Removal of spermatozoa with externalized phosphatidylserine from sperm preparation in human assisted medical procreation: effects on viability, motility and mitochondrial membrane potential

Human sperm quality is defined by the classical parameters, concentration, motility and morphology, according to standard World Health Organisation diagnostic semen analysis [1]. Nevertheless, hidden anomalies affecting spermatozoa membranes such as externalization of the phosphatidylserine (EPS), or spermatozoa organelles such as the disruption of mitochondrial membrane potential (MMP), caspases activation and/or DNA fragmentation, are common somatic features of apoptosis. Such features are not routinely detected in ejaculated spermatozoa but they have been proven to have a negative impact on assisted medical procreation (AMP) outcome [2‐8]. Indeed, successful assisted reproduction is mainly dependent on the quality of the sperm plasma membrane, requiring normal integrity and function to provide motility, acrosome reaction, and fertilization [9]. Spermatozoa with impaired membrane integrity occur more frequently in infertile men, partly explaining suboptimal results in AMP. However, striking modifications of sperm plasma membrane occur physiologically in ejaculated sperm. During capacitation, there is an AC/cAMP/PKA-dependent lipid remodelling of the sperm plasma membrane due to phospholipids translocation that lead to externalization of phosphatidylserine (PS) and phosphatidylethanolamine, and to an albumin mediated efflux of cholesterol resulting in an increase in membrane fluidity [10‐16].

Apoptosis plays a critical role in many normal biological processes such as embryogenesis and tissue homeostasis, as well as in pathological states and diseases. Apoptosis causes disruption of membrane, phospholipid asymmetry and translocation of PS, a signal for specific recognition and removal of apoptotic cells by phagocytosis [17‐20]. Annexin V, an anticoagulant protein of 35.8 kDa, binds outwardly exposed PS with high affinity in a calcium-dependent manner [21]. Thus, the combined use of annexin V and of a supravital fluorescent dye, such as Hoechst 33258, allows the simultaneous detection of apoptotic and necrotic cells [22]. Currently, it is known that germinal cells apoptosis is an underlying mechanism of normal spermatogenesis and an altered apoptotic process has been found to be closely associated with abnormal spermatozoa in semen and thus with male infertility [23‐26]. Indeed, Maeda et al. [27] inhibited sperm production in mice by annexin V microinjection into seminiferous tubules, showing that the phagocytic clearance of apoptotic spermatogenic cells by Sertoli cells is PS-mediated and required for a normal production of sperm. In contrast, apoptosis in ejaculated sperm is less well understood and remains controversial [28]. Few reports applied the detection of EPS, by annexin V-labeling, for the evaluation of cryopreservation techniques on ram [29, 30], boar [31, 32], bull [33‐35], and human [36‐43] spermatozoa. Other human sperm studies attempted to relate EPS, DNA fragmentation and/or apoptotic proteins with sperm quality parameters [6, 7, 15, 44‐56]. It comes out from the literature that EPS on ejaculated mature spermatozoa is either the result of a plasma membrane modification because of capacitation and/or acrosome reaction [11, 15, 54] or the sign of an early apoptotic phenotype [45, 57]. Sakkas et al. [2, 58] proposed that apoptosis in ejaculated sperm is the result of a spermatogenetic failure, thus an abortive apoptotic process that started before ejaculation [59]. It has also been described that inflammation [60‐62] and bacteria can induce EPS in human spermatozoa [63]. Nevertheless, the link between EPS and sperm apoptosis is still controversial, although it is accepted that annexin V binding to spermatozoa characterizes modified sperm plasma membrane. Interesting results on EPS in relationship with infertility problems in AMP are those of collaborators at the Reproductive Research Center of the Cleveland Clinic (USA), and at the Department of Dermatology and Andrology of Leipzig University (Germany) [39, 51, 64‐70] who adapted the technique of magnetic-activated cell sorting using annexin V-conjugated microbeads (MACS-ANMB) to sperm preparations in order to evaluate the impact of EPS on sperm parameters and fertility potential.

In the present study, we have characterized EPS on spermatozoa after DGC sperm preparation and tested its impact on the quality of sperm prepared for AMP, an essential element for successful fertilization and reproductive success. EPS is the main sperm surface apoptotic marker but its role in ejaculated sperm remains ambiguous. We have investigated the effects of removal of EPS-positive spermatozoa on the motility and MMP integrity of prepared spermatozoa in patients consulting for infertility. To this end, we have depleted sperm preparations of EPS spermatozoa by affinity to annexin V. The patient population studied, consulting for infertility, presented subobptimal semen characteristics and could benefit from removal of EPS sperm, using ANMB-MACS from sperm preparations. This study has tested the feasibility of such purification given the limited amounts of viable spermatozoa available in the ejaculates of these patients. These data allow to draw guidelines for the clinical application of this purification in low concentration and/or bad quality sperm.

Firstly, we determined the proportion of EPS-positive spermatozoa in noncapacitated and capacitated spermatozoa, and secondly we precisely localized PS on spermatozoa among sperms of patients consulting our infertility unit. Further, we compared raw sperm with two sperm preparations, DGC and DGC combined with MACS-ANMB, for percentages of EPS-positive spermatozoa, of viability, of progressive motility and of spermatozoa with disrupted MMP. Collectively, these data allowed to evaluate in details the quality of sperm preparations after elimination of EPS-positive spermatozoa by MACS-ANMB in our population of infertile patients in AMP conditions.

Several studies have shown that ejaculated spermatozoa do exhibit changes consistent with apoptosis in particular EPS, decrease in MMP integrity, DNA fragmentation and expression of a number of pro- and anti-apoptotic proteins [73]. Unfortunately, DGC preparation, which is the technique currently used for sperm preparations in AMP laboratories [74], does not eliminate all spermatozoa with apoptotic features. The present study was carried out to quantify EPS-positive spermatozoa and localize PS on spermatozoa in a population of infertile patients attending our in vitro Fertilization laboratory and to measure the impact of removing these EPS-positive spermatozoa by MACS-ANMB on sperm quality.

We determined in raw and DGC-prepared sperm from infertile patients the proportion of spermatozoa with compromised survival and fertility potential. In raw sperm, a mean of 20% (min 5%–max 68%) are annexin V-positive (Table 4). Following DGC, about 30% of the spermatozoa are still in late apoptotic or necrotic state and about 14% are annexin V-positive (Table 1).

The moderate increase in annexin-V-positive spermatozoa observed following incubation in capacitating conditions (involving only the A+/H+ subpopulation, Table 1) might be due to the in vitro incubation. Indeed, the number of live sperm drops quickly over time in samples from patients with poor semen quality. We cannot rule out the possibility that the A+/H- is a quick and transitory subpopulation being converted in the A+/H+ subpopulation [49], as also suggested by Sion et al. [43]. Kotwicka et al. [12] detected an increase from 15% to 35% annexin V-positive spermatozoa following incubation in capacitating medium. Martin et al. [54] showed that EPS is mainly related to the acrosome reaction because calcium ionophore A23187 induced a significant increase in the proportion of living EPS-positive spermatozoa from 7 to 48%.

Results from the literature are still controversial. Some authors reported that EPS was related to sperm capacitation process due to a bicarbonate-activated outward translocation of PS [11, 15, 54]. In contrast, other reports found correlations between EPS and apoptotic events such as DNA fragmentation, caspases activities and/or loss of MMP integrity [6, 7, 44‐47, 49, 50, 52, 53]. Taken together, EPS spermatozoa could be survivors of a testis abortive apoptotic process or the result of oxidative stresses initiated during the transit or storage in the male genital tract or be linked to a physiological event in the post-ejaculation period. We could also speculate that spermatozoa might enter an apoptotic-like pathway, giving a physiological role to EPS during capacitation/acrosome reaction, for the acquisition of fertilizing ability, in a natural mechanistic economy. Such mechanism would be dependent on a correct schedule for successful fertilization [75], or otherwise it would be converted in the apoptotic signal for elimination by phagocytic cells. Indeed, it is known that many boar spermatozoa are phagocytized after insemination, thus reducing the risk of eliciting a harmful immune reaction that may interfere with fertilization or embryos implantation [76]. Altogether, regardless of whether EPS represent, an apoptotic marker or a capacitated/acrosome reacted marker, EPS-positive spermatozoa in fresh ejaculates are potentially harmful in AMP being in apoptosis or off schedule for fertilization, respectively.

Concerning the localization of PS on human spermatozoa (Figure 1 and Table 2), we found several categories of annexin V-positive spermatozoa. Our results are similar to those of Shen et al. [49] who also reported annexin V-labeling around head, midpiece and certain parts of the tail. Following incubation in capacitating conditions we found an increase in both head and head plus midpiece categories. Kotwicka et al. [12] also found a change in the localization of PS during capacitation, firstly mainly in the midpiece and then in the acrosomal region of the head. de Vries et al. [15] reported that PS in living spermatozoa was restricted to the apical area of the head plasma membrane, whereas deteriorated spermatozoa presented PS at the midpiece. The various PS localizations on spermatozoa could thus be interpreted as different stages of the dynamic physiological process of capacitation/acrosome reaction or apoptosis versus necrosis status.

Numerous reports linked fertilization failures to apoptotic markers [8, 45, 77, 78]. Infertile patients have higher rates of spermatozoa with EPS, activated caspases and/or loss of MMP integrity [7, 45, 50]. Therefore MACS-ANMB, which eliminate EPS-positive spermatozoa, is adapted to human sperm in the context of seeking new techniques with better criteria of selection for sperm preparations in AMP [39]. Our study on sperm from infertile patients shows that DGC combined with MACS-ANMB is: 1) applicable to low amount, 43% of sperm tested with less than 5 million spermatozoa, and/or bad quality spermatozoa, 2) efficient in removing EPS spermatozoa and, 3) efficient in enhancing the quality of spermatozoa in term of MMP integrity, progressive motility and most importantly in terms of sperm survival. In our serie of experiments the sensitivity and specificity of MACS-ANMB technique for the separation of EPS-positive spermatozoa (Table 6) are very similar to that reported by Paasch et al. [50, 79], except that we labeled spermatozoa by annexin V-FITC and observed them by fluorescence microscope rather than labeling with FITC-conjugated anti-annexin V antibodies followed by FACS analysis. Indeed, in the apoptotic fraction a mean of 67.8% (n = 17 infertile patients, Table 4) of spermatozoa were recognized as EPS-positive compared to 72.2% (n = 30 with15 infertile and 15 fertile donors, [79] and in the non apoptotic fraction a mean of 6.7% (n = 21 infertile patients, Table 4) were recognized as EPS-positive compared to 5.2% [79]. Thus, DGC combined with MACS-ANMB was efficient in removing EPS-positive spermatozoa. MACS-ANMB allows a further decrease of 50% in EPS spermatozoa, with a mean of less than 7% EPS-positive spermatozoa in the non-apoptotic fraction.

We show that combining MACS-ANMB with DGC for sperm preparations had a positive effect on spermatozoa MMP integrity (Table 4). MACS-ANMB allowed a further reduction of 30% in MMP-disrupted spermatozoa, with a mean of 75% of spermatozoa with MPP integrity, compared to DGC alone. Removal of MMP-disrupted spermatozoa is of interest in AMP, since the functionality of mitochondria differentiates human spermatozoa with high and low fertilizing ability [80]. MACS-ANMB combined with DGC also enhanced spermatozoa progressive motility (76% compared to 69% in DGC, +7%) and most outstandingly sperm survival at 24 h (48% compared to 32% in DGC, +16%), however the data showed a great individual variability among sperm from minus 35% to plus 75% (Table 5). In a similar way, the use of the MACS-ANMB technique prior to sperm cryopreservation allowed a significantly higher cryosurvival rate expressed in motility following cryopreservation-thawing [51, 70, 81]. The better sperm survival at 24 h in DGC plus MACS-ANMB treated sperm suggested that in addition to a purely quantitative decrease of the percentage of EPS apoptotic spermatozoa, their removal might protect healthy spermatozoa from reactive oxygen species production. Indeed, excessive reactive oxygen species, produced either by the presence of immature and abnormal spermatozoa or seminal leukocytes result in altered semen parameters, including viability, motility and morphology [82‐90].

We have also undertaken the evaluation of the extent of spermatozoa loss using MACS-ANMB technique. The average number of lost spermatozoa was 24% (n = 28) which is considerably greater than the 15% obtained by Said et al. [69] following DGC plus MACS. This discrepancy may be explained by the use of low amount of spermatozoa from infertile sperm, which reflect the clinical situation in assisted medical procreation.

In conclusion, our results demonstrate in infertile sperm that MACS-ANMB is a simple, fast, low cost cell sorting system that can be easily adapted in AMP Laboratories to enhance classical sperm parameters and lower sperm apoptotic markers. These results confirm and expand the data from the groups of Cleveland and Leipzig using normal sperm [39, 65‐70, 81, 91, 92]. Additionnally, these authors reported that MACS-ANMB technique yielded a sperm population with higher normal morphology, reduced caspase-3 activation and DNA fragmentation. The cryosurvival rates and sperm fertilization potential were also improved as measured by higher oocyte penetration capacity in a zona free hamster sperm penetration assay [69, 70, 91]. Taken together, sperm preparations combining DGC with MACS-ANMB, should significantly enhance the outcome of AMP in terms of fertilization, embryo quality, pregnancies and birth rates. Even though, Varum et al.[93] suggested that elimination of defective spermatozoa using the surface marker annexin V seems unwarranted because annexin V and DNA nicks did not correlated with ubiquitin labeling. Further investigations need to be conducted to assess safety of the magnetic field and the eventual residual microbeads on spermatozoa [50, 70].

Implementation of MACS is very promising for the future of ART because MACS has many potential applications in diverse male infertilities due to the possibility of selecting the antibody of the ligand protein used for sperm selection [70]. Very recently, a newly developed solid phase molecular filtration system combining classical glass wool filtration with PS-binding properties of annexin V has been proven to enrich spermatozoa free of apoptosis markers to the same extent as MACS but without the potential inconvenient of an accidental transmission of supermagnetic microbeads into oocytes [94]. Overall, PS might become the prognostic marker of sperm fertility potential easy to use for sorting spermatozoa in ART. In consequence, discarding EPS spermatozoa may optimize AMP outcomes.