Our results suggest that basal sperm membrane potential (Em) is associated with fertilization success in non-normozoospermic patients undergoing conventional IVF, and to a lesser extent with ICSI. Specifically, sperm samples exhibiting more hyperpolarized (i.e., more negative) Em values were associated with significantly higher fertilization rates, whereas depolarized samples (i.e., more positive) were correlated with reduced fertilization success. This relationship was confirmed both by unsupervised clustering and by correlation analysis, highlighting Em as a potential functional biomarker to predict the sperm’s fertilizing capacity in non-normozoospermic patients. These findings are consistent with previous studies in normozoospermic samples showing that a more negative Em is associated with increased rates of hyperactivation, acrosome reaction, and ultimately, fertilization [20, 23].

The relationship between sperm Em and fertilization rates in ART is a developing area of research. Previous studies on normozoospermic samples have demonstrated that a more hyperpolarized Em is associated with higher fertilization rates [20, 23]. Conversely, a depolarized Em is commonly observed in subfertile men and has been linked to lower IVF success [20, 22, 23]. Most previous studies exploring the relationship between sperm Em and fertilization outcomes have been conducted exclusively in normozoospermic donors, limiting their translational relevance. In contrast, our study focuses specifically on non-normozoospermic patients, who represent a large portion of individuals undergoing ART in clinical settings [29]. For instance, in our fertility clinic, over 95% of semen samples are categorized as non-normozoospermic, with teratozoospermia being the most frequent diagnosis. According to WHO criteria, teratozoospermia is defined as < 4% morphologically normal spermatozoa and may be present in isolation or in combination with other abnormalities such as reduced motility (astenoteratozoospermia) or low semen volume (hypoteratozoospermia) [30,31,32]. By evaluating sperm Em in this clinically heterogeneous population, we demonstrated that Em hyperpolarization retains its predictive value even in patients who present abnormal semen parameters, particularly in the context of conventional IVF.

Capacitation in human sperm, as well as in that of several other species, involves significant modifications to the plasma membrane, one of which is membrane hyperpolarization. This process leads to an increase in the membrane negative potential, typically reaching values around − 60 to − 70 mV [12, 33]. The hyperpolarization is primarily driven by the activity of ion channels that mediate the efflux of H⁺ and the influx of K⁺ ions. Sperm that are excessively depolarized may have impaired regulation of these channels, potentially leading to suboptimal conditions for subsequent steps like hyperactivation of motility, the acrosome reaction, sperm–oocyte recognition and fusion, key events required for fertilization [10, 12, 34].

The presence of morphologically abnormal spermatozoa (teratozoospermia) is associated with a defective capacitation process or damage in the integrity of the plasma membrane [32, 35, 36]. Extremely hyperpolarized or premature states could lead to energy depletion (given the link to mitochondrial dysfunction in teratozoospermia) [37, 38] or the premature induction of the acrosome reaction, compromising functional competence before reaching the oocyte [38]. However, in our study, we did not observe a negative correlation in patients with sperm displaying high hyperpolarization regarding fertilization rates.

Measuring the Em using techniques such as flow cytometry provides an effective way to predict the function of teratozoospermic samples, overcoming the limitations of traditional semen analysis.

In this work, we evaluated Em of non-normozoospermic samples undergoing fertility treatment (either IVF or ICSI). Remarkably, using an unsupervised hierarchical clustering analysis of Em values, we identified four distinct patient subpopulations, reflecting substantial inter-individual variability. This variability is likely derived from several factors such as sperm maturity, capacitation status, and lifestyle of the patients, among others. Furthermore, it is known that only a subpopulation of human sperm undergoes Em hyperpolarization during capacitation [12]. Interestingly, we observed a positive association between more hyperpolarized Em values and higher fertilization rates in patients undergoing conventional IVF. However, this relationship was less clear with ICSI patients. This discrepancy is likely attributable to the ICSI procedure itself, which bypasses the physiological barriers that sperm must overcome, combined with the smaller sample for ICSI in our cohort. Furthermore, a potential source of experimental heterogeneity is the variable time elapsed between sample collection and Em measurement. Although this interval was dependent on clinical workflow (when surplus samples became available). Our rationale was that the measurement time was standardized in all cases (approximately 90 min).

Understanding the mechanisms behind sperm Em regulation is essential for comprehending sperm function during fertilization, potentially leading to advancements in male infertility diagnosis. Our study highlights the importance of sperm Em in male fertility and can be considered as a sperm’spredictive parameter of fertilization rates in ART even in non-normozoospermic patients. Notably, the latest WHO semen analysis manual [24] now recommends using complementary functional tests to assess male fertility. In this context, our findings reinforce the idea that Em hyperpolarization, a hallmark of capacitation, is a potential predictor of fertilization success, at least in conventional IVF cycles.

In mouse sperm, hyperpolarization of the sperm Em represents a key and decisive step during sperm capacitation, as it is both necessary and sufficient to prepare sperm for the acrosome reaction [10]. The study by De la Vega-Beltran et al. [10] shows that when the sperm plasma membrane becomes hyperpolarized, functional changes are enabled that allow proper Ca2⁺ signaling in response to physiological stimuli, which is essential for triggering the acrosome reaction. Conversely, preventing this hyperpolarization abolishes the sperm’s ability to respond appropriately, even when other capacitation-associated processes are present [10]. For humans, Em is also considered a critical biophysical marker of fertilizing capacity, as hyperpolarization is an absolute physiological prerequisite for activating essential signaling pathways in spermatozoa [20, 23]. A study demonstrated that a markedly depolarized sperm plasma membrane in subfertile men is strongly associated with reduced fertilization rates in IVF cycles. This condition, present in a significant proportion of patients, likely arises from abnormal potassium conductance or increased inward currents, highlighting defective ion channel function and membrane potential regulation as key contributors to male infertility and IVF failure [22]. In this study, findings derived from hierarchical clustering show that the patient group with the most depolarized profiles exhibits the lowest IVF success rates.

All these studies have demonstrated that depolarization of sperm Em is consistently associated with functional defects that impair fertilization, and Em does not merely reflect the cellular metabolic state but acts as an electrochemical switch that, when remaining in a “depolarized” state, predicts a functional failure in gametic interaction. These findings have helped consolidate current understanding, but the clinical application of Em as a predictive diagnostic marker remains limited due to substantial intra-individual variability across ejaculates [39]. In this context, Em is better regarded as a functional indicator of sperm fertilization competence when evaluated at the time of an ART cycle, as it provides a more accurate reflection of sperm physiological status during the procedure and can be considered a prognostic marker of IVF success.

Measuring Em using techniques such as flow cytometry offers an effective way to predict the function of teratozoospermic samples, overcoming the limitations of traditional semen analysis. Although our TLFC approach shows promise, standardizing Em assessment protocols is needed for broader clinical implementation. Additionally, exploring whether artificial modulation of sperm membrane potential can enhance fertility outcomes warrants further study. A limitation of this study is the lack of complementary diagnostics evaluating the male factor in relation to sperm Em. While our findings are promising, larger cohort studies are necessary to validate these findings and to investigate other sperm alterations, particularly in cases of idiopathic infertility, to better define the potential role of Em in reproductive medicine.