Membrane Potential Determined by Flow Cytometry Predicts Fertilizing Ability of Human Sperm

Abstract

Infertility affects 10 to 15% of couples worldwide, with a male factor contributing up to 50% of these cases. The primary tool for diagnosing male infertility is traditional semen analysis, which reveals sperm concentration, morphology, and motility. However, 25% of infertile men are diagnosed as normozoospermic, meaning that, in many cases, normalappearing sperm fail to fertilize an egg. Thus, new information regarding the mechanisms by which sperm acquire fertilizing ability is needed to develop a clinically feasible test that can predict sperm function failure. An important feature of sperm fertilization capability in many species is plasma membrane hyperpolarization (membrane potential becoming more negative inside) in response to signals from the egg or female genital tract. In mice, this hyperpolarization is necessary for sperm to undergo the changes in motility (hyperactivation) and acrosomal exocytosis required to fertilize an egg. Human sperm also hyperpolarize during capacitation, but the physiological relevance of this event has not been determined. Here, we used flow cytometry combined with a voltage-sensitive fluorescent probe to measure absolute values of human sperm membrane potential. We found that hyperpolarization of human sperm plasma membrane correlated positively with fertilizing ability. Hyperpolarized human sperm had higher in vitro fertilization (IVF) ratios and higher percentages of acrosomal exocytosis and hyperactivated motility than depolarized sperm. We propose that measurements of human sperm membrane potential could be used to diagnose men with idiopathic infertility and predict IVF success in normozoospermic infertile patients. Patients with depolarized values could be guided toward intracytoplasmic sperm injection, preventing unnecessary cycles of intrauterine insemination or IVF. Conversely, patients with hyperpolarized values of sperm membrane potential could undergo only conventional IVF, avoiding the risks and costs associated with intracytoplasmic sperm injection.

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

“…In this experiment, which was used as a representative example, the calculated resting E m value for the sperm subpopulation was −65 mV ( Figure 2 c, gray histogram). This resting E m value is consistent with previously reported data [ 26 , 27 ], strongly suggesting that our TLFC method is suitable for measuring absolute E m values in the human sperm model. As a proof of concept, we employed this method to evaluate the E m of sperm samples incubated in either non-capacitating (NC) or capacitating (CAP) conditions.…”

“…PM hyperpolarization occurs during sperm capacitation [ 15 , 35 , 36 ], and this physiological event has been suggested as a predictor of success output during ART implementation [ 26 , 27 ]. For this reason, we established a strategy to evaluate the E m of human sperm employing TLFC using 3,3′dipropylthiadicarbocyanine iodide (DiSC 3 (5), abbreviated to Disc), a cationic carbocyanine E m -sensitive probe.…”

“…Among these processes we can distinguish (a) changes in the plasma membrane’s ion permeability, which result in its hyperpolarization ([ 15 , 16 , 17 ], reviewed in [ 18 ]); (b) an increase in intracellular pH (pH i ) [ 19 , 20 , 21 ]; and (c) a rise in intracellular calcium concentration ([Ca 2+ ] i ) upon exposure to progesterone (Pg, a female hormone secreted by cumulus oophorus cells that surround the oocyte) [ 22 , 23 , 24 ]. These parameters have been independently analyzed in sperm samples from men with fertility issues, and results show that after capacitation induction, their sperm cells do not undergo pH i alkalinization, and they also fail to hyperpolarize (in some cases they depolarize) their plasma membrane (PM) [ 17 , 25 , 26 , 27 , 28 ]. Regarding [Ca 2+ ] i changes, some reports suggest that sperm cells from men with fertility issues present a decreased response to Pg [ 29 , 30 ].…”

“…These procedures present the disadvantage of only permitting the analysis of a limited number of cells, which can mask heterogeneous responses and therefore yield inaccurate results. Other methods include population-based approaches, such as spectrofluorometry [ 17 , 26 ] and flow cytometry [ 27 , 28 ], which analyze the behavior of an entire sperm cell sample population. Spectrofluorometry poses the disadvantage of only permitting the recording of average responses, and as a result, some non-homogeneous responses may also be masked.…”

Membrane Potential Assessment by Fluorimetry as a Predictor Tool of Human Sperm Fertilizing Capacity

“…During preparation of this manuscript, we contacted the group of Dr. Celia Santi at Washington University School of Medicine in Saint Louis, who independently achieved the same results using a different methodology. Both this manuscript and Santi's work are intended to be published concurrently (Puga Molina et al, 2020).…”