Gym lifestyle factors and male reproductive health: a study into young adult usage and perceptions.

RESEARCH QUESTION: What level of awareness do young adults have regarding the potential impacts of gym lifestyle factors and supplementation on male infertility? DESIGN: Between February and March 2023 a questionnaire (n = 153) was employed to gauge attitudes to and awareness of the effects of male reproductive health and gym lifestyles on male fertility. Two semi-structured focus groups (n = 10 total), stratified by sex assigned at birth, were conducted using a set of discussion topics. RESULTS: The survey revealed a statistically significant difference between male and female awareness of the potential impacts of some forms of high-intensity exercise and protein supplementation on male reproductive health (P = 0.045). Many men do not think about fertility unprompted; the survey revealed that fewer men have thought about their fertility compared with those who are curious about their fertility (P = 4.7 â€¯×  10-5) and those who believe their personal fertility is important to them (P = 8.1 â€¯×  10-6). Men were more likely to make a change in their behaviour if it had a long-term compared with a short-term effect on their fertility (P < 10-5). Five focus group themes surrounding awareness of male reproductive health were extracted. CONCLUSIONS: This work has shown that there is a significant lack of awareness and information surrounding the effects of gym lifestyles on male infertility in a young adult UK population. Crucially, levels of awareness differ significantly between men and women. Men have a potentially alarming lack of concern over their own fertility and how factors such as gym supplements can have negative long-term impacts.

Only the Best of the Bunch-Sperm Preparation Is Not Just about Numbers.

In this Seminar, we present an overview of the current and emerging methods and technologies for optimizing the man and the sperm sample for fertility treatment. We argue that sperms are the secret to success, and that there are many avenues for improving both treatment and basic understanding of their role in outcomes. These outcomes encompass not just whether treatment is successful or not, but the wider intergenerational health of the offspring. We discuss outstanding challenges and opportunities of new technologies such as microfluidics and artificial intelligence, including potential pitfalls and advantages. This article aims to provide a comprehensive overview of the importance of sperm in fertility treatment and suggests future directions for research and innovation.

Axonemal regulation by curvature explains sperm flagellar waveform modulation.

Flagellar motility is critical to natural and many forms of assisted reproduction. Rhythmic beating and wave propagation by the flagellum propels sperm through fluid and enables modulation between penetrative progressive motion, activated side-to-side yaw and hyperactivated motility associated with detachment from epithelial binding. These motility changes occur in response to the properties of the surrounding fluid environment, biochemical activation state, and physiological ligands, however, a parsimonious mechanistic explanation of flagellar beat generation that can explain motility modulation is lacking. In this paper, we present the Axonemal Regulation of Curvature, Hysteretic model, a curvature control-type theory based on switching of active moment by local curvature, embedded within a geometrically nonlinear elastic model of the flagellum exhibiting planar flagellar beats, together with nonlocal viscous fluid dynamics. The biophysical system is parameterized completely by four dimensionless parameter groupings. The effect of parameter variation is explored through computational simulation, revealing beat patterns that are qualitatively representative of penetrative (straight progressive), activated (highly yawing) and hyperactivated (nonprogressive) modes. Analysis of the flagellar limit cycles and associated swimming velocity reveals a cusp catastrophe between progressive and nonprogressive modes, and hysteresis in the response to changes in critical curvature parameter. Quantitative comparison to experimental data on human sperm exhibiting typical penetrative, activated and hyperactivated beats shows a good fit to the time-average absolute curvature profile along the flagellum, providing evidence that the model is capable of providing a framework for quantitative interpretation of imaging data.

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.

The ribosome inhibitor chloramphenicol induces motility deficits in human spermatozoa: A proteomic approach identifies potentially involved proteins.

Mature spermatozoa are almost completely devoid of cytoplasm; as such it has long been believed that they do not contain ribosomes and are therefore not capable of synthesising proteins. However, since the 1950s, various studies have shown translational activity within spermatozoa, particularly during their in vitro capacitation. But the type of ribosomes involved (cytoplasmic or mitochondrial) is still debated. Here, we investigate the presence and activity of the two types of ribosomes in mature human spermatozoa. By targeting ribosomal RNAs and proteins, we show that both types of ribosomes are localized in the midpiece as well as in the neck and the base of the head of the spermatozoa. We assessed the impact of cycloheximide (CHX) and chloramphenicol (CP), inhibitors of cytoplasmic and mitochondrial ribosomes, respectively, on different sperm parameters. Neither CHX, nor CP impacted sperm vitality, mitochondrial activity (measured through the ATP content), or capacitation (measured through the content in phosphotyrosines). However, increasing CP concentrations induced a decrease in total and progressive motilities as well as on some kinematic parameters while no effect was observed with CHX. A quantitative proteomic analysis was performed by mass spectrometry in SWATH mode to compare the proteomes of spermatozoa capacitated in the absence or presence of the two ribosome inhibitors. Among the ∼700 proteins identified in the different tested conditions, 3, 3 and 25 proteins presented a modified abundance in the presence of 1 and 2 mg/ml of CHX, and 1 mg/ml of CP, respectively. The observed abundance variations of some CP-down regulated proteins were validated using Multiple-Reaction Monitoring (MRM). Taken together, our results are in favor of an activity of mitochondrial ribosomes. Their inhibition by CP results in a decrease in the abundance of several proteins, at least FUNDC2 and QRICH2, and consequently induces sperm motility deficits.

'Genes versus children': if the goal is parenthood, are we using the optimal approach?

First medical contact for couples trying for a child will usually emphasise the array of assistance available to 'help them have their own child', usually with options involving ART, after diagnosis. For many poorer prognosis couples, this means repetitive unsuccessful cycles of invasive and stressful treatment. What is sometimes lost at this stage is a reflection on the likelihood of success of different options, which may lead patients to focus on hoping for their own 'genetic' progeny, but failing to consider the alternative and potentially more successful other options, including donation and adoption, for achieving parenthood of a child. Factors not only such as female age but also advanced requirements such as preimplantation genetic testing or even mitochondrial replacement therapies all have reduced chances of success but further tend to reinforce the importance of a genetic link. The financial, physical and psychosocial burden associated with cumulative failure also lead to a higher probability of dropout and consequently an even higher probability of remaining in involuntary childlessness. We advocate formulation of a detailed roadmap for discussion of parenthood, with reference explanation to genetics and epigenetics, which gives due consideration to the psychological effects from the beginning to end of the treatment process, alongside a balanced consideration of the likelihood of treatment success and discussion of other options. Only when we provide patients with the service of a clear and transparent discussion of these matters, we will really realise the true potential of our field, which may then be better considered as assisted families.

On-Demand Electrical Switching of Antibody-Antigen Binding on Surfaces.

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.

The Steroid Metabolome in the Isolated Ovarian Follicle and Its Response to Androgen Exposure and Antagonism.

The ovarian follicle is a major site of steroidogenesis, crucially required for normal ovarian function and female reproduction. Our understanding of androgen synthesis and metabolism in the developing follicle has been limited by the sensitivity and specificity issues of previously used assays. Here we used liquid chromatography-tandem mass spectrometry to map the stage-dependent endogenous steroid metabolome in an encapsulated in vitro follicle growth system, from murine secondary through antral follicles. Furthermore, follicles were cultured in the presence of androgen precursors, nonaromatizable active androgen, and androgen receptor (AR) antagonists to assess effects on steroidogenesis and follicle development. Cultured follicles showed a stage-dependent increase in endogenous androgen, estrogen, and progesterone production, and incubations with the sex steroid precursor dehydroepiandrosterone revealed the follicle as capable of active androgen synthesis at early developmental stages. Androgen exposure and antagonism demonstrated AR-mediated effects on follicle growth and antrum formation that followed a biphasic pattern, with low levels of androgens inducing more rapid follicle maturation and high doses inhibiting oocyte maturation and follicle growth. Crucially, our study provides evidence for an intrafollicular feedback circuit regulating steroidogenesis, with decreased follicle androgen synthesis after exogenous androgen exposure and increased androgen output after additional AR antagonist treatment. We propose that this feedback circuit helps maintain an equilibrium of androgen exposure in the developing follicle. The observed biphasic response of follicle growth and function in increasing androgen supplementations has implications for our understanding of polycystic ovary syndrome pathophysiology and the dose-dependent utility of androgens in in vitro fertilization settings.

Glyph-Based Video Visualization for Semen Analysis.

The existing efforts in computer assisted semen analysis have been focused on high speed imaging and automated image analysis of sperm motility. This results in a large amount of data, and it is extremely challenging for both clinical scientists and researchers to interpret, compare and correlate the multidimensional and time-varying measurements captured from video data. In this work, we use glyphs to encode a collection of numerical measurements taken at a regular interval and to summarize spatio-temporal motion characteristics using static visual representations. The design of the glyphs addresses the needs for (a) encoding some 20 variables using separable visual channels, (b) supporting scientific observation of the interrelationships between different measurements and comparison between different sperm cells and their flagella, and (c) facilitating the learning of the encoding scheme by making use of appropriate visual abstractions and metaphors. As a case study, we focus this work on video visualization for computer-aided semen analysis, which has a broad impact on both biological sciences and medical healthcare. We demonstrate that glyph-based visualization can serve as a means of external memorization of video data as well as an overview of a large set of spatiotemporal measurements. It enables domain scientists to make scientific observation in a cost-effective manner by reducing the burden of viewing videos repeatedly, while providing them with a new visual representation for conveying semen statistics.

Evolving minimum standards in responsible international sperm donor offspring quota.

An international working group was established with the aim of making recommendations on the number of offspring for a sperm donor that should be allowable in cases of international use of his sperm. Considerations from genetic, psychosocial, operational and ethical points of view were debated. For these considerations, it was assumed that current developments in genetic testing and Internet possibilities mean that, now, all donors are potentially identifiable by their offspring, so no distinction was made between anonymous and non-anonymous donation. Genetic considerations did not lead to restrictive limits (indicating that up to 200 offspring or more per donor may be acceptable except in isolated social-minority situations). Psychosocial considerations on the other hand led to proposals of rather restrictive limits (10 families per donor or less). Operational and ethical considerations did not lead to more or less concrete limits per donor, but seemed to lie in-between those resulting from the aforementioned ways of viewing the issue. In the end, no unifying agreed figure could be reached; however the consensus was that the number should never exceed 100 families. The conclusions of the group are summarized in three recommendations.

Sperm motility: is viscosity fundamental to progress?

The success of internal fertilization is reliant upon successful sperm migration through the female tract. Timely location of the oocyte in what is a complex three-dimensional, highly invaginated series of moist opposed surfaces is a challenge at which only tens of sperm ever succeed. In part this could be due to the differences in scale, with a 50 µm long cell facing a probable migration of well over 20 cm due to the complex architecture. Many groups have focused upon the role for a chemotactic 'attractive egg' effect in guiding sperm to increase numbers at the fertilization site. What most research has neglected to consider is the role that the viscosity of the mucous layers, which coat the entire tract and through which sperm must swim, plays in both sperm selection and ongoing modulation of their behaviour. From allowing sperm to enter through the cervix during the ovulation phase, to denying them entrance through action of the female contraceptive pill, viscous effects are fundamental in controlling the migrating sperm population throughout the tract. The physiological effects of viscosity are also crucial to consider when designing and extrapolating data from in vitro experiments to the in vivo situation.

Techniques for imaging Ca2+ signaling in human sperm.

Fluorescence microscopy of cells loaded with fluorescent, Ca(2+)-sensitive dyes is used for measurement of spatial and temporal aspects of Ca(2+) signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca(2+)-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca(2+)-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca(2+) response as % change in fluorescence is obtained.

Counting sperm does not add up any more: time for a new equation?

Although sperm dysfunction is the single most common cause of infertility, we have poor methods of diagnosis and surprisingly no effective treatment (excluding assisted reproductive technology). In this review, we challenge the usefulness of a basic semen analysis and argue that a new paradigm is required immediately. We discuss the use of at-home screening to potentially improve the diagnosis of the male and to streamline the management of the sub-fertile couple. Additionally, we outline the recent progress in the field, for example, in proteomics, which will allow the development of new biomarkers of sperm function. This new knowledge will transform our understanding of the spermatozoon as a machine and is likely to lead to non-ART treatments for men with sperm dysfunction.

Non-genomic steroid actions in human spermatozoa. "Persistent tickling from a laden environment".

As sperm traverse the female tract from vagina to oocyte, they experience a steroid milieu, which due to transcriptional inactivity, they can only respond to via non-genomic signaling. This environment mediates events including capacitation, changes in motility patterns, chemotaxis, and acrosome reaction. Current knowledge of the events, calcium signaling pathways, and potential identity of receptors involved is reviewed in light of recent data, with a context for further work in the field, and emphasizing the importance of steroids as a mixed stimulant. Progesterone receptor candidates are considered in light of recent findings, including novel classes of receptors such as a progesterone membrane receptor component-1 or -2 complex with serpine-1 mRNA binding protein, the best candidate so far for progesterone activity in human sperm. Given the number of other alternative candidates and the apparent diversity of the signaling pathways activated, the presence of multiple species of progesterone receptors should not be excluded. Given that sperm dysfunction is the most common defined cause of infertility, advances in our currently limited knowledge of these pathways and events are crucial to not only create better therapies but also improve rational diagnosis.

Kinetics of the progesterone-induced acrosome reaction and its relation to intracellular calcium responses in individual human spermatozoa.

Progesterone at 3 microM triggers a biphasic (transient and sustained) increase in intracellular calcium ([Ca(2+)](i)) in human sperm, which is believed to be a prerequisite for progesterone-induced acrosome reaction (AR). As very little is known about how AR occurrence, latency, and completion relate to the characteristics of the progesterone-induced [Ca(2+)](i) signal, we examined these events using fluorescence microscopy of individual living human sperm. Direct assessment of acrosomal status after calcium imaging showed no differences in kinetics or amplitude of the preceding progesterone-induced calcium responses in acrosome-reacted and acrosome-intact cells, which indicates that the amplitude of the [Ca(2+)](i) signal is not the critical determinant of AR. Chelation of extracellular calcium to arrest AR at varying times after progesterone stimulation revealed that maximal AR occurred immediately following progesterone stimulation, during the initial transient calcium influx rather than during the sustained calcium response. Attempts to follow acrosomal dispersal in real-time by staining with the acidic organelle probes LysoTracker DND-99 and dapoxyl (2-aminoethyl) sulphonamide (DAES) proved inconclusive due to heterogeneous labeling of the cell population. Surprisingly, the dye was often not confined to the acrosome but stained the whole sperm head, which suggests that only a subpopulation of human sperm cells contains a sufficiently acidic acrosome.

Slow calcium oscillations in human spermatozoa.

We have used single-cell imaging to investigate intracellular Ca2+ signalling in human spermatozoa stimulated with progesterone (3 microM). In approx. 9% of cells progesterone caused the activation of slow repetitive [Ca2+]i (intracellular Ca2+ concentration) oscillations, with a period of 1-4 min, which persisted for the duration of recording (20-30 min). Pretreatment with nifedipine, which blocks T- and L-type voltage-operated Ca2+ channels in spermatogenic cells, did not modify the characteristics of the oscillations, but reduced the proportion of cells in which they were observed. Stimulation with Bay K 8644 or FPL64176 induced [Ca2+]i oscillations in 5-10% of cells, but their frequency was low (period, 4-5 min). Application of valinomycin (1 microM) to clamp membrane potential at E(K) (equilibrium potential for potassium) did not modify activity in oscillating cells, showing that plasma membrane potential and activation of voltage-operated conductances are not involved in the mechanism by which sperm [Ca2+]i oscillations are generated.

Encoding of progesterone stimulus intensity by intracellular [Ca2+] ([Ca2+]i) in human spermatozoa.

Progesterone induces a biphasic Ca(2+) influx and consequent acrosome reaction in human spermatozoa. We have used two procedures to vary the stimulus (dosage and prior receptor desensitization) to investigate the encoding of stimulus strength by intracellular [Ca(2+)] ([Ca(2+)](i)). Acrosome reaction and amplitude (but not kinetics) of the transient [Ca(2+)](i) response (population measurement) showed sigmoidal dose sensitivity over the range 0.3 nM-3 microM, saturating at approximately 300 nM (ED(50) approximately 30 nM). The amplitude of the sustained response saturated at 3 microM. Single-cell imaging showed that the amplitudes of both transient and sustained [Ca(2+)](i) responses were highly dose-dependent, but that their frequency of occurrence and kinetics were largely dose-independent. Fluorimetric measurements confirmed that progesterone-induced [Ca(2+)](i) influx was subject to desensitization, with second and subsequent applications of 3 microM progesterone being ineffective. However, sequential additions of 3 nM, 30 nM and 3 microM progesterone generated transient [Ca(2+)](i) responses at each concentration, the amplitude and duration of the response to 3 microM progesterone being reduced compared with non-pretreated cells. Single-cell imaging revealed that pretreatment had no effect on the proportion of responsive cells, but single-cell responses, similarly to population responses, were smaller and markedly reduced in duration, consistent with an effect of desensitization on a late component of the [Ca(2+)](i) transient. We conclude that the strength of the progesterone stimulus, when varied by dosage or by desensitization, is encoded by an analogue [Ca(2+)](i) signal. Dose dependency of the acrosome reaction is therefore determined not by the number of progesterone-responsive cells but by variation in the probability of exocytosis in a 'constant' responsive population.