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Volunteer donors and patients were provided with written information about the study prior to giving informed consent. The study protocol was approved by the Bioethics Committee of the Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, protocol #564/2018. The studies are in compliance with the Declaration of Helsinki principles.

Semen samples were obtained by masturbation from healthy donors after 2–5 days of abstinence and analyzed following WHO recommendations (World Health Organization, 2010). Samples that fulfilled semen parameters (total fluid volume, sperm concentration, motility, viability and morphology) according to WHO normality criteria, were considered as normospermic and those that did not fulfilled any of them as non-normospermic. Samples were allowed to liquefy for 1 h at room temperature, then, sperm ejaculates were allowed to swim-up in non-capacitating media at 37°C for 1 h. The non-capacitating medium used was HEPES-buffered human tubal fluid (HTF) containing 90.7 mM NaCl, 4.7 mM KCl, 0.3 mM KH₂PO₄, 1.2 mM MgSO₄, 2.8 mM glucose, 3.4 mM sodium pyruvate, 1.6 mM CaCl₂, 60 mM sodium lactate and 23.8 mM HEPES (pH 7.4), which was supplemented with 20 mM bicarbonate and 5 mg/ml BSA to obtain the capacitating medium. Cells were left to capacitate at 37°C as detailed for each experiment, in the same procedure as that used to prepare spermatozoa for IVF.

In Fertya (Assisted Reproduction Medical Clinic), commercially available HTF media was used for sperm preparation. The spermatozoa were separated from semen by two-layer density gradient centrifugation (Irvine Scientific Isolate) and then washed and concentrated with Quinn’s Advantage Medium with HEPES (Sage) which was supplemented with 10% Serum Protein Substitute (Sage). Washed spermatozoa were incubated to capacitate at 37°C pH 7.4 for 3–4 h prior to performing the IVF.

Cells were loaded with 1 μM of the membrane-potential-sensitive dye DISC₃(5) (Molecular Probes) for at least 5 min. No mitochondrial un-couplers were used because their contribution to the resting potential has been determined to be insignificant (Guzmán-Grenfell et al., 2000). Sperm were then transferred to a gently stirred cuvette at 37°C, and the fluorescence was monitored with a Varian Cary Eclipse fluorescence spectrophotometer at 620/670 nm excitation/emission wavelengths. Recordings were initiated when steady-state fluorescence was reached and calibration was performed at the end of each measure by adding 1 μM valinomycin and sequential additions of KCl for internal calibration curves, as previously described (Ritagliati et al., 2018; Baro Graf et al., 2019). Sperm Em was obtained from the initial fluorescence (measured as Arbitrary Fluorescence Units) by linearly interpolating it in the theoretical Em values from the calibration curve against arbitrary fluorescence units of each trace. This internal calibration for each determination compensates for variables that influence the absolute fluorescence values.

After incubation in the respective conditions, progesterone (21 μM) was added and incubated for another 30 min. Alternatively, in the NC₀ condition, 1 μM valinomycin was added 5 min before the progesterone. Cells were seeded on eight-well glass slides. After air-drying, sperm were fixed with 3.7% formaldehyde in PBS for 15 min at room temperature, permeabilized with 0.5% Triton X-100 for 5 min, washed with PBS and incubated with PBS containing 1% BSA and FITC-conjugated pisum sativum lectin (1/200) for 1 h at room temperature. Before mounting, samples were washed with PBS (four times for 5 min each time). Epifluorescence microscopy was used to assess acrosome status. At least 200 sperm were analyzed in each condition.

Sperm suspensions were loaded on a 30-μm deep slide and placed on a microscope stage at 37°C. Sperm movements were examined using computer-assisted semen analysis (CASA) system (IVOS Sperm Analyzer, Hamilton Thorn). Thirty frames were acquired at a rate of 60 Hz. At least 200 sperm were analyzed in each condition. The following parameters were measured: mean path velocity (VAP, μm/sec), curvilinear velocity (VCL, μm/sec), straight-line velocity (VSL, μm/sec), linearity (LIN, %), amplitude of lateral head displacement (ALH, μm), and straightness (STR, %). Sperm were considered hyperactivated when presenting VCL ≥ 150 μm/sec, LIN < 50%, and ALH ≥ 5 μm. At the time of the analysis, 0.5 mg/ml BSA was added to non-capacitated conditions to avoid sperm adherence to the slide.

In vitro fertilization was performed 3–4 h post sperm preparation in 4-well dishes (Oosafe) with 0.5 ml of Quinn’s Advantage Fertilization medium supplemented with 10% Serum Protein Substitute (Sage) and covered with 0.5 ml of tissue culture oil (Sage). In order to assure good oocyte quality, this study only involved female patients with no detected female factors. All patients were below 38 years old. One thousand sperm were incubated per cumulus-oocyte complex. A maximum of five cumulus-oocyte complex were incubated per well in K-System G185 incubator (37°C, 6.2% CO₂, 5% O₂) during 4 h. After this period, oocytes were denudated and incubated in individual drops in QA Protein plus Cleavage medium (SAGE^®) covered with 0.5 ml of tissue culture oil (Sage) in the same conditions as described above. At 18 h post insemination, fertilization was analyzed with an inverted microscope. Successful fertilization was considered when two pronuclei (2PN) and two distinct or fragmented polar bodies were observed. When at least 60% of eggs were fertilized, the IVF procedure was classified as successful, according to local regulations.

Data are expressed as mean ± standard deviation (SD) or standard error of the mean (SEM), as indicated in each figure, of at least six independent experiments for all determinations. Statistical analyses were performed using the GraphPad Prism 6 software (La Jolla, CA, United States). Student’s t test was used to compare mean values between control and tested groups, while the difference between mean values of multiple groups was analyzed by one-way analysis of variance (ANOVA) with multiple comparison tests. A probability (p) value p < 0.05 was considered statistically significant.

In this work, we performed a fluorimetric population assay to determine the absolute Em values of non-capacitated and capacitated sperm. When constant concentrations of sperm and probe are used, this method provides a highly reproducible value of plasma membrane potential after calibration using valinomycin (a K⁺ ionophore) and sequential additions of KCl (Baro Graf et al., 2019) (Figure 1A). A total of 60 sperm samples were analyzed, among which 49 corresponded to normospermic and 11 to non-normospermic donors, as categorized on the basis of total sperm number, ejaculate volume and motility according to WHO guidelines (see section “Materials and Methods” for details). Consistent with previous observations using flow cytometry (López-González et al., 2014), we found a large heterogeneity among Em values of samples from different individuals. However, normal sperm samples exhibited a significant hyperpolarization after capacitation, while sub-normal samples did not (Figure 1B). The Em changes (Em_CAP – Em_NC) were also very variable between individuals. Both in normospermic and non-normospermic donors we could identify samples with depolarizing, hyperpolarizing or unchanged behaviors (Figure 1C). However, while in normospermic donors the majority of the samples (53.6%) hyperpolarized, among donors with sub-normal parameters, only 27.3% hyperpolarized and 63.6% depolarized (Figure 1D). When samples with different behaviors were pooled and further analyzed, Em changes became more evident. These results demonstrate that in most normospermic individuals there is a capacitation-induced hyperpolarization, with an initial non-capacitated Em value of −37.7 ± 9.9 mV, that shifts to –57.8 ± 12.9 mV upon capacitation (Figure 1E).

The AR is a key step in fertilization. In mammalian sperm, the AR is mediated by intracellular calcium fluxes. Since Ca²⁺ channels in sperm cells are voltage-dependent, it has been hypothesized that gating might be controlled by Em.

We evaluated the role of the Em in the acquisition of acrosomal responsiveness to progesterone (Pg) in samples from normospermic donors. Both Em and AR were evaluated immediately after swim-up (NC₀), as well as after 5 h in either non-capacitating (NC₅) or capacitating (CAP) media. For AR analysis, cells were further incubated for 30 min in the absence or presence of Pg. Non-capacitated sperm obtained after swim-up (NC₀) did not increase the percentage of acrosome reacted sperm in the presence of Pg. However, they acquired acrosomal responsiveness after pharmacological hyperpolarization with valinomycin (1 μM for 5 min) (Figures 2B,C). These results point toward the pharmacological hyperpolarization sufficiency for acrosomal responsiveness, in agreement to previous results in mouse sperm (De La Vega-Beltran et al., 2012). On the other hand, capacitated human sperm hyperpolarized (Figure 2A) and showed significantly increased induced AR (Figures 2B,C). Furthermore, a correlation analysis between Em change upon capacitation (Em_CAP – Em_NC0) and the induced AR (induced – spontaneous), although not statistically significant (p = 0.06), showed a clear negative correlation (r = −0.6337) (Figure 2D). Interestingly, as observed in the correlation analysis between absolute Em values and induced AR for each condition (Figures 2E,F), there is a strong negative correlation for sperm incubated in capacitating (r = −0.8234) media, but not in NC₀ (r = 0.0025).

Hyperactivation is essential for mammalian sperm to fertilize the oocyte (White and Aitken, 1989; Suarez et al., 1993); we therefore aimed to determine if hyperpolarization of human sperm would affect their motility. Hyperactivated motility is defined by an increase in the VCL and the ALH and a decreased LIN: VCL ≥ 150 μm/s, LIN ≤ 50% and ALH ≥ 5 μm/s, being VCL the most characteristic parameter. Figure 3 shows Em values (Figure 3A), percentage of hyperactivated sperm (Figure 3B) and VCL (Figure 3C) of sperm incubated in either non-capacitated or capacitated conditions at different time points. As can be observed, sperm progressively hyperpolarized in time with a maximum at 5 h (which was the longest time assayed), while the maximum HA rate and VCL were at 3 h (Figures 3A–C). In order to assess the role of hyperpolarization in sperm motility, we performed a correlation analysis of Em values with HA and VCL after 3 and 5 h of capacitation (Supplementary Figure S1). Though not significant, there is a tendency in the correlation between Em at 3 h of capacitation and VCL (Supplementary Figure S1A). On the other hand, the correlation was weaker at 5 h of capacitation (Supplementary Figure S1B), which can be explained by the fact that at 5 h sperm further hyperpolarized, while HA did not continue increasing (Figure 3). Finally, the analysis between Em change upon capacitation (Em_CAP3 – Em_NC0) and the VCL or HA values, showed a weak correlation (Supplementary Figure S1C).

Aiming to further investigate the biological role of Em on human sperm fertilization competence, we determined the Em of non-capacitated and capacitated sperm from IVF patients. One important drawback in correlation analyses of human sperm is the biological variation between samples obtained from the same donors on different days. Thus, in order to avoid this issue, the fluorimetric Em measurements were performed on aliquots of the ejaculate used on the day of the IVF treatment, allowing direct comparison of Em values with fertilization success.

In vitro fertilization patients were classified according to their fertilization rate, satisfying local clinical parameters for successful (fertilization rate ≥ 60%) and unsuccessful IVF procedures (fertilization rate < 60%). These two groups showed significant differences between their mean IVF success rates (Figure 4A). In a retrospective analysis, sperm samples with successful IVF rates exhibited a significant Em hyperpolarization during capacitation (Figure 4B). On the other hand, patients with IVF rates under 60% did not hyperpolarize (Figure 4B). As can be seen in Figure 4C, 78.6% of successful IVF samples hyperpolarized during capacitation. However, the majority of samples from poor IVF rate patients (66.7%) showed a depolarizing behavior. These results indicate that the IVF outcome might be dependent on a capacitation-associated hyperpolarization. To test this hypothesis, we performed correlation analysis of absolute Em values obtained after capacitation and IVF rates (Figure 4D), and also of Em changes upon capacitation (Em_CAP – Em_NC) against IVF rates (Figure 4E). In agreement with our previous results (Figure 2) where we observed a correlation between capacitated sperm Em and induced AR, there was a significant correlation between capacitated sperm Em (Em_CAP) and IVF rate. Interestingly, a predictive analysis showed that hyperpolarizing samples have significantly higher IVF rates in comparison with depolarizing samples, as depicted in Figure 4F. Finally, a ROC curve was constructed to assess the effectiveness of capacitated sperm Em in predicting IVF outcomes (Figure 5). The area under the curve was 0.8571 ± 0.098 (95% CI = 0.6647-1), indicating that capacitated sperm Em (Em_CAP) is a good parameter to discriminate between successful IVF rate (>60%) and unsuccessful IVF rate (<60%). The cut off value for Em_CAP with the highest sensitivity and specificity was −48.6 mV (100% sensitivity and 71.4% specificity). Considering our results, a depolarized Em could be related to IVF failure in idiopathic subfertile patients.