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Timing of sperm capacitation varies reproducibly among men

Sperm must mature functionally in the process of capacitation to become able to fertilize. Capacitation depends on membrane lipid changes, and can be quantitatively assessed by redistribution of the ganglioside GM1, the basis of the Cap‐Score™ sperm function test. Here, differences in Cap‐Score were compared among and within men at two time points. Ejaculates were liquefied, washed, and incubated for 3 hr under capacitating (Cap) conditions, then fixed and analyzed immediately (Day0); after being incubated 3 hr under Cap conditions then maintained 22–24 hr in fix (Day1‐fix); or after 22–24 hr incubation under Cap conditions prior to fixation (Day1). In all cases, a light fixative previously shown to allow membrane lipid movements was used. Day1‐fix and Day1 Cap‐Scores were greater than Day0 (p < 0.001; n = 25), whereas Day1‐fix and Day1 Cap‐Scores were equivalent (p = 0.43; n = 25). In 123 samples from 52 fertile men, Cap‐Score increased more than 1SD (7.7; calculated previously from a fertile cohort) from Day0 to Day1‐fix in 44% (54/123) of the samples. To test whether timing of capacitation was consistent within an individual, 52 samples from 11 fertile men were classified into either “early” or “late” capacitation groups. The average capacitation group concordance within a donor was 81%. Median absolute deviation (MAD; in Cap‐Score units) was used to assess the tightness of clustering of the difference from Day0 to Day1‐fix within individuals. The average (2.21) and median (1.98) MAD confirmed consistency within individuals. Together, these data show that the timing of capacitation differed among men and was consistent within men.

other species because of cost. Instead, the completion of capacitation at a single cell level has historically been assessed by a sperm's ability to undergo acrosome exocytosis (AE), or at a population level by looking at relative amounts of protein tyrosine phosphorylation events (Visconti & Kopf, 1998;. Recently, we defined representative patterns of localization of the ganglioside G M1 that could be used to identify capacitated human sperm at the single cell level  quantitatively provide information at the population level . Based on multiple lines of investigation regarding these patterns, including the observation that sperm having "capacitated" patterns were in fact those cells that could undergo AE, and demonstrations of the precision and repeatability of the assay , we defined the Cap-Score™ as the percentage of sperm having G M1 localization patterns consistent with capacitation in relation to the total number of sperm having G M1 localization patterns. These findings were consistent with earlier findings in the mouse and bull (Selvaraj et al., 2007), but more importantly, they had clinical relevance.
In an independently-performed clinical study that was included in the recent papers, Cap-Scores™ over a certain threshold were highly correlated with paternity by natural conception or within three or fewer cycles of intrauterine insemination, while those below that threshold were less likely to conceive . Because that study was performed at a tertiary care fertility clinic with a patient base atypical for most urologists and local fertility clinics, we performed a cohort comparison trial, comparing Cap-Scores™ in a population of 76 men with proven fertility versus 122 men questioning their fertility at a local urology center.
Significantly more men questioning their fertility had Cap-Scores™ at or below one standard deviation (SD) below the mean of the fertile men . These and other studies validated the use of the Cap-Score™ to assay sperm capacitation/function and to reflect male fertility.
Before one can determine how knowledge of capacitation status might best be used clinically, one must understand the process at a molecular level. It is known that capacitation is regulated both negatively and positively by interactions with factors in seminal plasma and the female reproductive tract. This modulation ensures that spermatozoa attain maximal fertilizing ability at the site of fertilization (Fraser, 1999). First, during epididymal transit and then ejaculation, the sperm surface interacts with components known as "decapacitation factors." These help keep the sperm in a non-capacitated state until they are progressively released during capacitation (Boué, Blais, & Sullivan, 1996;Fraser, Harrison, & Herod, 1990;Kawano, Yoshida, Iwamoto, & Yoshida, 2008). In concert with the release of decapacitation factors, exposure to extracellular Ca 2+ , HCO 3 − , and mediators of sterol efflux such as HDL and/or albumin must occur (Ehrenwald, Foote, & Parks, 1990;Langlais & Roberts, 1985;Travis & Kopf, 2002). These induce one or more signal transduction pathways involving cAMP, protein kinase A (PKA), and tyrosine kinases . In addition, removal of cholesterol decreases the cholesterol/phospholipid ratio, increasing membrane fluidity (Kopf, Visconti, & Galantino-Homer, 1999), stimulating phospholipase B (PLB) activity (Asano, Nelson-Harrington, & Travis, 2013), and enabling focal enrichment of G M1 (Cohen et al., 2014). In murine sperm, G M1 regulates Ca 2+ transients through the Ca V 2.3 channel, enabling AE in response to Ca 2+ waves induced by CatSper (Cohen et al., 2014).
These alterations in membrane lipids and downstream signaling are highly orchestrated, preventing premature AE and enabling fertilization competence at the right place/time. The importance of this timing of sperm capacitation cannot be overemphasized. Put simply, sperm must be present and capable of fertilizing when the oocyte is present, able to be fertilized, and of high enough quality to produce a viable embryo with high developmental potential (Wang & Sun, 2007). Oocytes arrested at the MII stage are usually fertilized shortly after ovulation, with the window for optimal fertilization differing among species: mouse (8-12 hr), rat (12-14 hr), and human (<24 hr) (Austin, 1974 (Giojalas, Rovasio, Fabro, Gakamsky, & Eisenbach, 2004). Conversely, the ability of rabbit sperm to undergo AE peaks at 16 hr after insemination-precisely matching the timing of the rabbit oocyte in this species characterized by induced ovulation (Giojalas et al., 2004).
In addition to these differences in capacitation timing among species, differences in timing of capacitation have been reported among individuals of the same species. In a comparison of eight men, it was shown that the ability of sperm to penetrate hamster zonae pellucidae was temporally dependent upon the whole cell cholesterol:phospholipid ratio, with lower ratios being consistent with faster capacitation times (Hoshi, Aita, Yanagida, Yoshimatsu, & Sato, 1990). Although of limited sample size, this finding has intriguing clinical implications. If fertilization does not occur within the optimal time window, unfertilized oocytes remaining in the oviduct or culture will undergo a time-dependent deterioration in quality, a process called "oocyte aging" (Miao, Kikuchi, Sun, & Schatten, 2009 Cap-Score™ was observed from Day0 to Day1-fix (p < 0.001; Figure 1) and from Day0 to Day1 (p < 0.001; Figure 1) in Cap samples. However, no difference was detected between Day1-fix and Day1 samples incubated under Cap conditions (p = 0.43; Figure 1). These observations confirmed that the changes in membrane lipids detected by the Cap-Score™ did occur in the presence of the light fixative, as described previously in murine sperm (Selvaraj et al., 2006), and showed that those changes occurred in the same percentage of cells as if they had just been incubated in the Cap medium.

| Effect of incubation on acrosome exocytosis
Acquiring the ability to undergo AE is a hallmark of capacitation. As we have shown previously in murine (Selvaraj et al., 2007) and human sperm , the Cap-Score™ goes down as capacitated cells undergo AE. This results from the loss of G M1 along with the hybrid plasma/outer acrosomal membrane vesicles, preventing labeling and/or resulting in atypical, "other" G M1 localization patterns, thereby decreasing Cap-Score™ Selvaraj et al., 2007). To confirm that our Cap conditions were indeed capacitating and that Cap-Score™ was accurately reflecting these cells, we compared sperm incubated under Cap conditions versus those incubated under Cap conditions and then treated with the calcium ionophore A23187. In these samples from 10 men, we saw an increase in Cap-Score™ from Day0 to Day1-fix (n = 10; p = 0.03; Figure 2), as was observed in Figure 1. We also saw no difference in Cap-Score™ between Day1-fix and Day1 (p = 0.19; Figure 2). Consistent with expectations from our prior experiments, in which single sperm were labeled with peanut agglutinin (PNA) to assess acrosomal status , we observed significant decreases in Cap-Score™ following treatment with A23187 versus sperm incubated under Cap conditions without ionophore in both Day0 (p = 0.02) and Day1 (p = 0.01) sperm ( Figure 2). Note that we did not assess AE in Day1-fix cells because of the presence of the fixative and its potential effect on proteins involved in membrane fusion.
Although the kinetics of AE triggered by calcium ionophore and progesterone are similar (Sosa et al., 2015), it is generally accepted that progesterone is more physiologically relevant. To further investigate ionophore, demonstrating that sperm with G M1 localization patterns associated with response to capacitating stimuli were indeed capacitated and capable of undergoing stimulated AE.

| Cap-Score™ changes differentially among men
To evaluate whether Cap-Score™ would change equally with time among men, we determined the Cap-Score™ for both Day0 and FIGURE 1 Changes in Cap-Score™ from Day0 to Day1 reflect membrane changes that are associated with capacitation. Samples from 25 fertile donors were incubated under non-capacitating (NonCap) and capacitating (Cap) conditions and then analyzed for Cap-Score™ after 3 hr of incubation (Day0; white bars), after overnight incubation in a light fixative (Day1-fix; grey bars) and after overnight incubation in physiologic media followed by fixation (Day1; patterned bars). The y-axis shows the average Cap-Score™ ± the standard error and the x-axis shows the different incubation treatments. There was a significant difference between Day0 and Day1-fix Cap samples (*p < 0.001) and between Day0 and Day1 (#p < 0.001). However, there was no difference between Day1-fix and Day1 Cap samples (p = 0.43). Differences among NonCap samples are illustrated by different letters (p < 0.05) and show an increase in spontaneous capacitation like events from Day0 to Day1-fix, which was not different than Day1. Samples were compared within a treatment using Fisher's LSD OSTERMEIER ET AL.. Cap-Score™ from Day0 to Day1-fix.

FIGURE 2
Evaluating capacitation status and acrosome exocytosis. Samples from 10 fertile men were exposed to both non-capacitating (NonCap; white bars) and capacitating (Cap; grey bars) media and then analyzed for Cap-Score™ after 3 hr incubation and fixation (Day0), after 3 hr incubation and then maintenance overnight in a light fixative (Day1-fix) and after overnight incubation followed by fixation (Day1).

| Cap-Rate™ consistency within men
Although differences in capacitation timing among individual men are known within the literature (Hoshi et al., 1990), it remains unclear whether these differences are consistent within repeated ejaculates from the same individual. For this evaluation, Cap-Rate™ was determined for 52 samples from 11 fertile donors, with at least four samples per donor. "Early" and "late" capacitation groups were defined based on the Cap-Rate™ being less than (early) or greater than (late) 1 SD (7.7) as defined previously in a population of 76 men of known fertility . The percent concordance in capacitation group was calculated for each donor ( Figure 5). The average (81%) and median (83%) concordance were greater than 50% (p < 0.001),  , showing consistency in timing as well as absolute score.
Although the number of men whose samples were analyzed in the study by Hoshi et al. (1990) was low (n = 8), they found that sperm with a high cholesterol to phospholipid ratio were slower to capacitate (as assessed by penetration of zona-free hamster oocytes) than sperm with a lower ratio. It is intriguing to consider whether relative lipid ratios might contribute to differences in timing of capacitation and through that, to infertility in humans.
In support of this possibility, the cholesterol to phospholipid ratio in sperm of patients with unexplained infertility is about twice that of fertile donors (Sugkraroek, Kates, Leader, & Tanphaichitr, 1991). Sperm of normospermic men who failed to fertilize eggs in vitro were found to be characterized by abnormally high cholesterol content, or by a slow loss, or even an increase in cholesterol during in vitro incubation (Benoff et al., 1993). Why/how the lipid composition of sperm varies among men is unclear. No correlation seems to exist between cholesterol or phospholipid levels found in blood serum and that found in sperm or seminal plasma, suggesting a local regulation within the male reproductive tract (Grizard, Sion, Jouanel, Benoit, & Boucher, 1995).
That a high relative cholesterol content might interfere with fertility makes sense in terms of directly making the plasma membrane more rigid and less fusogenic with the outer acrosomal membrane, or indirectly by reducing PLB activity and/or by reducing the ability of G M1 to regulate Ca 2+ influx, as described above. However, the timing of capacitation for the sperm of a given man might also impact his fertility status. It is essential that sperm and eggs both are fertilization competent and in the same place at the same time for successful conception. Because coitus in humans is not tightly linked to the time of ovulation, one would predict that there is no inherent benefit or deficit associated with having sperm capacitate relatively early or late.
Studies of timing of intercourse show that conception occurs in a 6-day window ending on the day of ovulation (Wilcox, Weinberg, & Baird, 1995), meaning that natural conception very likely occurs both with sperm that we would have defined as early and late capacitators. Indeed, here we found that in men with proven fertility and in those pursuing medical work-ups because they question their fertility, there were roughly similar ratios of individuals having sperm that capacitated relatively early or late. However, one might imagine that couples trying to time coitus relative to ovulation might introduce a systematic bias in their approach that effectively reduces their fertility if they do not have knowledge of the timing of capacitation for that individual man. This problem might be worsened in couples using assisted reproduction, in which timing of intrauterine insemination is usually standardized relative to ovulation, or in which the amount of time given for fertilization in classical in vitro fertilization (IVF) is also standardized.
Because the Cap-Rate™ was consistent among ejaculates within individuals, this information could potentially be used to optimize insemination timing in procedures such as IUI, IVF, and natural conception leading to personalized management of infertility ( Figure 7). Currently, IUI is most often performed within 24-36 hrs after hCG injection, and ovulation can occur over a window of time (26-46 hrs), with an average of approximately 38 hrs after the hCG injection (Andersen, Als-Nielsen, Hornnes, & Franch Andersen, 1995;Testart, Thebault, Souderes, & Frydman, 1982). Oocytes are believed to be fertilization competent for less than 24 hr after ovulation (Austin, 1974;Miao et al., 2009). Thus, inseminations occurring around or after the time of ovulation will introduce a selective bias against men whose sperm take longer to capacitate. Most work comparing the relative efficacy of different timings of IUI concentrate on times after the hCG injection (AboulGheit, 2010 (Fraser & Adeoya-Osiguwa, 2001;Fraser & Osiguwa, 2004). By accelerating the onset/ completion of capacitation in late capacitators, in theory, a greater proportion of the sperm could be functionally synchronized with the oocyte to achieve fertilization through IUI.
It is known that a significant proportion of couples fail this treatment repeatedly and are eventually directed to IVF/ICSI (Guzick et al., 2001;Ombelet et al., 1997;van der Steeg et al., 2011).

FIGURE 7
Current and future algorithms of the IUI process. Currently, IUI is most often performed 24-36 hr after hCG injection. However, few if any techniques are available to identify the significant proportion of couples that will fail this treatment repeatedly and eventually be directed to IVF/ICSI. Use of Cap-Score™ in conjunction with semen analysis (SA) can identify men that have defects in capacitation/sperm function and thus are destined to fail IUI. These individuals would be directed immediately to IVF/ICSI, preventing the emotional, physical, and financial costs associated with failed cycles. Those individuals that pass the Cap-Score™ could be further differentiated into "Early" or "Late" capacitation groups using Cap-Rate™. This classification could help determine whether the man's semen should be inseminated into the patient earlier than 24 hr after hCG injection (favoring late capacitators) or following the current protocol of inseminating 24-36 hr post hCG (favoring early capacitators). If it takes a patient's sperm 24 hr to reach optimal capacitation status (i.e., the man exhibits late capacitation), the common practice of inseminating at 36 hr, close to the time of ovulation, might have reduced success because by the time the sperm cells are fully capacitated, the egg may no longer be viable. Knowledge of the ability of sperm to capacitate and of the timing of capacitation, could thus be used to personalize both the most appropriate technology of assisted reproduction and the optimal timing with which that procedure would be performed

| Specimen collection
Semen samples were collected by manual masturbation from consenting men after a minimum of 2 and a maximum of 5 days of sexual abstinence. Any sample having fewer than 10 × 10 6 motile sperm was discarded. Those men with known fertility provided evidence of fathering a child within the past 3 years or having a currently pregnant partner. Men questioning their fertility had been referred to the New Jersey Urology group for evaluation; the fertility status of their partner was not known or considered.

| Standard sample processing
Samples were processed as previously described .

| Acrosome exocytosis experiments
Sperm were incubated with (Cap) or without (NonCap) CD. Day0-A23187 treatment sperm were incubated with CD for 2.5 hr and then calcium ionophore A23187 (Sigma-Aldrich, St. Louis, MO; catalogue # C7522) was added to a final concentration of 20 μM for 0.5 hr prior to fixation and evaluation. Day1-A23187 sperm were first incubated overnight with CD. The following day, A23187 was added to a final concentration of 20 μM and the cells were incubated for another 0.5 hr, fixed and evaluated.
A 6.36 mM solution of progesterone (P4; SigmaP8783) was prepared in pure ethanol (Sigma 459836), which was further diluted to create a 254.4 μM stock solution in mHTF. This P4 stock solution was stored at −20°C until use. Day0-P4 sperm were incubated with CD for 2.5 hr and then the P4 stock was added to yield a final concentration of 10 μM P4. Sperm were incubated in the presence of P4 for 0.5 hr prior to fixation and evaluation.
Day1-P4 sperm were incubated in the presence of CD overnight.
The following day, P4 was added to a final concentration of 10 μM and the cells were incubated for another 0.5 hr, fixed, and then evaluated.

| Sample labeling
Following incubation and fixation, samples were labeled with 2 μg/ml of Alexa Fluor 488-conjugated CTB (Thermo Fisher, Waltham, MA; catalogue # C34775). After 10 min, 5 μl of the labeled sperm were placed on a microscope slide, overlaid with a cover slip (50 mm no. 1), and moved to an imaging station.

| Cap-Score™ determination
Readers were trained to identify G M1 localization patterns associated with capacitation of human sperm and tested for proficiency as described .

ACKNOWLEDGMENTS
The work was funded by Androvia LifeSciences.