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.

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'.

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.

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.

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.

Potential Development of Vitrified Immature Human Oocytes: Influence of the Culture Medium and the Timing of Vitrification.

How does the in vitro maturation (IVM) medium and the vitrification procedure affect the survival of germinal vesicle (GV) oocytes obtained from stimulated cycles and their development to the blastocyst stage? In total, 1085 GV human oocytes were obtained after women underwent a cycle of controlled ovarian stimulation, and these oocytes were subjected to IVM before or after their vitrification. IVM was carried out in two commercial culture media not specifically designed for maturation. MII oocytes were then activated and embryo development until day 6 was evaluated. According to the results, a higher percentage of oocytes reach the MII stage if they are vitrified before they undergo IVM. Nevertheless, the medium used and the sample size determine whether these differences become significant or not. Similar survival rates and development to blastocysts were observed in all the conditions studied.

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.

Impact of Maturation and Vitrification Time of Human GV Oocytes on the Metaphase Plate Configuration.

The combination of in vitro maturation (IVM) techniques and oocyte vitrification (OV) could increase the number of useful oocytes in different types of patients. IVM and subsequent OV is the most widely used clinical strategy. Would the results improve if we reverse the order of the techniques? Here, we evaluated survival, in vitro maturation, time to extrude the first polar body (PB), and the metaphase plate configuration of human prophase I (GV) oocytes before or after their vitrification. Specific, 195 GV oocytes from 104 patients subjected to controlled ovarian stimulation cycles were included. We stablished three experimental groups: GV oocytes vitrified and IVM (Group GV-Vit), GV oocytes IVM and vitrified at MII stage (Group MII-Vit), and GV oocytes IVM (Group not-Vit). All of them were in vitro matured for a maximum of 48 h and fixed to study the metaphase plate by confocal microscopy. According to our results, the vitrification of immature oocytes and their subsequent maturation presented similar survival, maturation, and metaphase plate conformation rates, but a significantly higher percentage of normal spindle than the standard strategy. Additionally, the extension of IVM time to 48 h did not seem to negatively affect the oocyte metaphase plate configuration.

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.

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.

The effects of male social environment on sperm phenotype and genome integrity.

Sperm function and quality are primary determinants of male reproductive performance and hence fitness. The presence of rival males has been shown to affect ejaculate and sperm traits in a wide range of taxa. However, male physiological conditions may not only affect sperm phenotypic traits but also their genetic and epigenetic signatures, affecting the fitness of the resulting offspring. We investigated the effects of male-male competition on sperm quality using TUNEL assays and geometric morphometrics in the zebrafish, Danio rerio. We found that the sperm produced by males exposed to high male-male competition had smaller heads but larger midpiece and flagellum than sperm produced by males under low competition. Head and flagella also appeared less sensitive to the osmotic stress induced by activation with water. In addition, more sperm showed signals of DNA damage in ejaculates of males under high competition. These findings suggest that the presence of a rival male may have positive effects on sperm phenotypic traits but negative effects on sperm DNA integrity. Overall, males facing the presence of rival males may produce faster swimming and more competitive sperm but this may come at a cost for the next generation.

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.

Effectiveness of human spermatozoa biomarkers as indicators of structural damage during cryopreservation.

Human spermatozoa cryopreservation techniques are used to maintain and protect male fertility in cases such as infertility and malignancy treatments. However, during cryopreservation, the spermatozoa's metabolic rate is reduced and they undergo dramatic functional and structural changes owing to exposure to cryoprotectants and freezing-thawing procedures. While the effects of cryopreservation on cells are documented, to date the induced cryodamage on structural and/or functional sperm biomarkers is not well established at multivariate scale. To address this question, we performed basic sperm analysis, sperm DNA fragmentation assessment, spontaneous acrosome reaction measurement, and cytoskeleton evaluation after thawing samples from subjects with normal and low-quality semen. A cryodamage rate was used to determine the effects of the freeze-thaw process on spermatozoa. In addition, a Principal Component Analysis (PCA) was used for data reduction and to evaluate sperm-specific patterns during the cryopreservation process. We found that the vitality, progressive motility and sperm count from low-quality samples after cryopreservation show higher damage rates (≥40%) than in normal sperm samples. However, cytoskeleton, DNA, tail and mid-piece and acrosome display the highest cryodamage rates (∼50-99%) and are equally susceptible to cryopreservation-induced damage in both low- and normal-quality semen samples. Overall, the evaluation of these parameters provides meaningful information about different aspects of sperm functionality after cryopreservation.

Ultrastructural characteristics of human oocytes vitrified before and after in vitro maturation.

The development of an effective program that combines in vitro maturation (IVM) and cryopreservation for immature oocytes would represent a novel advance for in vitro fertilization (IVF), especially as a means to preserve the fertility of women in unique situations. The aim of this study was to analyze the ultrastructural characteristics of human oocytes, obtained after controlled ovarian stimulation, to determine whether IVM is best performed before or after vitrification. To this end, we analyzed the following features in a total of 22 MII oocytes: size, zona pellucida and perivitelline space, mitochondria number, M-SER (mitochondria-smooth endoplasmic reticulum) aggregates and M-V (mitochondria-vesicle) complexes, the number of cortical granules and microvilli, and the presence of vacuolization using transmission electron microscopy (TEM). Each oocyte presented a rounded shape, with an intact oolemma, and was surrounded by a continuous zona pellucida and perivitelline space. Statistical analysis comparing oocytes vitrified before or after IVM indicated that there were no significant differences between examined characteristics.

Metabolites involved in cellular communication among human cumulus-oocyte-complex and sperm during in vitro fertilization.

BACKGROUND: Fertilization is a key physiological process for the preservation of the species. Consequently, different mechanisms affecting the sperm and the oocyte have been developed to ensure a successful fertilization. Thus, sperm acrosome reaction is necessary for the egg coat penetration and sperm-oolema fusion. Several molecules are able to induce the sperm acrosome reaction; however, this process should be produced coordinately in time and in the space to allow the success of fertilization between gametes. The goal of this study was to analyze the metabolites secreted by cumulus-oocyte-complex (COC) to find out new components that could contribute to the induction of the human sperm acrosome reaction and other physiological processes at the time of gamete interaction and fertilization. METHODS: For the metabolomic analysis, eighteen aliquots of medium were used in each group, containing: a) only COC before insemination and after 3 h of incubation; b) COC and capacitated spermatozoa after insemination and incubated for 16-20 hours; c) only capacitated sperm after 16-20 h in culture and d) only fertilization medium as control. Six patients undergoing assisted reproduction whose male partners provided normozoospermic samples were included in the study. Seventy-two COC were inseminated. RESULTS: The metabolites identified were monoacylglycerol (MAG), lysophosphatidylcholine (LPC) and phytosphingosine (PHS). Analysis by PCR and in silico of the gene expression strongly suggests that the cumulus cells contribute to the formation of the PHS and LPC. CONCLUSIONS: LPC and PHS are secreted by cumulus cells during in vitro fertilization and they could be involved in the induction of human acrosome reaction (AR). The identification of new molecules with a paracrine effect on oocytes, cumulus cells and spermatozoa will provide a better understanding of gamete interaction.

Semen parameters can be predicted from environmental factors and lifestyle using artificial intelligence methods.

Fertility rates have dramatically decreased in the last two decades, especially in men. It has been described that environmental factors as well as life habits may affect semen quality. In this paper we use artificial intelligence techniques in order to predict semen characteristics resulting from environmental factors, life habits, and health status, with these techniques constituting a possible decision support system that can help in the study of male fertility potential. A total of 123 young, healthy volunteers provided a semen sample that was analyzed according to the World Health Organization 2010 criteria. They also were asked to complete a validated questionnaire about life habits and health status. Sperm concentration and percentage of motile sperm were related to sociodemographic data, environmental factors, health status, and life habits in order to determine the predictive accuracy of a multilayer perceptron network, a type of artificial neural network. In conclusion, we have developed an artificial neural network that can predict the results of the semen analysis based on the data collected by the questionnaire. The semen parameter that is best predicted using this methodology is the sperm concentration. Although the accuracy for motility is slightly lower than that for concentration, it is possible to predict it with a significant degree of accuracy. This methodology can be a useful tool in early diagnosis of patients with seminal disorders or in the selection of candidates to become semen donors.

Epigenetics Role in Spermatozoa Function: Implications in Health and Evolution-An Overview.

The unique properties of spermatozoa are established through the spermatogenesis and maturation processes concurrently with its epigenome. It is known that damage to epigenetic mechanisms can lead to reproductive problems. However, scientific reviews addressing the role of the spermatozoa epigenome during the reproductive process are scarce. Therefore, the aim of this review was to offer a detailed overview of current knowledge in the field of spermatozoa epigenetics and its consequent implications. A full search was performed through three databases by combining five keywords. Inclusion criteria were implemented to grant accessibility, relevance, and concretion. Besides, some articles were manually removed or added to obtain an adequate and complete collection of 485 scientific publications. This compilation was used to conduct the bibliometric analysis and the data review separately. Bibliometric results displayed that spermatozoa epigenetics is an active and growing research area. The bibliographic overview showed that sperm epigenome correlates with the development of its function, explaining the environmental influence on reproductive pathologies or abnormal inheritance. The main conclusions were that the normal performance of sperm is heavily reliant on its epigenetics and that this study area is burgeoning, with the potential ability to provide society with clinical innovations in a short-term period.

IZUMO1 Receptor Localization during Hyaluronic Acid Selection in Human Spermatozoa.

IZUMO1 is an acrosome transmembrane protein implicated in the adhesion and fusion of gametes. This study aims to describe the distribution of IZUMO1 in human sperm under different physiological conditions: before capacitation (NCS), at one-hour capacitation (CS1), after a hyaluronic acid (HA) selection test (mature, MS1 and immature, IS1), and induced acrosome reaction from one-hour-capacitated sperm (ARS1). The data obtained in NCS, CS1, and MS1 significantly highlight dotted fluorescence in the acrosomal region (P1) as the major staining pattern (~70%). Moreover, we describe a new distribution pattern (P2) with a dotted acrosomal region and a labelled equatorial region that significantly increases in HA-bound spermatozoa, suggesting the onset of the migration of IZUMO1. In contrast, unbound spermatozoa presented an increase in P3 (equatorial region labelled) and P4 (not labelled). Finally, costaining to observe IZUMO1 distribution and acrosome status was performed in ARS1. Interestingly, we reported a variety of combinations between the IZUMO1 staining patterns and the acrosomal stages. In conclusion, these data show as a novelty the diffusion of the IZUMO1 protein during different physiological conditions that could contribute to the improvement in sperm selection techniques.

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.

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.