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.

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halting assisted reproductive technology (ART) as the pandemic unfolded was ethical because public health goals superseded individual patient autonomy, the fertility sector now faces a greater challenge balancing ethical considerations in an era characterized by the ongoing threat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.

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.

Drug discovery for male subfertility using high-throughput screening: a new approach to an unsolved problem.

STUDY QUESTION: Can pharma drug discovery approaches be utilized to transform investigation into novel therapeutics for male infertility? SUMMARY ANSWER: High-throughput screening (HTS) is a viable approach to much-needed drug discovery for male factor infertility. WHAT IS KNOWN ALREADY: There is both huge demand and a genuine clinical need for new treatment options for infertile men. However, the time, effort and resources required for drug discovery are currently exorbitant, due to the unique challenges of the cellular, physical and functional properties of human spermatozoa and a lack of appropriate assay platform. STUDY DESIGN, SIZE, DURATION: Spermatozoa were obtained from healthy volunteer research donors and subfertile patients undergoing IVF/ICSI at a hospital-assisted reproductive techniques clinic between January 2012 and November 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: A HTS assay was developed and validated using intracellular calcium ([Ca2+]i) as a surrogate for motility in human spermatozoa. Calcium fluorescence was detected using a Flexstation microplate reader (384-well platform) and compared with responses evoked by progesterone, a compound known to modify a number of biologically relevant behaviours in human spermatozoa. Hit compounds identified following single point drug screen (10 µM) of an ion channel-focussed library assembled by the University of Dundee Drug Discovery Unit were rescreened to ensure potency using standard 10 point half-logarithm concentration curves, and tested for purity and integrity using liquid chromatography and mass spectrometry. Hit compounds were grouped by structure activity relationships and five representative compounds then further investigated for direct effects on spermatozoa, using computer-assisted sperm assessment, sperm penetration assay and whole-cell patch clamping. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 3242 ion channel library ligands screened, 384 compounds (11.8%) elicited a statistically significant increase in calcium fluorescence, with greater than 3× median absolute deviation above the baseline. Seventy-four compounds eliciting ≥50% increase in fluorescence in the primary screen were rescreened and evaluated further, resulting in 48 hit compounds that produced a concentration-dependent increase in [Ca2+]i. Sperm penetration studies confirmed in vitro exposure to two hit compounds (A and B) resulted in significant improvement in functional motility in spermatozoa from healthy volunteer donors (A: 1 cm penetration index 2.54, 2 cm penetration index 2.49; P < 0.005 and B: 1 cm penetration index 2.1, 2 cm penetration index 2.6; P < 0.005), but crucially, also in patient samples from those undergoing fertility treatment (A: 1 cm penetration index 2.4; P = 0.009, 2 cm penetration index 3.6; P = 0.02 and B: 1 cm penetration index 2.2; P = 0.0004, 2 cm penetration index 3.6; P = 0.002). This was primarily as a result of direct or indirect CatSper channel action, supported by evidence from electrophysiology studies of individual sperm. LIMITATIONS, REASONS FOR CAUTION: Increase and fluxes in [Ca2+]i are fundamental to the regulation of sperm motility and function, including acrosome reaction. The use of calcium signalling as a surrogate for sperm motility is acknowledged as a potential limitation in this study. WIDER IMPLICATIONS OF THE FINDINGS: We conclude that HTS can robustly, efficiently, identify novel compounds that increase [Ca2+]i in human spermatozoa and functionally modify motility, and propose its use as a cornerstone to build and transform much-needed drug discovery for male infertility. STUDY FUNDING/COMPETING INTEREST(S): The majority of the data were obtained using funding from TENOVUS Scotland and Chief Scientist Office NRS Fellowship. Additional funding was provided by NHS Tayside, MRC project grants (MR/K013343/1, MR/012492/1) and University of Abertay. The authors declare that there is no conflict of interest. TRAIL REGISTRATION NUMBER: N/A.

A spontaneous increase in intracellular Ca2+ in metaphase II human oocytes in vitro can be prevented by drugs targeting ATP-sensitive K+ channels.

STUDY QUESTION: Could drugs targeting ATP-sensitive K(+) (K(ATP)) channels prevent any spontaneous increase in intracellular Ca(2+) that may occur in human metaphase II (MII) oocytes under in vitro conditions? SUMMARY ANSWER: Pinacidil, a K(ATP) channel opener, and glibenclamide, a K(ATP) channel blocker, prevent a spontaneous increase in intracellular Ca(2+) in human MII oocytes. WHAT IS KNOWN ALREADY: The quality of the oocyte and maintenance of this quality during in vitro processing in the assisted reproductive technology (ART) laboratory is of critical importance to successful embryo development and a healthy live birth. Maintenance of Ca(2+) homeostasis is crucial for cell wellbeing and increased intracellular Ca(2+) levels is a well-established indicator of cell stress. STUDY DESIGN, SIZE, DURATION: Supernumerary human oocytes (n = 102) collected during IVF/ICSI treatment that failed to fertilize were used from October 2013 to July 2015. All experiments were performed on mature (MII) oocytes. Dynamics of intracellular Ca(2+) levels were monitored in oocytes in the following experimental groups: (i) Control, (ii) Dimethyl sulfoxide (DMSO; used to dissolve pinacidil, glibenclamide and 2,4-Dinitrophenol (DNP)), (iii) Pinacidil, (iv) Glibenclamide, (v) DNP: an inhibitor of oxidative phosphorylation, (vi) Pinacidil and DNP and (vii) Glibenclamide and DNP. PARTICIPANTS/MATERIALS/SETTINGS/METHODS: Oocytes were collected under sedation as part of routine treatment at an assisted conception unit from healthy women (mean ± SD) age 34.1 ± 0.6 years, n = 41. Those surplus to clinical use were donated for research. Oocytes were loaded with Fluo-3 Ca(2+)-sensitive dye, and monitored by laser confocal microscopy for 2 h at 10 min intervals. Time between oocyte collection and start of Ca(2+) monitoring was 80.4 ± 2.1 h. MAIN RESULTS AND THE ROLE OF CHANCE: Intracellular levels of Ca(2+) increased under in vitro conditions with no deliberate challenge, as shown by Fluo-3 fluorescence increasing from 61.0 ± 11.8 AU (AU = arbitrary units; n = 23) to 91.8 ± 14.0 AU (n = 19; P < 0.001) after 2 h of monitoring. Pinacidil (100 µM) inhibited this increase in Ca(2+) (85.3 ± 12.3 AU at the beginning of the experiment, 81.7 ± 11.0 AU at the end of the experiment; n = 13; P = 0.616). Glibenclamide (100 µM) also inhibited the increase in Ca(2+) (74.7 ± 10.6 AU at the beginning and 71.8 ± 10.9 AU at the end of the experiment; n = 13; P = 0.851. DNP (100 mM) induced an increase in intracellular Ca(2+) that was inhibited by glibenclamide (100 µM; n = 9) but not by pinacidil (100 µM; n = 5). LIMITATIONS, REASONS FOR CAUTION: Owing to clinical and ethical considerations, it was not possible to monitor Ca(2+) in MII oocytes immediately after retrieval. MII oocytes were available for our experimentation only after unsuccessful IVF or ICSI, which was, on average, 80.4 ± 2.1 h (n = 102 oocytes) after the moment of retrieval. As the MII oocytes used here were those that were not successfully fertilized, it is possible that they may have been abnormal with impaired Ca(2+) homeostasis and, furthermore, the altered Ca(2+) homeostasis might have been associated solely with the protracted incubation. WIDER IMPLICATIONS OF THE FINDINGS: These results show that maintenance of oocytes under in vitro conditions is associated with intracellular increase in Ca(2+), which can be counteracted by drugs targeting K(ATP) channels. As Ca(2+) homeostasis is crucial for contributing to a successful outcome of ART, these results suggest that K(ATP) channel openers and blockers should be tested as drugs for improving success rates of ART. STUDY FUNDING/COMPETING INTERESTS: University of Dundee, MRC (MR/K013343/1, MR/012492/1), NHS Tayside. Funding NHS fellowship (Dr Sarah Martins da Silva), NHS Scotland. The authors declare no conflicts of interest.

Clinically relevant enhancement of human sperm motility using compounds with reported phosphodiesterase inhibitor activity.

STUDY QUESTION: Can we identify compound(s) with reported phosphodiesterase inhibitor (PDEI) activity that could be added to human spermatozoa in vitro to enhance their motility without compromising other sperm functions? SUMMARY ANSWER: We have identified several compounds that produce robust and effective stimulation of sperm motility and, importantly, have a positive response on patient samples. WHAT IS KNOWN ALREADY: For >20 years, the use of non-selective PDEIs, such as pentoxifylline, has been known to influence the motility of human spermatozoa; however, conflicting results have been obtained. It is now clear that human sperm express several different phosphodiesterases and these are compartmentalized at different regions of the cells. By using type-specific PDEIs, differential modulation of sperm motility may be achieved without adversely affecting other functions such as the acrosome reaction (AR). STUDY DESIGN, SIZE, DURATION: This was a basic medical research study examining sperm samples from normozoospermic donors and subfertile patients attending the Assisted Conception Unit (ACU), Ninewells Hospital Dundee for diagnostic semen analysis, IVF and ICSI. Phase 1 screened 43 commercially available compounds with reported PDEI activity to identify lead compounds that stimulate sperm motility. Samples were exposed (20 min) to three concentrations (1, 10 and 100 µM) of compound, and selected candidates (n = 6) progressed to Phase 2, which provided a more comprehensive assessment using a battery of in vitro sperm function tests. PARTICIPANTS/MATERIALS, SETTING, METHODS: All healthy donors and subfertile patients were recruited at the Medical Research Institute, University of Dundee and ACU, Ninewells Hospital Dundee (ethical approval 08/S1402/6). In Phase 1, poor motility cells recovered from the 40% interface of the discontinuous density gradient were used as surrogates for patient samples. Pooled samples from three to four different donors were utilized in order to reduce variability and increase the number of cells available for simultaneous examination of multiple compounds. During Phase 2 testing, semen samples from 23 patients attending for either routine diagnostic andrology assessment or IVF/ICSI were prepared and exposed to selected compounds. Additionally, 48 aliquots of prepared samples, surplus to clinical use, were examined from IVF (n = 32) and ICSI (n = 16) patients to further determine the effects of selected compounds under clinical conditions of treatment. Effects of compounds on sperm motility were assessed by computer-assisted sperm analysis. A modified Kremer test using methyl cellulose was used to assess sperm functional ability to penetrate into viscous media. Sperm acrosome integrity and induction of apoptosis were assessed using the acrosomal content marker PSA-FITC and annexin V kit, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: In Phase 1, six compounds were found to have a strong effect on poor motility samples with a magnitude of response of ≥ 60% increase in percentage total motility. Under capacitating and non-capacitating conditions, these compounds significantly (P ≤ 0.05) increased the percentage of total and progressive motility. Furthermore, these compounds enhanced penetration into a cervical mucus substitute (P ≤ 0.05). Finally, the AR was not significantly induced and these compounds did not significantly increase the externalization of phosphatidylserine (P = 0.6, respectively). In general, the six compounds maintained the stimulation of motility over long periods of time (180 min) and their effects were still observed after their removal. In examinations of clinical samples, there was a general observation of a more significant stimulation of sperm motility in samples with lower baseline motility. In ICSI samples, compounds #26, #37 and #38 were the most effective at significantly increasing total motility (88, 81 and 79% of samples, respectively) and progressive motility (94, 93 and 81% of samples, respectively). In conclusion, using a two-phased drug discovery screening approach including the examination of clinical samples, 3/43 compounds were identified as promising candidates for further study. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study and caution must be taken when extrapolating the results. Data for patients were from one assessment and thus the robustness of responses needs to be established. The n values for ICSI samples were relatively small. WIDER IMPLICATIONS OF THE FINDINGS: We have systematically screened and identified several compounds that have robust and effective stimulation (i.e. functional significance with longevity and no toxicity) of total and progressive motility under clinical conditions of treatment. These compounds could be clinical candidates with possibilities in terms of assisted reproductive technology options for current or future patients affected by asthenozoospermia or oligoasthenozoospermia. STUDY FUNDING/COMPETING INTERESTS: This study was funded primarily by the MRC (DPFS) but with additional funding from the Wellcome Trust, Tenovus (Scotland), University of Dundee, NHS Tayside and Scottish Enterprise. The authors have no competing interests. A patent (#WO2013054111A1) has been published containing some of the information presented in this manuscript.

Differential expression and regulation by activin of the neurotrophins BDNF and NT4 during human and mouse ovarian development.

The tropomyosin-related kinase (Trk) B neurotrophin receptor is essential for ovarian germ cell survival and primordial follicle formation, but the contributions of its ligands, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4), are unknown. We have investigated their expression and regulation in developing human and mouse ovaries. BDNF expression increased with increasing gestation, expression of human NTF4 and of both Ntf5 and Bdnf in the mouse was unchanged. Bdnf expression was dramatically lower than Ntf5 in the mouse, but levels were comparable in the human. Human fetal ovarian somatic cells expressed BDNF. Activin A selectively regulated BDNF and Ntf5 expression in human and mouse, respectively, identifying an oocyte/somatic signaling pathway which might mediate the pro-survival effects of activin. These data reveal that expression and regulation of the TrkB ligands are differentially controlled in the developing ovaries of humans and mice, and identify BDNF as a potential regulator of germ cell fate in the human fetal ovary.

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.

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.

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.

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.

Increased expression of the FIGLA transcription factor is associated with primordial follicle formation in the human fetal ovary.

The process of primordial follicle formation is central to the determination of a woman's reproductive lifespan, and in humans occurs towards the end of mid-gestation. Gene knockout analysis in the mouse has shown that Figla, a transcription factor specifically expressed in germ cells, is essential for oocytes to survive and form primordial follicles. Our objective was to investigate whether a human homologue present in the genome database plays a similar role in human ovary development. Standard and real-time RT-PCR demonstrated that the human FIGLA gene is expressed in the fetal ovary but not by a range of other tissues, and that expression increases across mid-gestation, rising some 40-fold by the time of primordial follicle formation. The entire coding sequence was cloned and new exonic sequences identified. Electrophoretic mobility shift assays with in vitro-expressed human FIGLA protein showed that, as in the mouse, FIGLA can heterodimerize with E12 protein and bind to the E-box of the human ZP2 promoter. Similar mobility shifts were identified in human fetal ovary extracts. These results suggest that FIGLA is involved in continued oocyte survival as primordial follicles form in the human as in the rodent ovary.