Phospholipase C Zeta in Human Spermatozoa: A Systematic Review on Current Development and Clinical Application.

During fertilization, the fusion of the spermatozoa with the oocytes causes the release of calcium from the oocyte endoplasmatic reticulum. This, in turn, triggers a series of calcium ion (Ca2+) oscillations, a process known as oocyte activation. The sperm-specific factor responsible for oocyte activation is phospholipase C zeta (PLCζ). Men undergoing intracytoplasmic sperm injection (ICSI) with their spermatozoa lacking PLCζ are incapable of generating Ca2+ oscillation, leading to fertilization failure. The immunofluorescence assay is the most used technique to assess the expression and localization of PLCζ and to diagnose patients with reduced/absent ability to activate the oocytes. In these patients, the use of assisted oocyte activation (AOA) technique can help to yield successful ICSI results and shorten the time of pregnancy. However, the production of a stable PLCζ recombinant protein represents a new powerful therapeutic approach to treating individuals with this condition. We aim to conduct a systematic review focusing on the expression, level, and localization of PLCζ, discussing the novel genetic mutation associated with its impairment. In addition, we highlight the benefits of AOA, looking at new and less invasive methods to diagnose and treat cases with PLCζ dysfunction.

Composite material with enhanced recyclability as encapsulant for photovoltaic modules.

Encapsulation of photovoltaic cells was carried out using a transparent glass fiber reinforced composite with enhanced chemical recyclability based on a matrix of an epoxy resin containing cleavable functional groups. The current-voltage curves showed a decrease of 6.3% on the short-circuit current (Isc) after encapsulation of the cell, lower than the one observed for the reference non-recyclable standard epoxy composite. Its performance stability under thermal cycling, ultraviolet (UV), and damp-heat exposure was evaluated and compared with the one of the reference standard epoxy. Both resins showed good stability performance under UV exposure and thermal cycling accelerated aging. Moreover, a power loss below the 5% allowed by the photovoltaic standard was observed for the recyclable resin after 1000 h of damp-heat exposure, even the pronounced loss of 4.7% in power remains a concern. Regarding the recyclability, the composite was dissolved in acetic acid dissolution and glass fiber fabrics were successfully recovered. A new module was manufactured with these fabrics, showing this time a loss of 12% in Isc comparing with the non-encapsulated cell. Further work will consider improving the moisture barrier properties of the composite, and adjusting the recycling conditions to allow component recovery valid for new modules.

Human Sperm Head Vacuoles Are Related to Nuclear-Envelope Invaginations.

Nuclear vacuoles are specific structures present on the head of the human sperm of fertile and non-fertile men. Human sperm head vacuoles have been previously studied using motile sperm organelle morphology examination (MSOME) and their origin related to abnormal morphology, abnormal chromatin condensation and DNA fragmentation. However, other studies argued that human sperm vacuoles are physiological structures and consequently, to date, the nature and origin of the nuclear vacuoles remains to be elucidated. Here, we aim to define the incidence, position, morphology and molecular content of the human sperm vacuoles using transmission electron microscopy (TEM) and immunocytochemistry techniques. The results showed that ~50% of the analyzed human sperm cells (n = 1908; 17 normozoospermic human donors) contained vacuoles mainly located (80%) in the tip head region. A significant positive correlation was found between the sperm vacuole and nucleus areas. Furthermore, it was confirmed that nuclear vacuoles were invaginations of the nuclear envelope from the perinuclear theca and containing cytoskeletal proteins and cytoplasmic enzyme, discarding a nuclear or acrosomal origin. According to our findings, these human sperm head vacuoles are cellular structures originating from nuclear invaginations and contain perinuclear theca (PT) components, allowing us to define a new term of 'nuclear invaginations' rather than 'nuclear vacuoles'.

Analytical Determination of Heavy Metals in Human Seminal Plasma-A Systematic Review.

Infertility is a growing concerning health problem affecting around 15% of couples worldwide. Conventional semen parameters have limited accuracy for male infertility potential determination. Current advances in the understanding of male infertility indicate that environmental and occupational exposure to chemical contaminants are important etiological factors leading to infertility problems. In this context, some heavy metals (HMs) can be considered as endocrine-disrupting compounds (EDCs), thus altering the seminal quality. This systematic review aims to summarize the key points to detect and quantify HMs in human seminal plasma (SP) and the involved analytical tools. Our results showed that that for HM quantification, atomic absorption spectroscopy (AAS) and inductively coupled plasma (ICP) were the most employed techniques while Zn, Cd, Pb, and Cr were the analytes most often detected. Fast, reliable, and sensitive quantification of EDCs in SP could be important for the development of accurate diagnostic and preventive strategies to address male infertility towards providing personalized therapy.

Molecular Chaperone HSPA2 Distribution During Hyaluronic Acid Selection in Human Sperm.

During fertilization, sperm hyaluronidase activity is essential for spermatozoa to successfully penetrate the hyaluronic acid-enriched extracellular matrix of the cumulus cells. Since molecular chaperones, as the heat shock protein A2, are typically involved in bringing hyaluronic acid receptors to the cell surface, here we evaluated the presence and spatial location of HSPA2 on human spermatozoa based on its hyaluronic acid binding capacity. This study included 16 normozoospermic sperm samples from volunteering donors. The location of HSPA2 was studied in cells before and after 1-h incubation under capacitating conditions, as well as in spermatozoa selected according to their ability of binding to hyaluronic acid. Our results showed no significant differences in HSPA2 immunofluorescent cells before and after 1 h of incubation in capacitating conditions. Nevertheless, after hyaluronic acid selection, the percentage of HSPA2-labelled cells increased significantly, indicating that the interaction with hyaluronic acid may induce the unmasking of HSPA2 epitopes. Furthermore, after swim-up and hyaluronic acid selection, spermatozoa presented a highly immunostained equatorial band with a homogeneous fluorescence throughout the acrosomal region. This distribution has been previously suggested to have important implications in male fertility. Noteworthy, a homogeneous fluorescence among the acrosomal region with a more intense labelling at the apical region was observed only in hyaluronic acid bound sperm cells, which may be associated with primary gamete recognition. Our findings suggest that the hyaluronic acid selection technique and HSPA2 biomarker should be considered candidates to complement the classic seminal analysis before recommending an appropriate assisted reproduction technique.

Sperm Adhesion Molecule 1 (SPAM1) Distribution in Selected Human Sperm by Hyaluronic Acid Test.

The failures of binding to the oocyte zona pellucida are commonly attributed to defects in the sperm recognition, adhesion, and fusion molecules. SPAM1 (sperm adhesion molecule 1) is a hyaluronidase implicated in the dispersion of the cumulus-oocyte matrix. Therefore, the aim of this study was to characterize the SPAM1 distribution in the different physiological conditions of human sperm. Specifically, we evaluated the location of the SPAM1 protein in human sperm before capacitation, at one and four hours of capacitation and after hyaluronic acid (HA) selection test by fluorescence microscopy. Sperm bound to HA were considered mature and those that crossed it immature. Our results detected three SPAM1 fluorescent patterns: label throughout the head (P1), equatorial segment with acrosomal faith label (P2), and postacrosomal label (P3). The data obtained after recovering the mature sperm by the HA selection significantly (p < 0.05) highlighted the P1 in both capacitation times, being 79.74 and 81.48% after one hour and four hours, respectively. Thus, the HA test identified that human sperm require the presence of SPAM1 throughout the sperm head (P1) to properly contact the cumulus-oocyte matrix. Overall, our results provide novel insights into the physiological basis of sperm capacitation and could contribute to the improvement of selection techniques.

An artificial intelligence model correlated with morphological and genetic features of blastocyst quality improves ranking of viable embryos.

RESEARCH QUESTION: Can better methods be developed to evaluate the performance and characteristics of an artificial intelligence model for evaluating the likelihood of clinical pregnancy based on analysis of day-5 blastocyst-stage embryos, such that performance evaluation more closely reflects clinical use in IVF procedures, and correlations with known features of embryo quality are identified? DESIGN: De-identified images were provided retrospectively or collected prospectively by IVF clinics using the artificial intelligence model in clinical practice. A total of 9359 images were provided by 18 IVF clinics across six countries, from 4709 women who underwent IVF between 2011 and 2021. Main outcome measures included clinical pregnancy outcome (fetal heartbeat at first ultrasound scan), embryo morphology score, and/or pre-implantation genetic testing for aneuploidy (PGT-A) results. RESULTS: A positive linear correlation of artificial intelligence scores with pregnancy outcomes was found, and up to a 12.2% reduction in time to pregnancy (TTP) was observed when comparing the artificial intelligence model with standard morphological grading methods using a novel simulated cohort ranking method. Artificial intelligence scores were significantly correlated with known morphological features of embryo quality based on the Gardner score, and with previously unknown morphological features associated with embryo ploidy status, including chromosomal abnormalities indicative of severity when considering embryos for transfer during IVF. CONCLUSION: Improved methods for evaluating artificial intelligence for embryo selection were developed, and advantages of the artificial intelligence model over current grading approaches were highlighted, strongly supporting the use of the artificial intelligence model in a clinical setting.

The Role of Sperm Proteins IZUMO1 and TMEM95 in Mammalian Fertilization: A Systematic Review.

Gamete membrane fusion is a critical cellular event in sexual reproduction. In addition, the generation of knockout models has provided a powerful tool for testing the functional relevance of proteins thought to be involved in mammalian fertilization, suggesting IZUMO1 and TMEM95 (transmembrane protein 95) as essential proteins. However, the molecular mechanisms underlying the process remain largely unknown. Therefore, the aim of this study was to summarize the current knowledge about IZUMO1 and TMEM95 during mammalian fertilization. Hence, three distinct databases were consulted-PubMed, Scopus and Web of Science-using single keywords. As a result, a total of 429 articles were identified. Based on both inclusion and exclusion criteria, the final number of articles included in this study was 103. The results showed that IZUMO1 is mostly studied in rodents whereas TMEM95 is studied primarily in bovines. Despite the research, the topological localization of IZUMO1 remains controversial. IZUMO1 may be involved in organizing or stabilizing a multiprotein complex essential for the membrane fusion in which TMEM95 could act as a fusogen due to its possible interaction with IZUMO1. Overall, the expression of these two proteins is not sufficient for sperm-oocyte fusion; therefore, other molecules must be involved in the membrane fusion process.

Immunofluorescence and High-Resolution Microscopy Reveal New Insights in Human Globozoospermia.

Globozoospermia is a rare and severe type of teratozoospermia characterized by the presence of round-headed, acrosomeless spermatozoa with cytoskeleton defects. Current data support a negative relationship between globozoospermia and intracytoplasmic sperm injection (ICSI) outcomes, revealing the need to perform exhaustive studies on this type of sperm disorder. The aim of this study was to evaluate different structural, functional and molecular sperm biomarkers in total globozoospermia with proper embryo development after ICSI. The combination of field-emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) allowed us to identify and correlate eight morphological patterns with both types of microscopy. Additionally, results reported a high percentage of coiled forms, with cytoplasmic retentions around the head and midpiece. By fluorescent microscopy, we detected that most of the sperm showed tubulin in the terminal piece of the flagellum and less than 1% displayed tyrosine phosphorylation in the flagellum. Moreover, we did not detect chaperone Heat shock-related 70 kDa protein 2 (HSPA2) in 85% of the cells. Overall, these findings provide new insights into globozoospermia, which could have potential implications in improving sperm selection methods for assisted reproductive techniques.

Associations of paternal serum dioxin-like polychlorinated biphenyl concentrations with IVF success: A pilot study.

Dioxin-like polychlorinated biphenyls (DL-PCBs) are environmental pollutants that have been associated with impaired semen quality. However, research on the potential impact of paternal exposure to DL-PCBs and the risk of adverse pregnancy outcomes are limited. We examine the relationship between serum DL-PCB concentrations and IVF outcomes among 42 males seeking fertility treatment. Concentrations of 12 serum DL-PCBs were analyzed by high-resolution gas chromatography coupled to high-resolution mass spectrometry. Modified Poisson regressions, adjusted for confounders, were used to assess bivariate associations and to estimate risk ratios (RRs) between DL-PCBs and binary IVF outcomes. The median concentration (25th-75th percentiles) of the sum of the 12 DL-PCBs (∑DL-PCBs) obtained for the patients was 5.42 (3.78-7.78) ng/g lipid. No statistically significant association between DL-PCB levels and embryo quality was found. However, men with high serum PCB-77 concentrations present more probability of high-quality embryos (RR: 0.292; 95% CI: 0.090-0.942), whereas the opposite trend is observed for men with lower serum levels of PCB-156 (RR: 7.960; 95% CI: 1.020-62.100), who present increased odds of high-quality embryos. Serum concentrations of PCB-126 and PCB-114 were associated with decreased implantation rates (p < 0.05). Moreover, PCB-77 and ∑non-ortho PCBs were significantly associated with a lower likelihood of clinical pregnancy (p < 0.05). A lower likelihood of live birth was associated with higher levels of PCB-77, PCB-105, PCB-118, and recording significant differences for ∑non-ortho PCBs, ∑mono-ortho PCBs, and ∑DL-PCBs (p < 0.05). These findings suggest that paternal DL-PCB exposure before conception may be related to pregnancy endpoints. However, DL-PCB measurement were limited to male partners. Therefore, we propose that future studies with larger population sizes should include both maternal and paternal factors.

Quantification and Topographical Distribution of Terminal and Linked Fucose Residues in Human Spermatozoa by Using Field Emission Scanning Electron Microscopy (FE-SEM).

The modification of sperm glycocalyx is an essential process during sperm capacitation. The presence and redistribution of terminal and linked fucose have been described during in vitro capacitation in humans. However, the influence of the capacitation time on the quantification and localization of terminal and linked fucose is still unknown. In this study, the quantitative and qualitative changes in fucosyl residues during different in vitro capacitation times (1 and 4 h), are simultaneously characterized by using Aleuria aurantia (AAA) lectin-gold labelling and high-resolution field emission scanning electron microscopy (FE-SEM) in human sperm. A significant decrease was found in the number of terminal fucose registered in the whole sperm head during the in vitro capacitation. Nevertheless, the quantification of fucose residues after 1 h of in vitro capacitation was very similar to those found after 4 h. Therefore, the changes observed in terminal and linked fucose during capacitation were not time-dependent. Furthermore, the comprehensive analysis of the topographic distribution showed the preferential fucosyl location in the acrosomal region and the presence of distinct clusters distributed over the head in all the studied conditions. Overall, these findings corroborate the validity of FE-SEM combined with gold labelling to register changes in surface molecules during in vitro sperm capacitation.

Characterization of Human Spermatic Subpopulations by ConA-Binding Sites and Tyrosine Phosphorylation during in vitro Capacitation and Acrosome Reaction.

Spermatozoa capacitation is a time-dependent physiological process essential for acquiring the fertilizing capacity. In this context, reorganization of spermatozoa surface sugars and tyrosine phosphorylation are some of the most important biochemical changes involved in capacitation. However, the relationship between these 2 biomarkers remains poorly defined. By cytofluorescence we simultaneously characterized the head concanavalin A (ConA)-binding sites and the flagellar tyrosine phosphorylation before capacitation, during different capacitation times (1 and 4 h), and after acrosome reaction induction in human spermatozoa. The results showed a strong connection between ConA-label patterns and tyrosine phosphorylation according to the spermatozoa capacitation time and acrosomal status. Specifically, the spermatozoa subpopulation with phosphotyrosine presented proper sugar location (label in acrosomal and postacrosomal region) just after 1 h of capacitation, while cells without phosphotyrosine needed 4 h to do it. Moreover, after induction of spermatozoa acrosome reaction, phosphorylation was significantly correlated (p < 0.05) with the relocation of ConA-binding residues to the equatorial region, regardless of capacitation time. Overall, these observations provide novel insights regarding spermatozoa subpopulations based on essential physiological events like capacitation and acrosome reaction, which could have potential implications in the improvement of spermatozoa selection techniques.

Arylsulfatase A Remodeling during Human Sperm In Vitro Capacitation Using Field Emission Scanning Electron Microscopy (FE-SEM).

Capacitation drives sperm biophysical and biochemical changes for sperm-oocyte interactions. It is a well-known fact that the molecular complex arylsulfatase A (ARSA), hyaluronidase sperm adhesion molecule 1 (SPAM1), and heat shock protein 2 (HSPA2) plays a significant role in sperm-zona pellucida (ZP) binding. However, the time-dependent capacitation effects on the sperm surface ARSA presence and specific topographic distributions remain to be elucidated. Here, we quantified the ARSA density and specific membrane domain locations before (US) and after in vitro capacitation (one and four hours; CS1-CS4) in human sperm using high-resolution field emission scanning electron microscopy (FE-SEM) and immunogold labeling. Our results showed a significant and progressive capacitation-mediated increase of labeled spermatozoa from the US (37%) to CS4 (100%) physiological conditions. In addition, surface mapping revealed a close relationship between the ARSA residues and their acrosomal repositioning. Compared with the ARSA surface heterogeneous distribution found in US, the CS1-4 conditions exhibited clustering on the peri-acrosomal region, showing that time-dependent capacitation also induced a ARSA residue dramatic translocation on sperm surfaces. Our findings provide novel insights into the molecular remodeling events preceding sperm-oocyte interactions.

[Localization of Arylsulfatase A during in vitro incubation of human spermatozoa in capacitation media]. / Localización de arilsulfatasa A durante la incubación in vitro de espermatozoides humanos en medio de capacitación.

OBJECTIVE: To determine the localization and distribution of the ArylsulfataseA receptor (ARSA) in human spermatozoa before and after their incubation in capacitation medium for 1 and 4hours. MATERIAL AND METHODS: Semen samples were obtained from five normozoospermic donors. Capacitation was by swim-up technique using capacitation medium for 1 and 4hours. Localization of the ARSA receptor was assessed by indirect immunofluorescence using confocal microscopy. A minimum of 200cells were observed in each physiological condition. RESULTS: Before incubation, no representative pattern was observed among the cells positive for this biomarker (8.61%). This percentage increased significantly after incubation in the capacitation medium for 1 and 4hours (61.86% and 63.38% respectively). A majority pattern was observed among the capacitated cells, with intense labelling in the acrosomal region (27.11% and 28.20% after 1 and 4hours respectively). It should be noted that the pattern corresponding to fluorescence at the level of the periacrosomal region was not observed in the spermatozoa prior to incubation. Only after incubation in capacitation medium for 1 and 4hours, 9.13% and 12.78% of cells with such distribution were detected. CONCLUSIONS: In vitro capacitation, regardless of time, favours the immunolocalization of ARSA in the cephalic region of the spermatozoa. The most representative subpopulation after this process was the one in which ARSA was intensely and homogeneously distributed in the acrosome region, involved in primary gamete recognition.

Human sperm chaperone HSPA2 distribution during in vitro capacitation.

Human fertilization success depends on the ability of the spermatozoa to undergo capacitation. Even though this process can be conducted in vitro, the optimal time for a sperm cell to complete capacitation in vitro is still under discussion due to the lack of proper capacitation biomarkers. Here, we evaluated the influence of in vitro capacitation time on HSPA2 distribution over human sperm head testing this chaperone as a potential capacitation biomarker. The chaperone was assessed in human spermatozoa from 16 normozoospermic donors using indirect immunofluorescence in uncapacitated, one and four-hour capacitated spermatozoa. The percentage of HSPA2 immunofluorescent cells before and after one hour of capacitation did not differ significantly. However, after four hours of capacitation, we observed a significantly higher percentage of HSPA2 labelled cells. In fluorescent cells analysed before capacitation, we could not identify a predominant distribution pattern. Meanwhile, after capacitation, most sperm showed a highly labelled equatorial band accompanied by a homogeneous fluorescence throughout the acrosomal region. Our findings suggest that HSPA2 needs more than one hour of in vitro capacitation for being correctly distributed in the anterior region of the sperm head. In conclusion, the present study provides solid evidences for the utility of HSPA2 as a biomarker of human sperm in vitro capacitation. Due to its importance during egg-sperm recognition, the use of HSPA2 as a biomarker before an artificial reproduction technique may be suggested, in addition to a longer capacitation time during sperm preparation.

FE-SEM Characterization of α-Mannose Density and Surface Mapping Changes in Human Sperm Head During In Vitro Capacitation.

Sperm capacitation includes the reorganization of plasma membrane components and the outstanding modification of the glycocalyx. The α-mannose presence and location during in vitro capacitation have been commonly described in human spermatozoa using Concanavalin A (Con A) lectin. However, it is still unclear to date how in vitro capacitation time affects the α-mannose residues and their topographic spatial distribution on sperm membrane. Here, we characterized the α-mannose density and specific membrane domain locations before and after in vitro capacitation (1­4 h) using high-resolution field emission scanning electron microscopy (FE-SEM). Results showed that α-mannose residues were present preferably on the acrosome domains for all physiological conditions. Uncapacitated sperm comparatively exhibits significant highest labeling densities of α-mannose residues. In addition, as in vitro capacitation takes place, significant and progressive decreasing of sugar residues was combined with their relocation mostly affecting acrosomal domain apical areas. Our findings reveal that combined approach using FE-SEM and gold nanoparticle topographical mapping offers new human sperm biomolecular and structural details during capacitation events.

Proper cytoskeleton α-tubulin distribution is concomitant to tyrosine phosphorylation during in vitro capacitation and acrosomal reaction in human spermatozoa.

Spermatozoa motility is a key parameter during the fertilization process. In this context, spermatozoa tyrosine protein phosphorylation and an appropriate cytoskeleton α-tubulin distribution are some of the most important physiological events involved in motility. However, the relationship between these two biomarkers remains poorly defined. Here, we characterized simultaneously by immunocytochemistry the α-tubulin (TUBA4A) distribution and the tyrosine phosphorylation at flagellum before capacitation, during different capacitation times (1 and 4 hr), and after acrosome reaction induction in human spermatozoa. We found that the absence of spermatozoa phosphorylation in tyrosine residues positively and significantly correlated (p < 0.05) with the terminal piece α-tubulin flagellar distribution in all physiological conditions. Conversely, we observed a positive significant correlation (p < 0.01) between phosphorylated spermatozoa and continuous α-tubulin distribution in spermatozoa flagellum, independently of the physiological condition. Similarly, the subpopulation of spermatozoa with tyrosine phosphorylated and continuous α-tubulin increases with longer capacitation times and after the acrosome reaction induction. Overall, these findings provide novel insights into the post-transcriptional physiological events associated to α-tubulin and the tyrosine phosphorylation during fertilization, which present potential implications for the improvement of spermatozoa selection methods.

Comparison of ART outcomes in men with altered mRNA protamine 1/protamine 2 ratio undergoing intracytoplasmic sperm injection with ejaculated and testicular spermatozoa.

Assisted reproductive technologies involving the use of spermatozoa and eggs for in vitro fertilization (IVF) have come as the solution for many infertile couples to become parents. However, in some cases, the use of ejaculated spermatozoa delivers poor IVF performance. Some studies have suggested the use of testicular spermatozoa in severe male infertility cases, but no guidelines regarding their utilization are currently available. In the present study, we found the mRNA protamine 1/protamine 2 (P1/P2) ratio to be a valuable biomarker of poor sperm function that could be used as a diagnostic key for the identification of cases that would benefit from the use of testicular spermatozoa. A total of 23 couples undergoing egg donation cycles with at least one previous cycle failure were studied. All couples underwent two consecutive intracytoplasmic sperm injection (ICSI) cycles with either ejaculated or testicular spermatozoa (TESA). The sperm mRNA P1/P2 ratio, fertilization rate, blastocyst rate, and pregnancy and live birth rate were compared. Results showed improved ICSI and clinical outcomes in cycles with testicular spermatozoa in men with altered mRNA P1/P2 ratios. TESA cycles presented significantly higher rates of fertilization (mean ± standard deviation: 76.1% ± 15.1% vs 65.5% ± 18.8%), blastocyst formation (55.0% ± 20.3% vs 30.8% ± 23.8%), and good morphological quality blastocyst (28.9% ± 22.9% vs 13.5% ± 17.9%) and also improvements on pregnancy (60.9% vs 0%) and healthy birth rates (56.5% vs 0%) than EJACULATE cycles. The results described here suggest that in patients with previous IVF/ICSI failures and aberrant mRNA protamine ratios, the use of testicular spermatozoa may be a good alternative to improve clinical outcomes.

Influence of in vitro capacitation time on structural and functional human sperm parameters.

A cascade of dramatic physiological events is linked to the sperm acrosome reaction and binding to the oocyte's zona pellucida during human sperm capacitation. However, structural and functional sperm changes during capacitation currently remain poorly defined. Here, we performed a multibiomarker approach based on the utilization of sperm concentration, motility, viability, morphology, acrosome reaction, tyrosine phosphorylation, DNA fragmentation, and lectin-binding sites to analyze the impact caused by swim-up selection times (uncapacitated, 1 h capacitated, and 4 h capacitated) on sperm function and structure in normozoospermic samples. We found that a 4 h swim-up capacitation increased sperm quality, because a large number of cells with normal morphology and lower DNA fragmentation rates were recovered. Furthermore, the long-term capacitation induced a higher percentage of cells with tyrosine phosphorylation of the principal piece as well as a redistribution of lectin-binding sites. Overall, the multivariate biomarkers analyzed showed a less variable distribution on spermatozoa recovered after 4 h capacitation than that with the shorter capacitation time. These findings stress the importance of capacitation time as a relevant factor in sperm quality with potential biological reproductive implications both for basic research and in assisted reproduction techniques.

First birth of a healthy infant following intra-cytoplasmic sperm injection using a new permeable cryoprotectant-free sperm vitrification protocol.

PURPOSE: The purpose of this study is to present the first birth of healthy infant born following ICSI using the new permeable cryoprotectant-free sperm vitrification protocol Easy-Sperm®. PRINCIPAL RESULTS: A 39 years old woman and his 40 years old partner underwent egg donation treatment at IVF-Spain Alicante (Spain). Half of the mature oocytes obtained from a young and healthy donor were fertilized by ICSI, using slow-frozen spermatozoa and the other half with vitrified spermatozoa. A total of 5 blastocysts were obtained on day 5 (3 resulting from vitrified spermatozoa and 2 from frozen sperm). The best embryo, with AA quality (derived from one of the oocytes fertilized with vitrified sperm) was transferred. The woman conceived and, following a normal pregnancy, delivered a healthy boy. CONCLUSIONS: To the best of our knowledge, this is the first case report of a successful pregnancy and delivery of a healthy infant from ICSI with permeable vitrified spermatozoa in an oocyte donation program with transfer on blastocyst stage.