Normozoospermic Donors Research Articles

The efficacy and functional consequences of interactions between human spermatozoa and seminal fluid extracellular vesicles.

Seminal fluid extracellular vesicles (SFEVs) have previously been shown to interact with spermatozoa and influence their fertilisation capacity. Here, we sought to extend these studies by exploring the functional consequences of SFEV interactions with human spermatozoa. SFEVs were isolated from the seminal fluid of normozoospermic donors prior to assessing the kinetics of sperm-SFEV binding in vitro, as well as the effects of these interactions on sperm capacitation, acrosomal exocytosis, and motility profile. Biotin-labelled SFEV proteins were transferred primarily to the flagellum of spermatozoa within minutes of co-incubation, although additional foci of SFEV biotinylated proteins also labelled the mid-piece and head domain. Functional analyses of high-quality spermatozoa collected following liquefaction revealed that SFEVs did not influence sperm motility during incubation at pH 5, yet SFEVs induced subtle increases in total and progressive motility in sperm incubated with SFEVs at pH 7. Additional investigation of sperm motility kinematic parameters revealed that SFEVs significantly decreased beat cross frequency and increased distance straight line, linearity, straightness, straight line velocity, and wobble. SFEVs did not influence sperm capacitation status or the ability of sperm to undergo acrosomal exocytosis. Functional assessment of both high- and low-quality spermatozoa collected prior to liquefaction showed limited SFEV influence, with these vesicles inducing only subtle decreases in beat cross frequency in spermatozoa of both groups. These findings raise the prospect that, aside from subtle effects on sperm motility, the encapsulated SFEV cargo may be destined for physiological targets other than the male germline, notably the female reproductive tract. A male's influence over the biological processes of pregnancy extends beyond the provision of sperm. Molecular signals present in the ejaculate can influence the likelihood of pregnancy and healthy pregnancy progression, but the identity and function of these signals remain unclear. In this study, we wanted to understand if nano-sized particles present in the male ejaculate, called seminal fluid extracellular vesicles, can assist sperm in traversing the female reproductive tract to access the egg. To explore this, we isolated seminal fluid extracellular vesicles from human semen and incubated them with sperm. Our data showed that seminal fluid extracellular vesicles act to transfer molecular information to sperm, but this resulted in only subtle changes to the movement of sperm.

Decreased human sperm motility and vitality after fast gravity load changes in a parabolic flight

Little is known about the effects of low gravity on human gametes. The aim of this study was to analyze if fresh human sperm samples after fast gravity load changes suffered any detrimental effect in comparison to the splits maintained in Earth's gravity. Fifteen fresh samples from normozoospermic donors were analyzed. Statistically significant differences in vitality (69.7 ± 9.9 % vs 72.4 ± 9.7 %, [95 % CI: 0.002,0.07]); motile sperm concentration (23.7 ± 15.3 M/ml vs 31.5 ± 25.1 M/ml, [95 % CI: 1.03,14.65]); grade “a” sperm concentration (8.7 ± 6.5 M/ml vs 11.7 ± 9.9 M/ml, [95 % CI: 0.71,5.28]); percentage of progressive motility sperm (30 ± 12.9 % vs 36 ± 14.3 %, [95 % CI: 0.10,0.37]) and curvilinear velocity VCL: 45.7 ± 12.8 μm/s vs 47.7 ± 13.3 μm/s, [95 % CI: 0.79,3.22]) were observed. No statistical differences were observed in other sperm kinematic parameters, morphology, DNA fragmentation, apoptosis, and oxidative stress. In conclusion, even though it did not result in a total loss, heavy gravity load changes including microgravity causes a significant decrease in sperm vitality and motility suggesting that negative consequences would be even higher if the exposure were longer. The results obtained indicate that further research is really needed before Assisted Reproduction will be considered for the future human reproduction outside the Earth.

Mitochondrial function and reactive oxygen species production during human sperm capacitation: Unraveling key players.

Sperm capacitation is a critical process for male fertility. It involves a series of biochemical and physiological changes that occur in the female reproductive tract, rendering the sperm competent for successful fertilization. The precise mechanisms and, specifically, the role of mitochondria, in sperm capacitation remain incompletely understood. Previously, we revealed that in mouse sperm mitochondrial activity (e.g., oxygen consumption, membrane potential, ATP/ADP exchange, and mitochondrial Ca2+ ) increases during capacitation. Herein, we studied mitochondrial function by high-resolution respirometry (HRR) and reactive oxygen species production in capacitated (CAP) and non-capacitated (NC) human spermatozoa. We found that in capacitated sperm from normozoospermic donors, the respiratory control ratio increased by 36%, accompanied by a double oxygen consumption rate (OCR) in the presence of antimycin A. Extracellular hydrogen peroxide (H2 O2 ) detection was three times higher in CAP than in NC sperm cells. To confirm that H2 O2 production depends on mitochondrial superoxide ( ) formation, we evaluated mitochondrial aconitase (ACO2) amount, activity, and role in the metabolic flux from the sperm tricarboxylic acid cycle. We estimated that CAP cells produce, on average by individual, (59 ± 22)% more in the steady-state compared to NC cells. Finally, we analyzed two targets of oxidative stress: lipid peroxidation by western blot against 4-hydroxynonenal and succinate dehydrogenase (SDH) activity by HRR. We did not observe modifications in lipoperoxidation nor the activity of SDH, suggesting that during capacitation, the increase in mitochondrial H2 O2 production does not damage sperm and it is necessary for the normal CAP process.

Oxidative Stress Induces Changes in Molecular Markers Associated with Ferroptosis in Human Spermatozoa.

Ferroptosis is a type of iron-dependent regulated cell death characterized by increased bioavailability of redox-active iron, loss of GPX4 antioxidant capacity, and oxidation of polyunsaturated fatty acid-containing phospholipids mediated by reactive oxygen species (ROS). The aim of this study was to evaluate the effect of oxidative stress induced by arachidonic acid (AA) on ferroptotic cell death in human spermatozoa. Spermatozoa from normozoospermic donors were exposed to AA (5, 25, and 50 µM) for 1 hour at 37 ℃, including an untreated control. Oxidative stress was confirmed by evaluation of cytosolic and mitochondrial ROS production, viability, mitochondrial membrane potential (ΔΨm) and motility. Subsequently, molecular markers of ferroptosis including iron content, levels of GPX4, SLC7A11, ACSL4, IREB2 and lipid peroxidation were evaluated. The analyses were carried out using either flow cytometry, a microplate reader or confocal laser microscopy. AA-induced oxidative stress showed increased cytosolic and mitochondrial ROS production accompanied by impairedΔΨm, viability and motility in human spermatozoa. These results were associated with biochemical and molecular markers related to ferroptotic cell death including an increase in iron content in the form of ferrous (Fe2+) ions, SLC7A11, ACSL4, IREB2, a decrease in the level of GPX4, and an increase in the level of lipid peroxidation compared to the untreated control. This study revealed that AA-induced oxidative stress induces cell death with biochemical characteristics of ferroptosis in human spermatozoa, demonstrating another mechanism of alteration of sperm function induced by oxidative stress and could establish new therapeutic objectives to prevent the decrease in sperm quality mediated by oxidative stress.

P-094 Dynamics of progesterone-induced intracellular Ca2+ changes in sperm predicts zygote formation rate by IVF: Preliminary results

Abstract Study question Can the overall intracellular Ca2+ dynamics evoked by progesterone stimulation predict the fertilizing potential of a non-normozoospermic semen sample under fertility treatment? Summary answer Some kinetic parameters of the transient intracellular Ca2+ increase evoked by progesterone stimulation correlate with fertilization rates in conventional IVF but not with ICSI. What is known already Stimulation with female hormone progesterone induces the activation of the sperm-specific ion channel CatSper which in turn produces a fast increase in the intracellular Ca2+ concentration ([Ca2+]i). After reaching a maximum [Ca2+]i, different Ca2+ clearance mechanisms become active producing a transient followed by a plateau. We investigated whether the [Ca2+]i dynamics upon progesterone stimulus may be used to assess the fertilizing potential of sperm samples from men undergoing assisted reproduction. Study design, size, duration This prospective study employed unidentified semen samples from 39 non-normozoospermic men (age 38 ± 6 years old) undergoing fertility treatment from March 2022 to the present at CITMER Reproductive Medicine, Mexico City. Frozen semen samples and ART procedures with less than 3 oocytes were excluded. Ten normozoospermic semen samples were also analyzed. The progesterone-dependent [Ca2+]i changes were evaluated using time-lapse flow cytometry. Participants/materials, setting, methods Semen samples were obtained on the same day as oocyte retrieval. Sperm cells were washed and incubated in capacitating HTF media for further use in conventional IVF or ICSI procedures. When available, the surplus cells were stained with vitality and Ca2+-sensitive fluorescent dyes. Fluorescence changes were evaluated by AccuriC6 flow cytometer. Progesterone-induced [Ca2+]i transient as well as the correlations with the fertilization rates were calculated using custom-made R programming language code. Main results and the role of chance The progesterone (1 µM) stimulation evoked a fast and transitory increase in the [Ca2+]i. We then calculated six different kinetics parameters of such responses: basal Ca2+ level, increase and decrease in velocity, maximum Ca2+ level, total response duration, and the area under the curve (AUC). There were no significant differences in any of these parameters between normozoospermic donors (and non-normozoospermic patients. The former group of semen samples were divided into either conventional IVF (n = 17) or ICSI (n = 21). In IVF but not ICSI samples, the duration, and the area under the curve of the progesterone response tended to be higher than in normozoospermic (2.39±0.7 min, p = 0.03, and 113±30 AUF*min, p = 0.18, respectively). Furthermore, both parameters showed a tendency to correlate negatively with the conventional IVF (R= –0.45, p = 0.07 and R= –0. 5, p = 0.04, respectively) but not with ICSI fertilization ratio (number of zygotes/total number of mature oocytes). These results suggest that cells with a better Ca2+ recovery mechanism are well suited for IVF. Interestingly, we observed no difference between the magnitude of the [Ca2+]i increase after progesterone stimulus among donors, IVF, or ICSI samples, as was previously reported. Differences in the concentration of progesterone could explain such results. Limitations, reasons for caution The preliminary nature of the results implies a necessity to increase the number of analyzed semen samples (in progress). It is well known that progesterone induces variable responses even among the same donor cells. Our population-based analysis may be underestimating such single cells variations. Wider implications of the findings Current protocols for sperm analysis fail to successfully predict the fertilizing capability of semen samples from men seeking reproductive treatment. Further research into the molecular regulation of Ca2+ dynamics could offer promising alternatives for clinicians to appropriately choose between IVF and ICSI for each couple. Trial registration number .

P-069 A male factor panel of NGS can identify the genetic cause of more than half patients with seminal alterations

Abstract Study question Is Next Generation Sequencing (NGS) an effective diagnostic tool for sperm abnormalities? Summary answer NGS male factor gene panel can be a potent diagnostic tool for discovering pathogenic variants linked to sperm abnormalities. What is known already Male infertility, responsible for half of infertility cases, often shows as absence or decreased sperm count (azoospermia, criptozoospermia or oligozoospermia), poor motility (asthenozoospermia) or a higher proportion of morphologically abnormal sperm (teratozoospermia). While the spermiogram remains the gold standard for evaluating male fertility, the potential to identify the molecular basis of sperm abnormalities may make NGS a valuable approach for such patients. The aim of our study was to assess NGS panel's effectiveness in diagnosing patients with abnormal sperm count, motility and morphology. Study design, size, duration A prospective study was conducted from May 2021 to May 2022 in 82 patients including 20 patients with oligozoospermia, 19 with asthenozoospermia and 23 with teratozoospermia. No other sperm alterations were taken into account in each group. The control group consisted of 20 normozoospermic healthy donors selected on the basis of normal sperm parameters according to the WHO criteria (2010). Patients carrying Y-chromosome microdeletions or abnormal karyotype were excluded. Participants/materials, setting, methods Genomic DNA extraction from blood-EDTA of the patients was performed using the commercial MagMax DNA MultiSample Ultra kit and the King-Fisher automated extractor (ThermoFisher®). Next Generation Sequencing (NGS) was done using a panel with 426 genes involved in the spermatogenesis process. Panel sequencing for identification of genetic variants was performed using Nextera Enrichment technology (Illumina®). FASTAQ data were processed using BWA and GATK algorithms. VCF files were analysed using Variant Interpreter software and in silico predictors. Main results and the role of chance Data analysis showed that thirty-seven of the sixty-two patients were carriers of pathogenic mutations in at least one of the genes included in the panel (37/62,59.6%). In the oligozoospermic group, eleven patients (11/20,55%) were carriers of pathogenic mutations in:CFTR, CEP290, WDR66, ESR1, DNAI2, POLG, PIWIL3, GNRHR, MSH5 and CTNS. In the asthenozoospermic group, ten out of nineteen patients (10/19,52.6%) were carriers of pathogenic mutations in:HS6ST1, PMS2, CYP19A1, DNAI2, POLG, LRRC6, G6PD, CCDC39 and PIWIL3. In the teratozoospermic group, six of the twenty-three patients (6/23,26%) were carriers of pathogenic mutations in:CFTR, PIWIL3, CYP21A2, SRD5A. No pathogenic mutations were detected in the control group. The analysis showed comparable results among the various spermiogram alterations with no significant difference (p > 0.05). Each variant was found in a single patient per group, except POLG and CCDC39, which were found in two cases in the oligozoospermic and asthenozoospermic group, respectively, also CFTR and PIWIL3 in two teratozoospermic patients. Besides, it is remarkable that some of the pathogenic variants were present in genes in more than one group:CFTR (3/62), DNAI2 (2/62), POLG (3/62) and PIWIL3 (4/62). All identified variants were linked with processes of spermatogenesis such as dynein assembly and DNA integrity. Limitations, reasons for caution The main limitation of this study is the limited number of patients included. Functional studies with a larger cohort of males with seminal disorders is warranted to confidently correlate the genetic variants identified in this analysis with spermatogenic failure. Wider implications of the findings The gene list included in our panel represents a step-forward in the screening of males with altered sperm parameters. Our results may add in the knowledge of male factor infertility to provide etiologic factors towards a personalized treatment and adequate genetic counselling. Trial registration number Not applicable

FeTPPS, a Peroxynitrite Decomposition Catalyst, Ameliorates Nitrosative Stress in Human Spermatozoa.

Excessive levels of reactive nitrogen species (RNS), such as peroxynitrite, promote nitrosative stress, which is an important cause of impaired sperm function. The metalloporphyrin FeTPPS is highly effective in catalyzing the decomposition of peroxynitrite, reducing its toxic effects in vivo and in vitro. FeTPPS has significant therapeutic potential in peroxynitrite-related diseases; however, its effects on human spermatozoa under nitrosative stress have not been described. This work aimed to evaluate the in vitro effect of FeTPPS against peroxynitrite-mediated nitrosative stress in human spermatozoa. For this purpose, spermatozoa from normozoospermic donors were exposed to 3-morpholinosydnonimine, a molecule that generates peroxynitrite. First, the FeTPPS-mediated peroxynitrite decomposition catalysis was analyzed. Then, its individual effect on sperm quality parameters was evaluated. Finally, the effect of FeTPPS on ATP levels, motility, mitochondrial membrane potential, thiol oxidation, viability, and DNA fragmentation was analyzed in spermatozoa under nitrosative stress conditions. The results showed that FeTPPS effectively catalyzes the decomposition of peroxynitrite without affecting sperm viability at concentrations up to 50 μmol/L. Furthermore, FeTPPS mitigates the deleterious effects of nitrosative stress on all sperm parameters analyzed. These results highlight the therapeutic potential of FeTPPS in reducing the negative impact of nitrosative stress in semen samples with high RNS levels.

Human Sperm Head Vacuoles Are Related to Nuclear-Envelope Invaginations.

Nuclear vacuoles are specific structures present on the head of the human sperm of fertile and non-fertile men. Human sperm head vacuoles have been previously studied using motile sperm organelle morphology examination (MSOME) and their origin related to abnormal morphology, abnormal chromatin condensation and DNA fragmentation. However, other studies argued that human sperm vacuoles are physiological structures and consequently, to date, the nature and origin of the nuclear vacuoles remains to be elucidated. Here, we aim to define the incidence, position, morphology and molecular content of the human sperm vacuoles using transmission electron microscopy (TEM) and immunocytochemistry techniques. The results showed that ~50% of the analyzed human sperm cells (n = 1908; 17 normozoospermic human donors) contained vacuoles mainly located (80%) in the tip head region. A significant positive correlation was found between the sperm vacuole and nucleus areas. Furthermore, it was confirmed that nuclear vacuoles were invaginations of the nuclear envelope from the perinuclear theca and containing cytoskeletal proteins and cytoplasmic enzyme, discarding a nuclear or acrosomal origin. According to our findings, these human sperm head vacuoles are cellular structures originating from nuclear invaginations and contain perinuclear theca (PT) components, allowing us to define a new term of 'nuclear invaginations' rather than 'nuclear vacuoles'.

Seminal prolactin is associated with HSP90 transcript content in ejaculated spermatozoa.

Evidence indicates that an imbalance between the production of reactive oxygen species and defense ability of antioxidants has clinical significance in the pathophysiology of male infertility. To investigate the role of seminal prolactin (PRL) in the fertilizing capacity of men, the present study evaluated the associations of seminal PRL levels with semen parameters and heat shock protein 90 (HSP90) transcript abundance in ejaculated spermatozoa. We assessed seminal PRL levels and the abundance of HSP90 transcripts in ejaculated spermatozoa from normozoospermic donors (n=18) and infertile men (n=18). The transcript content of HSP90 in ejaculated spermatozoa was analyzed using real-time polymerase chain reaction. Seminal PRL concentrations in infertile patients were significantly lower (p=0.004) than in fertile controls. Seminal PRL showed relatively good diagnostic power for discriminating infertile men (area under the curve=0.776; 95% confidence interval, 0.568 to 0.934; p=0.005). Significant positive correlations were seen between seminal PRL levels and sperm count (r=0.400, p=0.016) and progressive motility (r=0.422, p=0.010). Infertile patients showed a significantly higher abundance of sperm HSP90 than fertile controls (p=0.040). Sperm HSP90 transcript abundance was negatively correlated with sperm progressive motility (r=0.394, p=0.018). Men with higher seminal PRL levels exhibited a lower abundance of sperm HSP90 transcripts. Our finding demonstrated associations among semen quality, seminal PRL levels, and the abundance of HSP90 transcripts in ejaculated spermatozoa. Seminal PRL may contribute to male fertility by maintaining the seminal antioxidant capacity and may have the potential to act as a diagnostic and prognostic biomarker.

Novel sperm chromatin dispersion test with artificial intelligence-aided halo evaluation: A comparison study with existing modalities.

Sperm chromatin dispersion test is a common and inexpensive technique to assess sperm DNA fragmentation, but its subjectivity in assessing a small number of spermatozoa is a disadvantage. To study the efficacy of a new sperm chromatin dispersion test kit (R10) combined with an artificial intelligence-aided halo-evaluation platform (X12) and compare the results to those of existing sperm DNA fragmentation testing methods. Semen samples from normozoospermic donors (n=10) and infertile men with abnormal semen parameters (n=10) were enrolled. DNA fragmentation indices were examined by multiple assays, including R10, Halosperm G2 (G2), sperm chromatin structure assay, and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick end labeling. In R10 assay, the DNA fragmentation indices were obtained both manually (manual R10) and by X12 (AI-R10). The obtained DNA fragmentation indices were analyzed by agreement analyses. The DNA fragmentation indices obtained by manual R10 and those obtained by AI-R10 showed a strong significant correlation (r=0.97, p<0.001) and agreement. The number of spermatozoa evaluated by AI-R10 was 2078 (680-5831). The DNA fragmentation indices obtained by manual R10 and AI-R10 both correlated with those of G2 (r=0.90, p<0.001; r=0.88, p<0.001). Between the AI-R10 and G2 results, Passing-Bablok regression showed no systematic or proportional difference, and Bland-Altman plots revealed overall agreement and a mean bias of 6.3% with an SD of 6.9% (95% limit of agreement: -7.2% to 19.9%). AI-R10 and sperm chromatin structure assays showed systematic differences with a mean bias of -1.9%, while AI-R10 and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick end labeling revealed proportional differences with a mean bias of -10.7%. The novel sperm chromatin dispersion kit and artificial intelligence-aided platform demonstrated significant correlation and agreement with existing sperm chromatin dispersion methods by assessing greater number of spermatozoa. This technique has the potential to provide a rapid and accurate assessment of sperm DNA fragmentation without technical expertise or flow cytometry.

Ketone Body β-Hydroxy-Butyrate Sustains Progressive Motility in Capacitated Human Spermatozoa: A Possible Role in Natural Fertility.

Background Calorie restriction is recognized as a useful nutritional approach to improve the endocrine derangements and low fertility profile associated with increased body weight. This is particularly the case for dietary regimens involving ketosis, resulting in increased serum levels of ketone bodies such as β-hydroxy-butyrate (β-HB). In addition to serum, β-HB is detected in several biofluids and β-HB levels in the follicular fluid are strictly correlated with the reproductive outcome in infertile females. However, a possible direct role of ketone bodies on sperm function has not been addressed so far. Methods Semen samples were obtained from 10 normozoospermic healthy donors attending the University Andrology Unit as participants in an infertility survey programme. The effect of β-HB on cell motility in vitro was evaluated on isolated spermatozoa according to their migratory activity in a swim-up selection procedure. The effect of β-HB on spermatozoa undergone to capacitation was also assessed. Results Two hours of exposure to β-HB, 1 mM or 4 mM, proved to be ineffective in modifying the motility of freshly ejaculated spermatozoa isolated according to the migratory activity in a swim-up procedure (all p values > 0.05). Differently, sperm maintenance in 4 mM β-HB after capacitation was associated with a significantly higher percentage of sperm cells with progressive motility compared to β-HB-lacking control (respectively, 67.6 ± 3.5% vs. 55.3 ± 6.5%, p = 0.0158). Succinyl-CoA transferase inhibitor abolished the effect on motility exerted by β-HB, underpinning a major role for this enzyme. Conclusion Our results suggest a possible physiological role for β-HB that could represent an energy metabolite in support of cell motility on capacitated spermatozoa right before encountering the oocyte.

Effects of in vitro repaglinide supplementation on improving sperm motility parameters, viability, and DNA integrity in normozoospermic and asthenozoospermic men.

Human sperm motility and hyperactivation (HA) are induced by different factors such as intracellular calcium concentration. Repaglinide is an antidiabetic drug that, via the blocking of ATP-sensitive potassium channels (K-ATP channels), depolarization of the β-cell membrane, and opening of the voltage-gated calcium channels leads to an increase in intracellular calcium. The present study aimed to examine the effects of repaglinide on in vitro sperm motility parameters, viability, and DNA integrity in normozoospermic and asthenozoospermic men. Semen samples were collected from two groups of normozoospermic donors and asthenozoospermic patients. The samples were washed free of seminal plasma and then treated with medium alone (control) or with 100 nM and 1µM concentrations of repaglinide. After 1 h of incubation, percent sperm motility and hyperactivation were assessed; after 2 h of incubation, sperm viability and DNA fragmentation rate were evaluated by the Eosin-Y and acridine orange staining, respectively. In both groups, repaglinide at a concentration of 100 nM and 1µM significantly improved percent sperm motility, hyperactivation, and vital sperms with normal DNA; in specimens from normozoospermic men, the 1µM concentration had a noticeable effect on progressive motility; in samples from asthenozoospermic men, the highest hyperactivation rate was seen at a concentration of 100 nM as compared with the 1µM concentration and controls (p<0.05). Our results suggest that repaglinide can improve sperm motility, hyperactivity, viability, and DNA integrity in both normozoospermic and asthenozoospermic men.

Relationship between serum vitamin D concentration and parameters of gonadal function in infertile male patients.

Vitamin D (vitD) deficiency could affect male reproductive function. Our objective was to investigate the relationship between serum vitD concentrations and hormonal and seminal parameters in infertile patients and to compare the results with those in healthy controls. Infertile patients (n = 29) and normozoospermic healthy donors (n = 27) were recruited for the study. Serum concentrations of vitD, total testosterone, estradiol, and sex hormone-binding globulin were determined using chemiluminescence assays, and free testosterone concentration was determined by radioimmunoassay. Semen analysis was performed as suggested by the World Health Organization. Statistical analysis was conducted using Student's t test, contingency tables, and linear regression studies. VitD concentrations were lower in patients than in controls (p < 0.001). A significant association (p < 0.001) was observed between vitD concentrations <20ng/mL and infertility. In the control group, significant correlations were reported between vitD concentrations >30 ng/mL and the concentrations of testosterone (p < 0.05), free testosterone (p < 0.01), and estradiol (p < 0.05). A direct correlation was found between vitD concentration and percentage of sperm vitality (p = 0.01). VitD also positively correlated with the percentage of progressive sperm motility (p <0.05) and sex hormone-binding globulin concentrations (p < 0.01). VitD may affect male reproductive parameters, and its deficiency could be associated with infertility.

P-024 Identification of spermatogenic infertility phenotypes using next generation sequencing

Abstract Study question Can next generation sequencing (NGS) contribute to diagnoses male idiopathic infertility? Summary answer A male factor gene panel identifies pathogenic variants associated to spermatogenic failure in oligozoospermia and cryptozoospermia patients. What is known already In 50% of cases, infertility is due to a male factor problem. Although the causes of male infertility are heterogeneous, genetic causes account for approximately 30% of cases. Some phenotypes have been associated with specific genetic disorders such as chromosomal abnormalities and chromosome Y microdeletions. However, current genetic studies explain only 4% of cases, whilst most cases of male factor infertility remain without a clear diagnosis. Therefore, new techniques that explain the cause of male infertility are needed. Advances in NGS allowed us to study a large number of genes involved in spermatogenesis process in patients with idiopathic infertility. Study design, size, duration A retrospective study was performed from July 2020 until May 2021. A total of 30 patients with abnormal seminal count parameters (oligozoospermic and cryptozoospermic) were included in the male factor gene panel study. Patients carrying Y-chromosome microdeletions or abnormal karyotype were excluded. The control group included 20 normozoospermic healthy donors selected on the basis of normal semen parameters according to the WHO criteria (2010). Participants/materials, setting, methods Genomic DNA extraction from blood-EDTA of the patients was performed using the commercial MagMax DNA MultiSample Ultra kit and the King-Fisher automated extractor (ThermoFisher®). Next Generation Sequencing (NGS) was done using a panel with 426 genes involved in the spermatogenesis process. Panel sequencing for identification of genetic variants was performed using Nextera Enrichment technology (Illumina®). FASTAQ data were processed using BWA and GATK algorithms. VCF files were analyzed using Variant Interpreter software. Main results and the role of chance After data analysis, we observed that eight of the thirty patients studied were carriers of mutations in least one of the genes included in the panel (8/30, 26.7%). We identified the following pathogenic variants: a missense mutation (Phe1052Val) and a deletion (Phe508del) of CFTR gene (2/30, 6.6%), two frameshifts (Asp128GlufsTer34 and Lys1299Ter) of CEP290 (2/30, 6.6%), a missense mutation (Tyr284Cys) of GNRHR gene (1/30, 3.3%), a missense mutation (Tyr416Cys) of SCN5A gene (1/30, 3.3%), a deletion (Ser83del) of NANOS1 gene (1/30, 3.3%), a stop gained in splice region Arg341Ter of TEX14 gene (1/30, 3.3%), a splicing donor c.362 + 2T&amp;gt;C of ESR2 gene (1/30, 3.3%) and a missense mutation (Ser321Leu) of DNAH5 gene (1/30, 3.3%), which are related to spermatogenesis failure. Additionally, some variants classified as benign have been identified, which are not associated with pathogenicity. All the variants identified are related with male infertility, affecting spermatogenesis process, such as congenital bilateral absence of the vas deferens (CFTR), reproductive system syndrome (CEP190), endocrine disorder (GNRHR, hypogonadotropic hypogonadism), testis expressed (SCN5A), spermatogenic failure (NANOS1, TEX14 and ESR2) and syndromic infertility (DNAH5). Nevertheless, no pathogenic mutations associated to spermatogenic failure were observed in the control group. Limitations, reasons for caution The main limitation of this study is the small number of patients included. Further studies including a higher number of males with idiopathic infertility are warranted to confidently link the genetic variants included in our gene panel to spermatogenic failure. Wider implications of the findings The gene list included in our panel represents a step-forward in the diagnosis screening of males with altered sperm parameters. Our results may add in the knowledge of male factor infertility in order to provide etiologic factors towards a personalized treatment and adequate genetic counselling. Trial registration number Not applicable

P-073 SARS-CoV2 infection in human testis and sperm: in vivo and in vitro studies

Study questionCan severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) enter somatic and germinal cells of human testis or ejaculated sperm, thus affecting male reproductive function?Summary answerThis research provides a biological background of the potential route for infection of SARS-CoV-2 and may enable rapid deciphering of COVID-19-induced male-related reproductive disorders.What is known alreadyEpidemiologic studies suggest a significant male sex susceptibility for severe COVID19 symptoms. SARS-CoV-2 is known to affect certain cell types based on their expression of angiotensin-converting enzyme 2 (ACE2) and Transmembrane serine protease2 (TMPRSS2). ACE2 makes available the binding site for the Spike protein of SARS-CoV-2. TMPRSS2 facilitates virus entry by cleaving the S antigen into S1 (the active binding site). Several studies reported the presence of ACE2 in Leydig and Sertoli cells as well as in germ cells from spermatogonia to spermatozoa. These data suggest that the human testis and gametes are a target for SARS-CoV-2.Study design, size, durationTo address this question, we examined the gene expression profile of SARS-CoV-2-associated receptors and proteases (ACE2-TMPRSS2) as well as their protein expression and localization in testicular tissue of males undergoing diagnostic surgery and in sperm of healthy, normozoospermic donors referring to the Unit of Assisted Reproduction, Siena University Hospital, from April 2020 to January 2022.Participants/materials, setting, methodsAssays were performed on tissue biopsies (n = 3) or on freshly ejaculated sperm of men (n = 6) undergoing routine semen analysis after granting informed consent. To this end, tissue biopsies and ejaculated sperm have been co-cultivated for 5-12h with infected VERO E6 cells. Immunoelectron microscopy, in situ hybridization, and highly sensitive digital droplet PCR analysis, have been used to assess if particles containing SARS-CoV- 2 antigens can be detected in testicular tissue or spermatozoa in vitro.Main results and the role of chanceWe carefully investigated the presence of the SARS-CoV-2 virus and the possible effect on male fertility, both at the molecular and ultrastructural levels. To elucidate the mechanisms underlying virus infection in germ cells and male gametes, we analyzed the expression of both ACE2 and TMPRSS2. Both genes are expressed in testes and ejaculated sperm, confirming the possibility that SARS-CoV2 can enter these cells. We also provided evidence of the expression of the corresponding proteins in both testis and sperm by immunofluorescence assays. The immunofluorescence staining of paraffin-embedded slices of testicular tissue with anti-ACE2 antibody revealed a strong signal in Leydig cells. However, ACE2 staining was also present in human Sertoli cells and was concentrated in the adluminal half of the cell, i.e. surrounding spermatocytes and spermatids.The analysis of TRPMSS2 showed similar results. Indeed, this protease is localized mainly in the interstitium, at the level of Leydig cells. This is an intriguing datum, since, while Leydig cells may be considered as a high-risk cells because of the co-expression of TMPRSS2 and ACE2, germ cells may not be at increased risk of ACE2and TMPRSS2-mediated viral entry and spread, given the lack of co-expression in these testicular cell type.Limitations, reasons for cautionThe low number of analyzed samples may limit the statistical power of this study. Whether the SARS-CoV-2 infection change the gene expression profile of other SARS-CoV-2-associated proteases is under investigationWider implications of the findingsThis validated RT-PCR assay allows reliable screening of SARS-CoV-2 in sperm, useful for investigating the presence of the virus in patients undergoing ART, as well as for explaining the molecular and cellular mechanisms related to the gender specificity of the more severe infection-related symptoms.Trial registration numberNot applicable

Comprehensive chromosome FISH assessment of sperm aneuploidy in normozoospermic males.

Sperm chromosomal abnormalities impact male fertility and pregnancy outcomes. However, the proportion of sperm with chromosomal abnormalities in normozoospermic men remains unclear. Herein, we evaluated sperm aneuploidy for 23 chromosomes to elucidate its incidence in normozoospermic men. Sperm from ten normozoospermic donors were obtained from a human sperm bank and analyzed using fluorescence in situ hybridization. The frequencies of nullisomy, disomy, and diploidy were analyzed along with trisomy, triploidy, tetraploidy, and other numerical abnormalities per chromosome and per donor levels. A total of 248,811 sperm cells were analyzed (average: 24,881 ± 381 cells/donor), of which 246, 658 were haploid, 818 nullisomic, 393 disomic, 894 diploid, 13 triploid, 8 tetraploid, 3 trisomic, and 24 harbored multiple aneuploidies. Among the 22 autosomal and 2 sex chromosomes, the mean frequency of aneuploidy per chromosome was 0.49 ± 0.16%, including 0.33 ± 0.16% for nullisomy and 0.16 ± 0.08% for disomy. The mean frequencies of nullisomy, disomy, and aneuploidy per donor were 0.33 ± 0.13%, 0.16 ± 0.05%, and 0.49 ± 0.13%, respectively. The total frequencies of nullisomy, disomy, diploidy, and aneuploidy per donor were 7.62 ± 3.06%, 3.63 ± 1.12%, 0.36 ± 0.15%, and 11.25 ± 3.05%, respectively. The dominant chromosome numerical abnormalities in normozoospermic men are nullisomy, disomy, and diploidy. Generally, the frequency of nullisomy is higher than that of disomy. The disomy or nullisomy frequencies for each chromosome being gained or lost were not unified and varied; some chromosomes (e.g., chromosomes 21 and 22 and sex chromosomes) are more prone to disomy while some others (e.g., chromosome 3) are more prone to nullisomy.

Autophagy is activated in human spermatozoa subjected to oxidative stress and its inhibition impairs sperm quality and promotes cell death.

Does oxidative stress (OS) activate autophagy in human sperm? Human spermatozoa subjected to OS activate an autophagic response. Autophagy is a regulated pathway of lysosomal degradation which helps eukaryotic cells to maintain or restore homeostasis, being a cellular stress response mechanism. OS is a main cause of impaired sperm function and is linked to male infertility; however, whether OS activates autophagy in human spermatozoa is unknown. Human spermatozoa were exposed separately to ionomycin and hydrogen peroxide in order to induce OS. An untreated control group was included. Sperm cells under OS were then exposed to chloroquine in order to block autophagy. An untreated control and a control incubated only with the OS inducer were included in each experimental setting. For this study, semen samples from normozoospermic donors were used and motile sperm cells were selected by the swim up technique. First, the generation of OS under our experimental conditions was demonstrated by analyzing sperm parameters including viability, reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm) motility and thiol oxidation. Then, proteins involved in autophagy, including the microtubule-associated protein light chain 3 (LC3), particularly LC3-I and LC3-II, autophagy-related 5 (ATG5) and autophagy-related 16 (ATG16) proteins as well as the phosphorylated form of AMP-activated protein kinase (pAMPK) were evaluated in spermatozoa exposed to OS and compared to the untreated control. Finally, the impact of autophagy blocking by chloroquine treatment on sperm quality, metabolic parameters, including glycolysis and oxidative phosphorylation, as well as the cell death markers phosphatidylserine externalization and caspase activation was analyzed. Sperm quality parameters, cell death markers and autophagy-related proteins were analyzed by flow cytometry. Motility was evaluated by the computer-assisted sperm analysis system and metabolic parameters were analyzed using an extracellular flux analyzer. Exposure to ionomycin and hydrogen peroxide promotes OS resulting in increased ROS production and decreased viability, ΔΨm and motility, while increasing thiol oxidation. These alterations were accompanied by a decrease in LC3-I, indicating that autophagy was activated upon OS exposure. Ionomycin also caused an increase in LC3-II, ATG5, ATG16 and pAMPK content. Autophagy blocking of sperm exposed to OS caused deterioration in sperm quality and metabolic parameters as well as an increase in cell death markers. N/A. The study was carried out in vitro using motile sperm from normozoospermic donors; tests on sperm from infertile patients were not carried out. The autophagy blocking plus OS might generate a non-specific response to a highly stressful situation leading to the induction of cell death. Human spermatozoa subjected to OS activate an autophagic response and its blockage results in increased oxidative damage and commits spermatozoa to cell death. These results suggest a crucial role of autophagy as a stress response by male gametes, which contributes to maintaining the functionality and lifespan of ejaculated sperm cells. Detection of autophagy activation in sperm cells ex vivo could be included in semen analysis as a marker of OS, especially in men displaying high levels of seminal ROS. Novel strategies that aim to activate this cellular stress response could improve sperm quality/functionality under natural ejaculate conditions in which increased ROS levels are expected. This work was supported by the Fondo Nacional de Investigación Científica y Tecnológica, Chile (ANID/FONDECYT, Grant number 11170758 to P.U.); the Comisión Nacional de Investigación Científica y Tecnológica, Chile (ANID/CONICYT, Grant number PAI79160030 to P.U.) and the Dirección de Investigación, Universidad de La Frontera. The authors disclose no potential conflicts of interest.

Expression of Apoptosis-Related Genes in Cat Testicular Tissue in Relation to Sperm Morphology and Seasonality-A Preliminary Study.

Simple SummaryProgrammed cell death (apoptosis) is a crucial process in spermatogenesis, responsible for the elimination of abnormal sperm cells and for the reduction of testicular volume outside the breeding season. Poor sperm morphology (teratozoospermia) and lower semen quality out of season are commonly observed in domestic cats, but the exact reasons and mechanisms are not known. The aim of this study was to use gene expression analysis to identify which apoptotic processes and pathways are expressed in the phenomenon of teratozoospermia and seasonality in the domestic cat. The results showed a higher expression of two antiapoptotic genes and one proapoptotic gene during the non-reproductive season, with no differences noted between normozoospermic and teratozoospermic cats. We hypothesize that during the non-breeding season there is a potential detrimental factor which activates a cascade of caspases, against which germ cells mount a defense by producing anti-apoptotic proteins. Further identification of this factor may help in the amelioration of semen quality of cats and improve feline breeding.Apoptosis is a crucial process in spermatogenesis, responsible for the elimination of abnormal sperm cells and testicular regression out of breeding season. The aim of this study was to assess if the expression of apoptosis-related genes in testicular tissue of domestic cats differed: (1) between normozoospermic and teratozoospermic donors, and (2) between reproductive and non-reproductive season. The expression of genes: BCL2L1, BCL2, BAX, BAD, FAS, FASLG, and caspases (CASP3, CASP8, CASP9, and CASP10) was analyzed by qRT-PCR in testicular tissue samples. During non-reproductive season significantly higher expression of two anti-apoptotic genes (BCL2L1 and BCL2) was observed. Additionally, there was a significant higher expression of CASP10 in teratozoospermic cats during non-reproductive than during reproductive season. No differences were noted between normozoospermic and teratozoospermic groups. Upregulation of some genes during the non-reproductive season indicates engagement of apoptotic mechanisms in the seasonal changes of semen quality in cats, however further studies on protein levels and analysis of changes on distinct testicular germinal layers are required. At the same time, teratozoospermia in the general population of cats seems to be not connected with dysregulation of apoptosis in the testes.

Time-Lapse Flow Cytometry: A Robust Tool to Assess Physiological Parameters Related to the Fertilizing Capability of Human Sperm.

Plasma membrane (PM) hyperpolarization, increased intracellular pH (pHi), and changes in intracellular calcium concentration ([Ca2+]i) are physiological events that occur during human sperm capacitation. These parameters are potential predictors of successful outcomes for men undergoing artificial reproduction techniques (ARTs), but methods currently available for their determination pose various technical challenges and limitations. Here, we developed a novel strategy employing time-lapse flow cytometry (TLFC) to determine capacitation-related membrane potential (Em) and pHi changes, and progesterone-induced [Ca2+]i increases. Our results show that TLFC is a robust method to measure absolute Em and pHi values and to qualitatively evaluate [Ca2+]i changes. To support the usefulness of our methodology, we used sperm from two types of normozoospermic donors: known paternity (subjects with self-reported paternity) and no-known paternity (subjects without self-reported paternity and no known fertility problems). We found relevant differences between them. The incidences of membrane hyperpolarization, pHi alkalinization, and increased [Ca2+]i were consistently high among known paternity samples (100%, 100%, and 86%, respectively), while they varied widely among no-known paternity samples (44%, 17%, and 45%, respectively). Our results indicate that TLFC is a powerful tool to analyze key physiological parameters of human sperm, which pending clinical validation, could potentially be employed as fertility predictors.

Localización de arilsulfatasa A durante la incubación in vitro de espermatozoides humanos en medio de capacitación

ObjectiveTo determine the localization and distribution of the ArylsulfataseA receptor (ARSA) in human spermatozoa before and after their incubation in capacitation medium for 1 and 4hours. Material and methodsSemen samples were obtained from five normozoospermic donors. Capacitation was by swim-up technique using capacitation medium for 1 and 4hours. Localization of the ARSA receptor was assessed by indirect immunofluorescence using confocal microscopy. A minimum of 200cells were observed in each physiological condition. ResultsBefore incubation, no representative pattern was observed among the cells positive for this biomarker (8.61%). This percentage increased significantly after incubation in the capacitation medium for 1 and 4hours (61.86% and 63.38% respectively). A majority pattern was observed among the capacitated cells, with intense labelling in the acrosomal region (27.11% and 28.20% after 1 and 4hours respectively). It should be noted that the pattern corresponding to fluorescence at the level of the periacrosomal region was not observed in the spermatozoa prior to incubation. Only after incubation in capacitation medium for 1 and 4hours, 9.13% and 12.78% of cells with such distribution were detected. ConclusionsIn vitro capacitation, regardless of time, favours the immunolocalization of ARSA in the cephalic region of the spermatozoa. The most representative subpopulation after this process was the one in which ARSA was intensely and homogeneously distributed in the acrosome region, involved in primary gamete recognition.