Association between embryo morphokinetic development and intracytoplasmic sperm injection with epididymal sperm via time-lapse imaging.

The objective of this study was to investigate the impact of sperm source on embryo morphokinetics and the clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles by considering the clustering of data (multiple embryos per patient that share a comparable developmental timing). This matched cohort study was performed at a private university-affiliated in vitro fertilization center. Women who underwent ICSI with epididymal sperm between January 2019 and December 2020 (the percutaneous epididymal sperm aspiration group, n = 32 cycles) were matched with women who underwent ICSI with ejaculated sperm because of idiopathic male factor infertility (the male factor infertility [MFI] group, n = 32 cycles) or female infertility (the control group, n = 32 cycles). Embryos were cultured in a time-lapse imaging incubator, and morphokinetic development was recorded and compared among the groups. Significantly slower divisions were observed in embryos derived from epididymal sperm than in those derived from the MFI and control groups. Embryos derived from epididymal sperm had a significantly lower KIDScore (3.1 ± 0.2) than did those derived from ejaculated spermatozoa from the MFI (5.4 ± 0.1) and control (5.6 ± 0.2, p < 0.001) groups. Epididymal sperm-derived embryos showed a significantly greater occurrence of multinucleation (23.2%) than did those derived from ejaculated sperm from the MFI and control groups (2.8% and 3.7%, p < 0.001, respectively). Epididymal sperm-derived embryos were significantly more likely to undergo direct or reverse cleavage (11.1%) than ejaculated sperm-derived embryos in the control group (4.3%, p = 0.001). In conclusion, delayed cell cleavage and increased incidences of blastomere multinucleation and abnormal cleavage patterns are observed when epididymal-derived sperm are used for ICSI.

Development of a thermotaxis and rheotaxis microfluidic device for motile spermatozoa sorting.

Male infertility is a pervasive global reproductive challenge, primarily attributed to a decline in semen quality. Addressing this concern, there has been a growing focus on spermatozoa sorting in assisted reproductive technology. This study introduces a groundbreaking development in the form of a thermotaxis and rheotaxis microfluidic (TRMC) device designed for efficient motile spermatozoa sorting within a short 15-min timeframe. The TRMC device mimics the natural sperm sorting mechanism of the oviduct, selecting spermatozoa with superior motility and DNA integrity. The experimental outcomes demonstrate a remarkable enhancement in the percentage of progressive spermatozoa following sorting, soaring from 3.90% to an impressive 96.11% when subjected to a temperature decrease from 38 °C to 35 °C. Notably, sperm motility exhibited a substantial 69% improvement. The TRMC device exhibited a commendable recovery rate of 60.93%, surpassing current clinical requirements. Furthermore, the sorted spermatozoa displayed a notable reduction in the DNA fragmentation index to 6.94%, signifying a substantial 90% enhancement in DNA integrity. This remarkable advancement positions the TRMC device as highly suitable for applications in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), offering a promising solution to male infertility challenges.

Embryonic development through in vitro fertilization using high-quality bovine sperm separated in a biomimetic cervix environment.

This study is the first to identify bovine blastocysts through in vitro fertilization (IVF) of matured oocytes with a large quantity of high-quality sperm separated from a biomimetic cervix environment. We obtained high-quality sperm in large quantities using an IVF sperm sorting chip (SSC), which could mimic the viscous environment of the bovine cervix during ovulation and facilitates isolation of progressively motile sperm from semen. The viscous environment-on-a-chip was realized by formulating and implementing polyvinylpyrrolidone (PVP)-based solutions for the SSC medium. Sperm separated from the IVF-SSC containing PVP 1.5% showed high motility, normal morphology and high DNA integrity. As a result of IVF, a higher rate of hatching blastocysts, which is the pre-implantation stage, were observed, compared to the conventional swim-up method. Our results may significantly contribute to improving livestock with superior male and female genetic traits, thus overcoming the limitation of artificial insemination based on the superior genetic traits of existing males.

Histological aspects of the renal sexual segment of Brazilian snakes of the Boidae family.

Snakes represent a wide and diverse group of species and have anatomical particularities, such as the renal sexual segment (RSS), a structure located in the kidneys and formed from the hypertrophy of the urinary ducts and nephrons. This study aims at describing the histological aspects of the RSS of Boa constrictor, Epicrates cenchria and Corallus hortulanus, all of which are Brazilian snake species from the Boidae family. The reproductive system and kidneys of five male specimens of E. cenchria, three male specimens of C. hortulanus and two male specimens of B. constrictor were obtained. Tissue samples were processed histologically and different stains used (Toluidine Blue, Alcian Blue and Periodic Acid Schiff). The histological evaluation of the RSS of E. cenchria, C. hortulanus and B. constrictor shows that the RSS in these species varies when comparing individuals in the reproductive period with those which are not. It also allows for the observation of the segment's secretory activity in animals in the reproductive stage (mature sperm in the lumen of the seminiferous tubules) as well as in those which are not. Finally, the histological evaluation also reveals the variation of the secretion product in individuals in the reproductive period, in those which are not, and also among individuals within the same reproductive stage.

Corallus hortulanus testes histology: morphological and reproductive aspects.

In general snakes show differentiate anatomical, biological and behavioral particularities compared to other species. Basic information about the snakes anatomy, physiology and reproductive biology is scarce in several species, making the reproduction a challenge. Thus, the present work aims to evaluate morphological aspects of the Corallus hortulanus testes, correlating these findings with environmental factors and reproductive aspects. The testes of three specimens of Corallus hortulanus were cut to a thickness of 3µm in microtome, stained with 1% toluidine blue, photo documented and described. Seasonality was observed in the sperm production of Corallus hortulanus, with the presence of mature spermatozoa in the wettest and hottest periods of the year, as well as the largest testicular volume in these periods.

Microfluidics as an emerging paradigm for assisted reproductive technology: A sperm separation perspective.

Millions of people are subject to infertility worldwide and one in every six people, regardless of gender, experiences infertility at some period in their life, according to the World Health Organization. Assisted reproductive technologies are defined as a set of procedures that can address the infertility issue among couples, culminating in the alleviation of the condition. However, the costly conventional procedures of assisted reproduction and the inherent vagaries of the processes involved represent a setback for its successful implementation. Microfluidics, an emerging tool for processing low-volume samples, have recently started to play a role in infertility diagnosis and treatment. Given its host of benefits, including manipulating cells at the microscale, repeatability, automation, and superior biocompatibility, microfluidics have been adopted for various procedures in assisted reproduction, ranging from sperm sorting and analysis to more advanced processes such as IVF-on-a-chip. In this review, we try to adopt a more holistic approach and cover different uses of microfluidics for a variety of applications, specifically aimed at sperm separation and analysis. We present various sperm separation microfluidic techniques, categorized as natural and non-natural methods. A few of the recent developments in on-chip fertilization are also discussed.

Agreement and internal quality assurance of the Neubauer hemocytometer and Makler chamber for human sperm concentration determination.

OBJECTIVE: The Neubauer hemocytometer, as well as the Makler chamber, are devices commonly used in andrology laboratories. The present study aimed to verify if both methods yield comparable results, and whether they can be used interchangeably to determine sperm concentration. METHODS: Sperm and latex beads concentration measurements were performed with the Neubauer hemocytometer and the Makler chamber. Fixed and proportional biases were estimated, and the method agreement was determined by assessing sperm concentration results with the Bland and Altman plot. The Coefficient of Variation (CV) and relative bias were calculated as an index of precision and accuracy, respectively, by measuring latex beads target concentrations in both chambers. RESULTS: The Makler chamber systematically overestimated the Neubauer hemocytometer concentration measurements by a mean of -7.99%, with limits of agreement (LOA) between -41% to 25.61% (p<0.001). The fixed bias was found for concentration values inferior to 40 x 106/ml range (p<0.001), but not higher concentration results (p>0.05). Measurements with the Neubauer hemocytometer showed the greatest consistency in the study with the CV ranging from 3.01% to 6.67%; while the CV with the Makler chamber ranged from 8.46% to 25.64%. The relative bias for the Neubauer hemocytometer determinations varied from 0.12% to 8.40%, while for the Makler chamber varied from 7.6% to an overestimation of 38.0%. CONCLUSIONS: Measurements made with the Makler chamber demonstrated more variability and a higher degree of overestimation. The Makler chamber is a poor substitute to the Neubauer hemocytometer for evaluation of oligozoospermic samples, although both chambers render similar results for highly concentrated samples.

Sperm Preparation with Microfluidic Sperm Sorting Chip May Improve Intracytoplasmic Sperm Injection Outcomes Compared to Density Gradient Centrifugation.

Does sperm preparation using the FERTILE PLUS™ Sperm Sorting Chip improve fertilization rates, blastocyst formation, utilization, and euploidy rates in patients undergoing intracytoplasmic sperm injection (ICSI), compared with density gradient centrifugation (DGC)? A single-cohort, retrospective data review including data from 53 couples who underwent ICSI cycles within a 12-month period. For each couple, the two closest, consecutive cycles were identified, where one used the standard technique of sperm preparation (DGC) and the subsequent used FERTILE PLUS™, therefore, couples acted as their own controls. Paired samples t-test was used to compare means for the outcomes (fertilization, blastocyst formation, utilization, and euploidy rates). Binary logistic regression analysis assessed the relationship between female age, the presence of male factor infertility, and euploidy rates. Blastocyst, utilization, and euploidy rates were significantly higher for cycles using FERTILE PLUS™ compared to DGC (76% vs 56%, p = 0.002; 60% vs 41%, p = 0.005, and 40% vs 20%, p = 0.001, respectively). Although there was an increase in fertilization rates for cycles using FERTILE PLUS™, this was not significant (72% vs 68%, p = 0.449). The euploidy rates of females ≤ 35 years were significantly increased when the FERTILE PLUS™ sperm preparation method was used, compared to the older age group (OR 2.31, p = 0.007). No significant association was found between the presence or absence of male factor infertility and euploidy rates between the two cycles. This study provides tentative evidence that the FERTILE PLUS™ microfluidic sorting device for sperm selection can improve blastocyst formation, utilization, and euploidy rates following ICSI in comparison to the DGC method.

C9orf131 and C10orf120 are not essential for male fertility in humans or mice.

Male infertility affects approximately 7% of childbearing couples and is a major health issue. Although nearly 50% idiopathic infertile men are assumed to have a genetic basis, the underlying causes remain largely unknown in most infertility cases. Here, we report two rare homozygous variants in two previously uncharacterized genes, C9orf131 and C10orf120, identified in two unrelated men with asthenozoospermia. Both genes were predominantly expressed in the testes. Furthermore, C9orf131 and C10orf120 knockout mice were successfully generated using the CRISPR-Cas9 technology. However, both C9orf131-/- and C10orf120-/- adult male mice were fertile, with testis-to-body weight ratios comparable to those of wild-type mice. No overt differences were found between wild-type, C9orf131-/-, and C10orf120-/- mice regarding testicular/epididymal tissue morphology, sperm count, sperm motility, or sperm morphology. Moreover, TUNEL assays indicated that the number of apoptotic germ cells in testes was not significantly different between the three groups. In summary, these findings suggest that C9orf131 and C10orf120 are redundant genes in male infertility.

Management of dog sperm parameters and gut microbiota composition with Lactobacillus rhamnosus supplementation.

The effects of probiotics supplementation on the reproductive function have been evaluated in many species, but no study has evaluated the changes in the gut microbiome along with the sperm quality changes simultaneously. This study evaluated the effects of dietary supplementation with probiotics on the gut microbiome, sperm quality and gene expression, along with possible correlations between these parameters in dogs. The dogs were supplemented with Lactobacillus rhamnosus for six weeks, and fecal and semen samples were collected at 0, 3, and 6 weeks. Fecal samples were assessed using 16S Metagenomic Sequencing for gut microbiome analysis; and semen samples were analyzed using computer-assisted sperm analysis, DNA and acrosome integrity assessment, viability and morphology assessment, and real-time PCR. The analyses suggested that probiotic supplementation improved kinematic parameters, viability, DNA and acrosome integrity, and morphology of sperms. The mRNA levels of genes associated with fertility, DNA repair and integrity, and antioxidation were also upregulated. The sperm parameters were positively correlated with the relative abundance of Actinobacteria, Allobaculum, Phascolarctobacterium and Catenibacterium, and negatively correlated with Faecalibacterium and Streptococcus. Taken together, the sperm quality enhancement through the gut-testis axis may be due to a change in the gut microorganisms populations.

Differentiation-linked changes in the biosynthesis and turnover of sphingomyelins in rat male germ cells: Genes involved and effects of testosterone.

In rodents, sphingomyelins (SMs) species with very-long-chain polyunsaturated fatty acid (VLCPUFA) are required for normal spermatogenesis. Data on the expression of enzymes with roles in their biosynthesis and turnover during germ cell differentiation and on possible effects on such expression of testosterone (Tes), known to promote this biological process, were lacking. Here we quantified, in isolated pachytene spermatocytes (PtS), round spermatids (RS), and later spermatids (LS), the mRNA levels from genes encoding ceramide (Cer), glucosylceramide (GlcCer), and SM synthases (Cers3, Gcs, Sms1, and Sms2) and sphingomyelinases (aSmase, nSmase) and assessed products of their activity in cells in culture using nitrobenzoxadiazole (NBD)-labeled substrates and [3H]palmitate as precursor. Transcript levels from Cers3 and Gcs were maximal in PtS. While mRNA levels from Sms1 increased with differentiation in the direction PtS→RS→LS, those from Sms2 increased between PtS and RS but decreased in LS. In turn, the nSmase transcript increased in the PtS→RS→LS order. During incubations with NBD-Cer, spermatocytes produced more GlcCer and SM than did spermatids. In total germ cells cultured for up to 25 h with NBD-SM, not only abundant NBD-Cer but also NBD-GlcCer were formed, demonstrating SM→Cer turnover and Cer recycling. After 20 h with [3H]palmitate, PtS produced [3H]SM and RS formed [3H]SM and [3H]Cer, all containing VLCPUFA, and Tes increased their labeling. In total germ cells, Tes augmented in 5 h the expression of genes with roles in VLCPUFA synthesis, decreased the mRNA from Sms2, and increased that from nSmase. Thus, Tes enhanced or accelerated the metabolic changes occurring to VLCPUFA-SM during germ cell differentiation.

A novel, patented method for semen collection in dromedary camel (Camelus dromedarius).

In the current article, a developed, patented method denoted the 'Camel Semen Collection Kit-CSCK', was designed to solve the problem of semen collection in dromedary camels. CSCK is composed of three main parts: (1) Semen collection sac: made from supersensitive flexible low-density polyethylene- (LDPE); (2) Metal stainless steel applicator: designed to introduce the collection sac intravaginally and fixate it to the vaginal wall of a female camel through air insufflation; (3) Fixation sticker: a cushion sheet sticker is used to secure the outer portion of the collection sac to the female's perineal area. Semen was collected twice a week from eight dromedary bulls by using electroejaculation (EJ), artificial vagina (AV) and CSCK. Successful semen collections were 81.3%, 84.4% and 43.8% using EJ, CSCK and AV techniques respectively. Semen obtained by EJ technique showed lower semen volume, gross activity, sperm concentration, total sperm motility and percentage of live sperm cells compared to the other two techniques. Semen collected by CSCK showed a longer collection period and higher volume, gross activity, sperm motility and percentage of live spermatozoa and a lower rate of visible contamination compared to AV technique. The advantages and disadvantages of the three techniques were compared and discussed. In conclusion, CSCK represents a practical and easy method to reliably collect high-quality semen from any untrained male dromedary camel and may facilitate the widespread application of assisted reproductive technologies (ARTs) on a large scale in this species.

PDCL2 is essential for sperm acrosome formation and male fertility in mice.

BACKGROUND: Each year, infertility affects 15% of couples worldwide, with 50% of cases attributed to men. Globozoospermia is an uncommon cause of male factor infertility, characterized by defects in sperm acrosome formation, leading to round-headed spermatozoa. OBJECTIVE: We generated Pdcl2 knockout mice to investigate the essential roles of PDCL2 in mammalian reproduction. MATERIALS AND METHODS: We used reverse transcription-polymerase chain reaction to demonstrate that PDCL2 was expressed exclusively in the male reproductive tract in mice and humans. We created Pdcl2 knockout mice using the CRISPR-Cas9 system and analyzed their fertility. Pdcl2 null spermatozoa underwent further evaluation using computer-assisted sperm analysis, light microscopy, and ultrastructural microscopy. We used immunoblot analysis and immunofluorescence to elucidate relationships between PDCL2 and other acrosomal proteins. RESULTS: The PDC family is highly conserved in eukaryotes. Mouse and human PDCL2 are testis enriched and localized to the testicular endoplasmic reticulum. Loss of the protein causes sterility because of abnormal acrosome biogenesis during spermiogenesis and immotility. Furthermore, Pdcl2 null spermatozoa have rounded heads, similar to globozoospermia in humans. Observation of the knockout testis shows a lack of acrosomal cap formation, aberrant localization of mitochondria in the sperm head, and misshapen nuclei. CONCLUSION: PDCL2 is essential for sperm acrosome development and male fertility in mice and is a putative contraceptive target in men.

A male germ-cell-specific ribosome controls male fertility.

Ribosomes are highly sophisticated translation machines that have been demonstrated to be heterogeneous in the regulation of protein synthesis1,2. Male germ cell development involves complex translational regulation during sperm formation3. However, it remains unclear whether translation during sperm formation is performed by a specific ribosome. Here we report a ribosome with a specialized nascent polypeptide exit tunnel, RibosomeST, that is assembled with the male germ-cell-specific protein RPL39L, the paralogue of core ribosome (RibosomeCore) protein RPL39. Deletion of RibosomeST in mice causes defective sperm formation, resulting in substantially reduced fertility. Our comparison of single-particle cryo-electron microscopy structures of ribosomes from mouse kidneys and testes indicates that RibosomeST features a ribosomal polypeptide exit tunnel of distinct size and charge states compared with RibosomeCore. RibosomeST predominantly cotranslationally regulates the folding of a subset of male germ-cell-specific proteins that are essential for the formation of sperm. Moreover, we found that specialized functions of RibosomeST were not replaceable by RibosomeCore. Taken together, identification of this sperm-specific ribosome should greatly expand our understanding of ribosome function and tissue-specific regulation of protein expression pattern in mammals.

Polarized epithelium-sperm co-culture system reveals stimulatory factors for the secretion of mouse epididymal quiescin sulfhydryl oxidase 1.

Spermatozoa acquire fertilization ability through post-translational modifications. These membrane surface alterations occur in various segments of the epididymis. Quiescin sulfhydryl oxidases, which catalyze thiol-oxidation reactions, are involved in disulfide bond formation, which is essential for sperm maturation, upon transition and migration in the epididymis. Using castration and azoospermia transgenic mouse models, in the present study, we showed that quiescin sulfhydryl oxidase 1 (QSOX1) protein expression and secretion are positively correlated with the presence of testosterone and sperm cells. A two-dimensional in vitro epithelium-sperm co-culture system provided further evidence in support of the notion that both testosterone and its dominant metabolite, 5α-dihydrotestosterone, promote epididymal QSOX1 secretion. We also demonstrated that immature caput spermatozoa, but not mature cauda sperm cells, exhibited great potential to stimulate QSOX1 secretion in vitro, suggesting that sperm maturation is a key regulatory factor for mouse epididymal QSOX1 secretion. Proteomic analysis identified 582 secretory proteins from the co-culture supernatant, of which 258 were sperm-specific and 154 were of epididymal epithelium-origin. Gene Ontology analysis indicated that these secreted proteins exhibit functions known to facilitate sperm membrane organization, cellular activity, and sperm-egg recognition. Taken together, our data demonstrated that testosterone and sperm maturation status are key regulators of mouse epididymal QSOX1 protein expression and secretion.

Energy Metabolism and Hyperactivation of Spermatozoa from Three Mouse Species under Capacitating Conditions.

Mammalian sperm differ widely in sperm morphology, and several explanations have been presented to account for this diversity. Less is known about variation in sperm physiology and cellular processes that can give sperm cells an advantage when competing to fertilize oocytes. Capacitation of spermatozoa, a process essential for mammalian fertilization, correlates with changes in motility that result in a characteristic swimming pattern known as hyperactivation. Previous studies revealed that sperm motility and velocity depend on the amount of ATP available and, therefore, changes in sperm movement occurring during capacitation and hyperactivation may involve changes in sperm bioenergetics. Here, we examine differences in ATP levels of sperm from three mouse species (genus Mus), differing in sperm competition levels, incubated under non-capacitating and capacitating conditions, to analyse relationships between energetics, capacitation, and swimming patterns. We found that, in general terms, the amount of sperm ATP decreased more rapidly under capacitating conditions. This descent was related to the development of a hyperactivated pattern of movement in two species (M. musculus and M. spicilegus) but not in the other (M. spretus), suggesting that, in the latter, temporal dynamics and energetic demands of capacitation and hyperactivation may be decoupled or that the hyperactivation pattern differs. The decrease in ATP levels during capacitation was steeper in species with higher levels of sperm competition than in those with lower levels. Our results suggest that, during capacitation, sperm consume more ATP than under non-capacitating conditions. This higher ATP consumption may be linked to higher velocity and lateral head displacement, which are associated with hyperactivated motility.

Sperm Numbers as a Paternity Guard in a Wild Bird.

Sperm competition is thought to impose strong selection on males to produce competitive ejaculates to outcompete rival males under competitive mating conditions. Our understanding of how different sperm traits influence fertilization success, however, remains limited, especially in wild populations. Recent literature highlights the importance of incorporating multiple ejaculate traits and pre-copulatory sexually selected traits in analyses aimed at understanding how selection acts on sperm traits. However, variation in a male's ability to gain fertilization success may also depend upon a range of social and ecological factors that determine the opportunity for mating events both within and outside of the social pair-bond. Here, we test for an effect of sperm quantity and sperm size on male reproductive success in the red-back fairy-wren (Malurus melanocephalus) while simultaneously accounting for pre-copulatory sexual selection and potential socio-ecological correlates of male mating success. We found that sperm number (i.e., cloacal protuberance volume), but not sperm morphology, was associated with reproductive success in male red-backed fairy-wrens. Most notably, males with large numbers of sperm available for copulation achieved greater within-pair paternity success. Our results suggest that males use large sperm numbers as a defensive strategy to guard within-pair paternity success in a system where there is a high risk of sperm competition and female control of copulation. Finally, our work highlights the importance of accounting for socio-ecological factors that may influence male mating opportunities when examining the role of sperm traits in determining male reproductive success.

Mitochondrial regulation during male germ cell development.

Mitochondria tailor their morphology to execute their specialized functions in different cell types and/or different environments. During spermatogenesis, mitochondria undergo continuous morphological and distributional changes with germ cell development. Deficiencies in these processes lead to mitochondrial dysfunction and abnormal spermatogenesis, thereby causing male infertility. In recent years, mitochondria have attracted considerable attention because of their unique role in the regulation of piRNA biogenesis in male germ cells. In this review, we describe the varied characters of mitochondria and focus on key mitochondrial factors that play pivotal roles in the regulation of spermatogenesis, from primordial germ cells to spermatozoa, especially concerning metabolic shift, stemness and reprogramming, mitochondrial transformation and rearrangement, and mitochondrial defects in human sperm. Further, we discuss the molecular mechanisms underlying these processes.

Morphometric and morphokinetic differences in the sperm- and oocyte-originated pronuclei of male and female human zygotes: a time-lapse study.

PURPOSE: To study the morphometric and morphokinetic profiles of pronuclei (PN) between male and female human zygotes. METHOD(S): This retrospective cohort study included 94 consecutive autologous single day 5 transfer cycles leading to a singleton live birth. All oocytes were placed in the EmbryoScope + incubator post-sperm injection with all annotations performed retrospectively by one embryologist (L-SO). Timing parameters included 2nd polar body extrusion (tPB2), sperm-originated PN (tSPNa) or oocyte-originated PN (tOPNa) appearance, and PN fading (tPNF). Morphometrics were evaluated at 8 (stage 1), 4 (stage 2), and 0 h before PNF (stage 3), measuring PN area (um2), PN juxtaposition, and nucleolar precursor bodies (NPB) arrangement. RESULTS: Male zygotes had longer time intervals of tPB2_tSPNa than female zygotes (4.8 ± 0.2 vs 4.2 ± 0.1 h, OR = 1.442, 95% CI 1.009-2.061, p = 0.044). SPN increased in size from stage 1 through 2 to 3 (435.3 ± 7.2, 506.7 ± 8.0, and 556.3 ± 8.9 um2, p = 0.000) and OPN did similarly (399.0 ± 6.1, 464.3 ± 6.7, and 513.8 ± 6.5 um2, p = 0.000), with SPN being significantly larger than OPN at each stage (p < 0.05 respectively). More male than female zygotes reached central PN juxtaposition at stage 1 (76.7% vs 51.0%, p = 0.010), stage 2 (97.7% vs 86.3%, p = 0.048), and stage 3 (97.7% vs 86.3%, p = 0.048). More OPN showed aligned NPBs than in SPN at stage 1 only (44.7% vs 28.7%, p = 0.023). CONCLUSION(S): Embryos with different sexes display different morphokinetic and morphometric features at the zygotic stage. Embryo selection using such parameters may lead to unbalanced sex ratio in resulting offspring.