Abstract
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.
Highlights
Sexual reproduction involves the successful fusion of the female and male gametes, a process called fertilization
Plasma membrane (PM) hyperpolarization occurs during sperm capacitation [15,35,36], and this physiological event has been suggested as a predictor of success output during artificial reproduction techniques (ARTs) implementation [26,27]
We established a strategy to evaluate the Em of human sperm employing time-lapse flow cytometry (TLFC) using 3,3 dipropylthiadicarbocyanine iodide (DiSC3(5), abbreviated to Disc), a cationic carbocyanine Em-sensitive probe. This fluorescent dye partitions into the sperm PM according to its Em—when the PM becomes hyperpolarized, Disc will accumulate in the PM due to its cationic nature, while a depolarization favors the dye’s efflux from the cell, resulting in decreased fluorescence [37]
Summary
Sexual reproduction involves the successful fusion of the female (egg or oocyte) and male (sperm) gametes, a process called fertilization. Sperm dysfunctions are considered to be the most frequent etiology of fertility issues [4]. The World Health Organization (WHO) updated the reference values for the seminogram analysis in 2010 [6]. Men who fulfill these reference values are considered normozoospermic and are presumed to be fertile. The usefulness of these parameters as predictors of reproductive outcomes has been on debate for seven decades, given that the reference values observed in fertile men usually overlap with those obtained in men with fertility issues [6,7,8]. The sole evaluation of seminogram parameters is not sufficient to unequivocally establish whether a male individual is fertile or infertile [9,10]
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