In vitro exposure of porcine sperm to functionalized superparamagnetic nanoparticles.

Nanotechnology and its applications have advanced significantly in recent decades, contributing to various fields, including reproduction. This study introduces a novel method to label porcine oocytes with nanoparticles (NPs) bound to oviductin (OVGP1, Ov) for use in Assisted Reproductive Technologies (ARTs). Despite promising developments, concerns about NP toxicity in gametes necessitate thorough investigation. This research aims to assess the impact of functionalized NPs (NPOv) on sperm functionality. Boar sperm were co-incubated with NPOv for 0, 0.5 and 1 h in two media: BTS (semen dilution and conservation) and TALP (sperm capacitation and in vitro fertilization-IVF). Sperm quality parameters (viability, motility and kinematics) showed no significant differences in TALP medium (p > .05). In BTS, although some kinetic parameters were altered, motility, progressive motility and viability remained unaffected (p > .05). Additionally, NPs presence on the zona pellucida (ZP) of oocytes did not affect sperm attachment (p > .05). In conclusion, in vitro exposure of boar sperm to OVGP1-functionalized NPs in IVF medium or attached to the ZP surface of matured oocytes does not impair sperm functionality, including their binding ability to the ZP.

The Effect of Synthetic Polyamine BPA-C8 on the Fertilization Process of Intact and Denuded Sea Urchin Eggs.

Sea urchin eggs are covered with layers of extracellular matrix, namely, the vitelline layer (VL) and jelly coat (JC). It has been shown that sea urchin eggs' JC components serve as chemoattractants or ligands for the receptor on the fertilizing sperm to promote the acrosome reaction. Moreover, the egg's VL provides receptors for conspecific sperm to bind, and, to date, at least two sperm receptors have been identified on the surface of sea urchin eggs. Interestingly, however, according to our previous work, denuded sea urchin eggs devoid of the JC and VL do not fail to become fertilized by sperm. Instead, they are bound and penetratedby multiple sperm, raising the possibility that an alternative pathway independent of the VL-residing sperm receptor may be at work. In this research, we studied the roles of the JC and VL using intact and denuded eggs and the synthetic polyamine BPA-C8. BPA-C8 is known to bind to the negatively charged macromolecular complexes in the cells, such as the JC, VL, and the plasma membrane of echinoderm eggs, as well as to the actin filaments in fibroblasts. Our results showed that, when added to seawater, BPA-C8 significantly repressed the Ca2+ wave in the intact P. lividus eggs at fertilization. In eggs deprived of the VL and JC, BPA-C8 binds to the plasma membrane and increases fibrous structures connecting microvilli, thereby allowing the denuded eggs to revert towards monospermy at fertilization. However, the reduced Ca2+ signal in denuded eggs was nullified compared to the intact eggs because removing the JC and VL already decreased the Ca2+ wave. BPA-C8 does not cross the VL and the cell membrane of unfertilized sea urchin eggs to diffuse into the cytoplasm at variance with the fibroblasts. Indeed, the jasplakinolide-induced polymerization of subplasmalemmal actin filaments was inhibited in the eggs microinjected with BPA-C8, but not in the ones bath-incubated with the same dose of BPA-C8.

Coevolution between eggs and sperm of insects.

Sexual selection is known to play a major role in the evolution of insect sperm size, whereas natural selection is thought to be a major driver of insect egg size. Despite these differing forms of selection operating, it is possible coevolution between male and female gametes can occur owing to their vital interactions during fertilization. We tested egg-sperm coevolution in insects and found that longer sperm correlated to longer and wider eggs. Moreover, the size of the entry point of sperm into insect eggs (micropyles), was positively related to the diameter of sperm, on average being approximately three times the diameter of the sperm. This suggests a function in reducing and channelling sperm entry, but potentially still leaving space for movement. Our work suggests that greater attention needs to be paid to egg-sperm interactions prior to the point of fertilization as they may influence the evolution of gametes.

The relationship of the male's proctodeal gland size to sperm-egg interaction and the duration of fertility in Japanese quail.

In avian species, male fertility significantly impacts reproductive success. This study investigates the relationship between proctodeal gland size in male Japanese quails and sperm function, as well as female fertility duration. Six hundred adult Japanese quails were selected and housed in individual cages. Males (n = 300) were divided into 6 groups (50/group) based on the size of their proctodeal glands. Females (n = 300) were randomly assigned to corresponding groups. After acclimatization, sperm kinematics and the frequency and weight of foam discharge were evaluated. Males were paired with females for 24 h, and eggs were collected for 20 d postcopulation. Eggs were incubated to assess fertility duration. Fresh eggs (n = 20/group/d) were used to assess sperm penetration into the perivitelline membrane on the 2nd, 9th, and 16th d postinsemination. Plasma testosterone levels and the testes' relative weight were determined. The results indicated a significant increase (p < 0.0001) in sperm concentration, total and progressive motility, and nearly all sperm kinematic parameters such as VSL, VCL, VAP, LIN, WOB, and STR values as the size of the proctodeal gland increased. Quails copulated with males having a small and average proctodeal gland area (<400 mm2) laid fertile eggs for a shorter period and had significantly fewer sperm penetration holes than those mated with males having a larger proctodeal gland area (>400 mm2). The proctodeal gland size was positively correlated with testicular weight, plasma testosterone concentrations, and sperm kinetic variables. The results indicate that the size of the proctodeal gland in males can be used to predict sperm function and the duration of fertility in Japanese quail.

Sperm functionality is differentially regulated by porcine oviductal extracellular vesicles from the distinct phases of the estrous cycle.

Context Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P Conclusions poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm-oocyte interaction. Implications The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm-oocyte interaction.

Deep learning insights into the architecture of the mammalian egg-sperm fusion synapse.

A crucial event in sexual reproduction is when haploid sperm and egg fuse to form a new diploid organism at fertilization. In mammals, direct interaction between egg JUNO and sperm IZUMO1 mediates gamete membrane adhesion, yet their role in fusion remains enigmatic. We used AlphaFold to predict the structure of other extracellular proteins essential for fertilization to determine if they could form a complex that may mediate fusion. We first identified TMEM81, whose gene is expressed by mouse and human spermatids, as a protein having structural homologies with both IZUMO1 and another sperm molecule essential for gamete fusion, SPACA6. Using a set of proteins known to be important for fertilization and TMEM81, we then systematically searched for predicted binary interactions using an unguided approach and identified a pentameric complex involving sperm IZUMO1, SPACA6, TMEM81 and egg JUNO, CD9. This complex is structurally consistent with both the expected topology on opposing gamete membranes and the location of predicted N-glycans not modeled by AlphaFold-Multimer, suggesting that its components could organize into a synapse-like assembly at the point of fusion. Finally, the structural modeling approach described here could be more generally useful to gain insights into transient protein complexes difficult to detect experimentally.

Female fertility and the mammalian egg's zona pellucida.

All mammalian eggs are surrounded by a relatively thick extracellular matrix (ECM) or zona pellucida (ZP) to which free-swimming sperm bind in a species-restricted manner during fertilization. The ZP consists of either three (e.g., Mus musculus) or four (e.g., Homo sapiens) glycosylated proteins, called ZP1-4. These proteins are unlike those found in somatic cell ECM, are encoded by single-copy genes on different chromosomes, and are well conserved among different mammals. Mammalian ZP proteins are synthesized as polypeptide precursors by growing oocytes that will become ovulated, unfertilized eggs. These precursors are processed to remove a signal-sequence and carboxy-terminal propeptide and are secreted into the extracellular space. Secreted ZP proteins assemble into long, crosslinked fibrils that exhibit a structural repeat due to the presence of ZP2-ZP3 dimers every 140 Å or so along fibrils. Fibrils are crosslinked by ZP1 and are oriented either perpendicular, parallel, or randomly to the plasma membrane of eggs depending on their position in the ZP. Free-swimming mouse sperm recognize and bind to ZP2 or ZP3 that serve as sperm receptors. Acrosome-intact sperm bind to ZP3 oligosaccharides and acrosome-reacted sperm bind to ZP2 polypeptide. ZP fibrils fail to assemble in the absence of either nascent ZP2 or ZP3 and results in mouse eggs that lack a ZP and female infertility. Gene sequence variations due to point, missense, or frameshift mutations in genes encoding ZP1-4 result in human eggs that lack a ZP or have an abnormal ZP and female infertility. These and other features of the mouse and human egg's ZP are discussed here.

Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism.

Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry; albumin, CD81, HSP90ß). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO2 in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism.

Expression of IZUMO1 and JUNO in the gonads of domestic cats (Felis catus).

Because of the time-consuming nature of surgical neutering and the rapid rate of reproduction among domestic cats, it is crucial to investigate alternative, nonsurgical methods of contraception for this species. Sperm protein IZUMO1 and its oocyte receptor JUNO have been proposed as potential targets for nonsurgical contraceptives. This study aimed to demonstrate (1) the protein coding sequence of feline IZUMO1 and JUNO, (2) gene expression in specific organs by measuring mRNA levels in different visceral tissues, and (3) the expression of IZUMO1 and JUNO during sperm maturation and folliculogenesis, respectively. Amplification for sequencing of feline IZUMO1 and JUNO was performed using the RT-PCR method. Levels of gene expression in different tissues were evaluated using real-time PCR. In situ hybridization was performed to localize JUNO mRNA in ovarian tissues. The complete coding sequences of IZUMO1 and JUNO were obtained and analyzed. A comparison between protein orthologs demonstrated the conservation of IZUMO1 and JUNO in Felidae. The real-time PCR results from various visceral organs indicated that IZUMO1 was significantly higher in the testis than in other organs, whereas JUNO was significantly higher in the ovary than in other organs. Expression of IZUMO1 was found to be higher in the testes than in the caput, corpus, and cauda of epididymides. In situ hybridization revealed that JUNO mRNA was in the ooplasm and nucleus of the primordial, primary, secondary, and antral follicles. Importantly, this was the first study to demonstrate the IZUMO1 and JUNO genes in the testis and ovary of cats. The results are useful for future research related to these genes and for developing contraceptives against these targets.

Spem2, a novel testis-enriched gene, is required for spermiogenesis and fertilization in mice.

Spermiogenesis is considered to be crucial for the production of haploid spermatozoa with normal morphology, structure and function, but the mechanisms underlying this process remain largely unclear. Here, we demonstrate that SPEM family member 2 (Spem2), as a novel testis-enriched gene, is essential for spermiogenesis and male fertility. Spem2 is predominantly expressed in the haploid male germ cells and is highly conserved across mammals. Mice deficient for Spem2 develop male infertility associated with spermiogenesis impairment. Specifically, the insufficient sperm individualization, failure of excess cytoplasm shedding, and defects in acrosome formation are evident in Spem2-null sperm. Sperm counts and motility are also significantly reduced compared to controls. In vivo fertilization assays have shown that Spem2-null sperm are unable to fertilize oocytes, possibly due to their impaired ability to migrate from the uterus into the oviduct. However, the infertility of Spem2-/- males cannot be rescued by in vitro fertilization, suggesting that defective sperm-egg interaction may also be a contributing factor. Furthermore, SPEM2 is detected to interact with ZPBP, PRSS21, PRSS54, PRSS55, ADAM2 and ADAM3 and is also required for their processing and maturation in epididymal sperm. Our findings establish SPEM2 as an essential regulator of spermiogenesis and fertilization in mice, possibly in mammals including humans. Understanding the molecular role of SPEM2 could provide new insights into future therapeutic treatment of human male infertility and development of non-hormonal male contraceptives.

No evidence for a direct extracellular interaction between human Fc receptor-like 3 (MAIA) and the sperm ligand IZUMO1.

Fertilization involves the recognition and fusion of sperm and egg to form a previously unidentified organism. In mammals, surface molecules on the sperm and egg have central roles, and while adhesion is mediated by the IZUMO1-JUNO sperm-egg ligand-receptor pair, the molecule/s responsible for membrane fusion remain mysterious. Recently, MAIA/FCRL3 was identified as a mammalian egg receptor, which bound IZUMO1 and JUNO and might therefore have a bridging role in gamete recognition and fusion. Here, we use sensitive assays designed to detect extracellular protein binding to investigate the interactions between MAIA and both IZUMO1 and JUNO. Despite using reagents with demonstrable biochemical activity, we did not identify any direct binding between MAIA/FCRL3 and either IZUMO1 or JUNO. We also observed no fusogenic activity of MAIA/FCRL3 in a cell-based membrane fusion assay. Our findings encourage caution in further investigations on the role played by MAIA/FCRL3 in fertilization.

Positive selection in gamete interaction proteins in Carnivora.

The absence of robust interspecific isolation barriers among pantherines, including the iconic South American jaguar (Panthera onca), led us to study molecular evolution of typically rapidly evolving reproductive proteins within this subfamily and related groups. In this study, we delved into the evolutionary forces acting on the zona pellucida (ZP) gamete interaction protein family and the sperm-oocyte fusion protein pair IZUMO1-JUNO across the Carnivora order, distinguishing between Caniformia and Feliformia suborders and anticipating few significant diversifying changes in the Pantherinae subfamily. A chromosome-resolved jaguar genome assembly facilitated coding sequences, enabling the reconstruction of protein evolutionary histories. Examining sequence variability across more than 30 Carnivora species revealed that Feliformia exhibited significantly lower diversity compared to its sister taxa, Caniformia. Molecular evolution analyses of ZP2 and ZP3, subunits directly involved in sperm-recognition, unveiled diversifying positive selection in Feliformia, Caniformia and Pantherinae, although no significant changes were linked to sperm binding. Structural cross-linking ZP subunits, ZP4 and ZP1 exhibited lower levels or complete absence of positive selection. Notably, the fusion protein IZUMO1 displayed prominent positive selection signatures and sites in basal lineages of both Caniformia and Feliformia, extending along the Caniformia subtree but absent in Pantherinae. Conversely, JUNO did not exhibit any positive selection signatures across tested lineages and clades. Eight Caniformia-specific positive selected sites in IZUMO1 were detected within two JUNO-interaction clusters. Our findings provide for the first time insights into the evolutionary trajectories of ZP proteins and the IZUMO1-JUNO gamete interaction pair within the Carnivora order.

MARC-3, a membrane-associated ubiquitin ligase, is required for fast polyspermy block in Caenorhabditis elegans.

In many sexually reproducing organisms, oocytes are fundamentally fertilized with one sperm. In Caenorhabditis elegans, chitin layer formation after fertilization by the EGG complex is one of the mechanisms of polyspermy block, but other mechanisms remain unknown. Here, we demonstrate that MARC-3, a membrane-associated RING-CH-type ubiquitin ligase that localizes to the plasma membrane and cortical puncta in oocytes, is involved in fast polyspermy block. During polyspermy, the second sperm entry occurs within approximately 10 s after fertilization in MARC-3-deficient zygotes, whereas it occurs approximately 200 s after fertilization in egg-3 mutant zygotes defective in the chitin layer formation. MARC-3 also functions in the selective degradation of maternal plasma membrane proteins and the transient accumulation of endosomal lysine 63-linked polyubiquitin after fertilization. The RING-finger domain of MARC-3 is required for its in vitro ubiquitination activity and polyspermy block, suggesting that a ubiquitination-mediated mechanism sequentially regulates fast polyspermy block and maternal membrane protein degradation during the oocyte-to-embryo transition.

Sperm induction of somatic cell-cell fusion as a novel functional test.

The fusion of mammalian gametes requires the interaction between IZUMO1 on the sperm and JUNO on the oocyte. We have recently shown that ectopic expression of mouse IZUMO1 induces cell-cell fusion and that sperm can fuse to fibroblasts expressing JUNO. Here, we found that the incubation of mouse sperm with hamster fibroblasts or human epithelial cells in culture induces the fusion between these somatic cells and the formation of syncytia, a pattern previously observed with some animal viruses. This sperm-induced cell-cell fusion requires a species-matching JUNO on both fusing cells, can be blocked by an antibody against IZUMO1, and does not rely on the synthesis of new proteins. The fusion is dependent on the sperm's fusogenic capacity, making this a reliable, fast, and simple method for predicting sperm function during the diagnosis of male infertility.

Has the concept of polyspermy prevention been invented in the laboratory?

There is no evidence, nor need, for a fast block to polyspermy in animal oocytes. The idea that oocytes have evolved a mechanism to allow the entry of one spermatozoon and repel all others has, however, gained consensus over the last century. The main culprit is the sea urchin, which has been used for over a century in in vitro studies of the fertilization process. Images of sea urchin oocytes with thousands of sperm attached to the surface are commonplace in textbooks and appeal to the nature of the reader implying an intriguing surface mechanism of sperm selection despite these oocytes being fixed for photography (Figure ). The abundance of gametes in this marine invertebrate and the ease of experimentation have given us the possibility to elucidate many aspects of the mechanism of fertilization, but has also led to ongoing controversies in reproductive biology, one being polyspermy prevention. Kinetic experiments by Rothschild and colleagues in the 1950s led to the hypothesis of a fast partial block to polyspermy in sea urchin oocytes that reduced the probability of a second spermatozoon from entering the oocyte by 1/20th. In the 1970s, Jaffe and colleagues suggested, with circumstantial evidence, that this partial block was due to the sperm-induced depolarization of the oocyte plasma membrane. However, the fate of supernumerary spermatozoa is determined well before the plasma membrane of the oocyte depolarizes. Transmembrane voltage does not serve to regulate sperm entry. Scholastic texts have inadvertently promulgated this concept across the animal kingdom with no logical correlation or experimentation and, as of today, a molecular mechanism to regulate sperm entry in oocytes has not been identified.

The strong anti-hyaluronidase effect of ellagic acid markedly decreases polyspermy during in vitro fertilization, resulting in sustainment of the developmental potency in porcine oocytes.

The present study investigated the effects of ellagic acid, a type of polyphenol that does not have a glycan and is composed of four hydroxyl groups and two lactone functional groups, on porcine in vitro fertilization (IVF) by focusing on its anti-hyaluronidase activity. A comparative analysis of ellagic acid and apigenin, which is commonly used as a hyaluronidase inhibitor, was performed. It compared the effects of ellagic acid and apigenin on hyaluronidase activity at different concentrations. The results showed that 10, 20, and 40 µM ellagic acid strongly reduced hyaluronidase activity (P < 0.05). The addition of 20 µM ellagic acid, but not apigenin, to porcine IVF medium effectively reduced polyspermy without decreasing sperm penetration or the formation rates of male pronuclei in cumulus-free oocytes. However, neither ellagic acid nor apigenin affected the number of sperm that bound to zona pellucida (ZP) or the induction of zona hardening and protease resistance. The percentage of acrosome-reacting sperm that bound to the ZP was markedly lower in the presence of 20 µM ellagic acid than in the untreated and apigenin-treated groups, even though the antioxidant capacity of ellagic acid was weaker than that of apigenin. Furthermore, a markedly higher percentage of embryos developed to the blastocyst stage in the ellagic acid-treated group, and the apoptotic indexes of expanded blastocysts produced by the ellagic acid treatment during IVF were significantly low. Therefore, the anti-hyaluronidase effect of ellagic acid markedly suppressed the induction of the acrosome reaction in sperm that bound to the ZP, resulting in a marked decrease in polyspermy under conditions that maintained high sperm penetrability during IVF and sustainment of the developmental potency in porcine oocytes.

Molecular dynamics of JUNO-IZUMO1 complexation suggests biologically relevant mechanisms in fertilization.

JUNO-IZUMO1 binding is the first known physical link created between the sperm and egg membranes in fertilization, however, how this initiates sperm-egg fusion remains elusive. As advanced structural insights will help to combat the infertility crisis, or advance fertility control, we employed all-atom Molecular Dynamics (MD) to derive dynamic structural insights that are difficult to obtain experimentally. We found that the hydrated JUNO-IZUMO1 interface is composed of a large set of short-lived non-covalent interactions. The contact interface is destabilized by strategically located point mutations, as well as by Zn2+ ions, which shift IZUMO1 into the non-binding "boomerang" conformation. We hypothesize that the latter might explain how the transient zinc spark, as released after sperm entry into the oocyte, might contribute to block polyspermy. To address a second mystery, we performed another set of simulations, as it was previously suggested that JUNO in solution is unable to bind to folate despite it belonging to the folate receptor family. MD now suggests that JUNO complexation with IZUMO1 opens up the binding pocket thereby enabling folate insertion. Our MD simulations thus provide crucial new hypotheses how the dynamics of the JUNO-IZUMO1 complex upon solvation might regulate fertility.

Exposure to Cumulus Cell Secretome Improves Sperm Function: New Perspectives for Sperm Selection In Vitro.

In the literature, there is a well-known correlation between poor semen quality and DNA sperm integrity, which can turn into negative outcomes in terms of embryo development and clinical pregnancy. Sperm selection plays a pivotal role in clinical practice, and the most widely used methods are mainly based on sperm motility and morphology. The cumulus oophorus complex (COC) during natural fertilization represents a barrier that spermatozoa must overcome to reach the zona pellucida and fertilize the oocyte. Spermatozoa that can pass through the COC have better structural and metabolic characteristics as well as enhanced acrosome reaction (AR). The present study aimed to evaluate the exposure of sperm to cumulus cell secretome during swim-up treatment (SUC) compared with the routinely used swim-up method (SU). To determine the effectiveness of this method, biological factors critical for the ability of sperm to fertilize an oocyte, including capacitation, AR, tyrosine phosphorylation signature, DNA integrity, and mitochondrial functionality, were assessed. The SUC selection assures recovery of high-quality spermatozoa, with enhanced mitochondrial functionality and motility compared with both SU-selected and unselected (U) sperm. Furthermore, using this modified swim-up procedure, significantly reduced sperm DNA damage (p < 0.05) was detected. In conclusion, the SUC approach is a more physiological and integrated method for sperm selection that deserves further investigation for its translation into clinical practice.