized by the ongoing threat of COVID-19. This article discusses justice and autonomy in the context of ART, potential conflicts and resolutions.

Mechanistic Insight into the Regulation of Lipoxygenase-Driven Lipid Peroxidation Events in Human Spermatozoa and Their Impact on Male Fertility.

A prevalent cause of sperm dysfunction in male infertility patients is the overproduction of reactive oxygen species, an attendant increase in lipid peroxidation and the production of cytotoxic reactive carbonyl species such as 4-hydroxynonenal. Our previous studies have implicated arachidonate 15-lipoxygenase (ALOX15) in the production of 4-hydroxynonenal in developing germ cells. Here, we have aimed to develop a further mechanistic understanding of the lipoxygenase-lipid peroxidation pathway in human spermatozoa. Through pharmacological inhibition studies, we identified a protective role for phospholipase enzymes in the liberation of peroxidised polyunsaturated fatty acids from the human sperm membrane. Our results also revealed that arachidonic acid, linoleic acid and docosahexanoic acid are key polyunsaturated fatty acid substrates for ALOX15. Upon examination of ALOX15 in the spermatozoa of infertile patients compared to their normozoospermic counterparts, we observed significantly elevated levels of ALOX15 protein abundance in the infertile population and an increase in 4-hydroxynonenal adducts. Collectively, these data confirm the involvement of ALOX15 in the oxidative stress cascade of human spermatozoa and support the notion that increased ALOX15 abundance in sperm cells may accentuate membrane lipid peroxidation and cellular dysfunction, ultimately contributing to male infertility.

l-carnitine and l-acetylcarnitine supplementation for idiopathic male infertility.

Fifteen percent of couples are globally estimated to be infertile, with up to half of these cases attributed to male infertility. Reactive oxidative species (ROS) are known to damage sperm leading to impaired quantity and quality. Although not routinely assessed, oxidative stress is a common underlying pathology in infertile men. Antioxidants have been shown to improve semen analysis parameters by reducing ROS and facilitating repair of damage caused by oxidative stress, but it remains unclear whether they improve fertility. Carnitines are naturally occurring antioxidants in mammals and are normally abundant in the epididymal luminal fluid of men. We conducted a systematic review and meta-analysis to evaluate the safety and efficacy of carnitine supplementation for idiopathic male infertility. We searched ClinicalKey, ClinicalTrials.gov, Cochrane Central Register of Controlled Trials (CENTRAL), EMBASE, MEDLINE, PubMed and ScienceDirect for relevant studies published from 1 January 2000 to 30 April 2020. Of the articles retrieved, only eight randomised controlled trials were identified and included. Analysis showed that carnitines significantly improve total sperm motility, progressive sperm motility and sperm morphology, but without effect on sperm concentration. There was no demonstrable effect on clinical pregnancy rate in the five studies that included that outcome, although patient numbers were limited. Therefore, the use of carnitines in male infertility appears to improve some sperm parameters but without evidence of an increase in the chance of natural conception. LAY SUMMARY: Although male infertility affects 1:15 men, there is no obvious reason in the vast majority of cases. Reactive oxidative species (ROS) are highly active molecules containing oxygen and are natural byproducts of normal metabolism. However, high concentrations of ROS have been shown to damage sperm, which negatively impacts a couple's ability to conceive. Carnitines are natural antioxidants found in the body that counterbalance the damaging effects of ROS. We conducted a comprehensive review of published studies to assess whether carnitine supplements are safe and effective in improving sperm quality and pregnancy rates. Our analysis shows that carnitines improve sperm swimming and production of normal-shaped sperm cells but do not affect sperm count or pregnancy rates, although there are only a few studies and scientific evidence is limited. Whilst it is possible that carnitines may benefit male infertility, more evidence is required regarding chances of pregnancy after carnitine therapy.

Male infertility and antioxidants: one small step for man, no giant leap for andrology?

Oxidative stress is detrimental to spermatozoa and is acknowledged to be a common pathology in infertile men. Antioxidant supplements, therefore, represent a logical therapeutic approach, although the recent Cochrane review recommends cautious interpretation of publications and findings to date. This commentary considers whether male fertility supplements have a place in current reproductive medicine practice. Importantly, although sperm selection for intracytoplasmic sperm injection is a common research theme, survey data show that men would prefer medication to achieve natural conception, rather than treatment to improve assisted reproductive technology (ART) success. A total of 27.1% (n = 112), 26.6% (n = 110) and 24.5% (n = 101) respondents indicated they (or their male partner) would undertake medical treatment to attempt natural conception for up to 6 months, 12 months and 2 years, respectively. A total of 63% indicated that they would be prepared to participate in a clinical trial and 57% would defer ART by 6 months to do so. This information represents the beginnings of a dialogue with patients and stakeholders and should be used to shape research efforts.

Human sperm ion channel (dys)function: implications for fertilization.

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.

Novel pharmacological actions of trequinsin hydrochloride improve human sperm cell motility and function.

BACKGROUND AND PURPOSE: Asthenozoospermia is a leading cause of male infertility, but development of pharmacological agents to improve sperm motility is hindered by the lack of effective screening platforms and knowledge of suitable molecular targets. We have demonstrated that a high-throughput screening (HTS) strategy and established in vitro tests can identify and characterise compounds that improve sperm motility. Here, we applied HTS to identify new compounds from a novel small molecule library that increase intracellular calcium ([Ca2+ ]i ), promote human sperm cell motility, and systematically determine the mechanism of action. EXPERIMENTAL APPROACH: A validated HTS fluorometric [Ca2+ ]i assay was used to screen an in-house library of compounds. Trequinsin hydrochloride (a PDE3 inhibitor) was selected for detailed molecular (plate reader assays, electrophysiology, and cyclic nucleotide measurement) and functional (motility and acrosome reaction) testing in sperm from healthy volunteer donors and, where possible, patients. KEY RESULTS: Fluorometric assays identified trequinsin as an efficacious agonist of [Ca2+ ]i , although less potent than progesterone. Functionally, trequinsin significantly increased cell hyperactivation and penetration into viscous medium in all donor sperm samples and cell hyperactivation in 22/25 (88%) patient sperm samples. Trequinsin-induced [Ca2+ ]i responses were cross-desensitised consistently by PGE1 but not progesterone. Whole-cell patch clamp electrophysiology confirmed that trequinsin activated CatSper and partly inhibited potassium channel activity. Trequinsin also increased intracellular cGMP. CONCLUSION AND IMPLICATIONS: Trequinsin exhibits a novel pharmacological profile in human sperm and may be a suitable lead compound for the development of new agents to improve patient sperm function and fertilisation potential.

Homozygous in-frame deletion in CATSPERE in a man producing spermatozoa with loss of CatSper function and compromised fertilizing capacity.

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 FUNDING/COMPETING INTEREST(S): This study was funded by project grants from the MRC (MR/K013343/1 and MR/012492/1), Chief Scientist Office/NHS research Scotland. This work was also supported by NIH R01GM111802, Pew Biomedical Scholars Award 00028642 and Packer Wentz Endowment Will to P.V.L. C.L.R.B is the editor-in-chief of Molecular Human Reproduction, has received lecturing fees from Merck and Ferring, and is on the Scientific Advisory Panel for Ohana BioSciences. C.L.R.B was chair of the World Health Organization Expert Synthesis Group on Diagnosis of Male infertility (2012-2016).

Single-cell analysis of [Ca2+]i signalling in sub-fertile men: characteristics and relation to fertilization outcome.

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 ≈ 2 and plots for most IVF(+ve) samples closely resembled the donor distribution. However, in a subset (≈ 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 oscillations when challenged with progesterone but in IVF-FF samples only ≈ 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.