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Over the past decade, there has been an increasing trend in the use of assisted reproductive technologies, which have significantly expanded the opportunities to overcome the problem of infertility. However, the problem of increasing the effectiveness of in vitro fertilization remains open. Isolation of germ cells from animals is a necessary process for various experimental studies. Animal germ cells can be used in experiments to study physical, chemical, genetic, immunological, and microbiological factors affecting reproduction efficiency and for the development of techniques that increase the effectiveness of in vitro fertilization. All of the above determines the relevance of studying existing methods of oocyte and sperm isolation for experimental in vitro studies. Here we discuss the existing methods of sperm and oocyte isolation from animals and their advantages and disadvantages, and also substantiate priority methods for use., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

In mammalian reproduction, testis-specific protein IZUMO1 and its receptor JUNO on the oocyte surface are essential for sperm-oocyte recognition, binding, and membrane fusion. However, these factors alone are insufficient to accomplish cytoplasmic membrane fusion. It is believed that other gametic proteins interact with them to facilitate sperm-oocyte interaction on the head and mid-tail of rat spermatozoa as well as on the surface of oocytes. In this study, Copper Transport Protein 2 (CTR2) has been identified on the head and mid-tail of rat spermatozoa as well as on the surface of oocytes. CTR2 directly interacts with both IZUMO1 and JUNO, colocalizing with IZUMO1 on the sperm head and with JUNO on the oocyte membrane. Treatment of the capacitated sperm and zona pellucida-free oocytes with anti-CTR2 antibody resulted in a significant decrease in fertilization rates in IVF experiments. These findings suggest that CTR2 plays an important role in mammalian fertilization by interacting with IZUMO1 and JUNO, providing new insights into the molecular mechanisms of mammalian sperm-oocyte adhesion and fusion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Traditionally, sperm and embryos were studied using microscopy to assess morphology and motility. However, OMICS technologies, especially transcriptomic analysis, are now being used to screen the molecular dynamics of fertility markers at cellular and molecular levels, with high sensitivity. Transcriptomics is the study of the transcriptome - RNA transcripts produced by the genome - using high-throughput methods to understand how the RNAs are expressed. In this review, we have discussed gene contributions to sperm structure and function and their role in fertilization and early embryo development. Further, we identified miRNAs shared by sperm, oocytes, and early embryos and their roles in fertilization and early embryo development., Competing Interests: Declaration of Competing Interest The authors have nothing to declare., (Published by Elsevier B.V.)

Epigenetic mechanisms, including DNA methylation and histone modifications, govern chromatin arrangement in sperm, enhancing motility and safeguarding DNA integrity for accurate epigenetic inheritance. Abnormal methylation is linked to poor sperm quality and fertility issues, underscoring the need to study sperm DNA methylation and its impact on sperm function and embryo development in assisted reproductive technology. In this study, processed spermatozoa from 75 normozoospermic and 15 abnormal ejaculates were examined for sperm global DNA methylation levels using a colourimetric absorbance method. Although semen characteristics were poor in abnormal ejaculates, no significant correlation was found between sperm global DNA methylation levels and sperm characteristics in either normozoospermic or abnormal cohorts. However, mean global DNA methylation levels were significantly lowered in abnormal sperm samples compared to normozoospermic samples ( p  < 0.05). Furthermore, injecting spermatozoa from these patients (N = 50) into donor oocytes did not show a significant relationship between sperm global DNA methylation and embryo developmental competence. These findings highlight the limitation of sperm global DNA methylation as a biomarker for embryo development and quality.

We evaluated the quality and fertilizing ability of frozen-thawed porcine sperm that were selected using a commercially available device (MIGLIS, Menicon Life Science) consisting of three parts: an outer lid, an inner lid, and a tube. Firstly, to determine an adequate concentration of caffeine for separation, frozen-thawed sperm were incubated with different concentrations of caffeine (0, 1, 2.5, 5, and 10 mM) in a MIGLIS device. To determine the appropriate incubation time for separating sperm in the MIGLIS device, frozen-thawed sperm were incubated with 2.5 mM caffeine for 5, 10, 15, or 20 min. To evaluate the fertilization and embryo development of oocytes fertilized with frozen-thawed sperm separated into two regions (outer and inner) in the MIGLIS device, the separated sperm from the three boars was used to fertilize in vitro-matured oocytes and cultured in vitro for 7 days. Sperm quality parameters of sperm collected from the inner tube after incubation with 2.5 mM caffeine were superior to sperm incubated without caffeine. Moreover, sperm collected from the inner tube after incubation for 10 min had a higher progressive motility. The rate of blastocyst produced from spermatozoa collected from the inner tube after incubation with 2.5 mM caffeine for 10 min significantly increased compared to that produced from spermatozoa from the outer tube, regardless of the boar. In conclusion, sperm sorting using the MIGLIS device may be useful for separating high-quality sperm after incubation with 2.5 mM caffeine for 10 min to improve blastocyst formation., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Background: The intracytoplasmic sperm injection (ICSI) technique has low efficiency in cattle. This has mainly been attributed to the oocyte activation failure due to oocyte and/or sperm factors., Aim: Our aim was to evaluate the effect of conventional ICSI and Piezo-ICSI with bull or human sperm on bovine oocyte activation and embryo development and to assess its relationship with the phospholipase C zeta (PLCɀ) activity of both species., Methods: In vitro matured bovine oocytes were randomly divided into five groups and were fertilized as follows: conventional ICSI using bovine sperm with chemical activation (control), conventional ICSI using bovine sperm, Piezo-ICSI using bovine sperm, conventional ICSI using human sperm, and Piezo-ICSI using human sperm. PLCɀ activity was determined in bull and human sperm samples., Results: Within the groups using bull sperm, the oocytes fertilized by conventional ICSI had the lowest values of 2 pronuclei (PN) formation and cleavage, Piezo-ICSI increased both percentages and ICSI + chemical activation presented the highest 2 PN, cleavage, and blastocyst rates ( p < 0.05). Within the groups using human sperm, the oocytes fertilized by Piezo-ICSI presented higher 2 PN and cleavage rates than those activated by conventional ICSI ( p < 0.05). Piezo-ICSI with human sperm increased bovine oocyte activation as much as conventional ICSI + chemical activation with bovine sperm ( p < 0.05). Higher values of PLCɀ activity were found in human sperm compared with bovine sperm ( p < 0.05)., Conclusion: Our results suggest that the higher stability of the bovine sperm in combination with its relatively low content of PLCɀ impairs bovine oocyte activation after ICSI., Competing Interests: The authors have declared that there is no conflict of interest concerning this study.

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.

Fertilization failure refers to the failure in the pronucleus formation, evaluating 16-18 h post in vitro fertilization or intracytoplasmic sperm injection. It can be caused by sperm, oocytes, and sperm-oocyte interaction and lead to great financial and physical stress to the patients. Recent advancements in genetics, molecular biology, and clinical-assisted reproductive technology have greatly enhanced research into the causes and treatment of fertilization failure. Here, we review the causes that have been reported to lead to fertilization failure in fertilization processes, including the sperm acrosome reaction, penetration of the cumulus and zona pellucida, recognition and fusion of the sperm and oocyte membranes, oocyte activation, and pronucleus formation. Additionally, we summarize the progress of corresponding treatment methods of fertilization failure. This review will provide the latest research advances in the genetic aspects of fertilization failure and will benefit both researchers and clinical practitioners in reproduction and genetics., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

As the time of ovulation draws near, mouse spermatozoa move out of the isthmic reservoir, which is a prerequisite for fertilization. However, the molecular mechanism remains unclear. The present study revealed that mouse cumulus cells of oocytes-cumulus complexes (OCCs) expressed transforming growth factor-β ligand 1 (TGFB1), whereas ampullary epithelial cells expressed the TGF-β receptors, TGFBR1 and TGFBR2, and all were upregulated by luteinizing hormone (LH)/human chorionic gonadotropin (hCG). OCCs and TGFB1 increased natriuretic peptide type C (NPPC) expression in cultured ampullae via TGF-β signaling, and NPPC treatment promoted spermatozoa moving out of the isthmic reservoir of the preovulatory oviducts. Deletion of Tgfb1 in cumulus cells and Tgfbr2 in ampullary epithelial cells blocked OCC-induced NPPC expression and spermatozoa moving out of the isthmic reservoir, resulting in compromised fertilization and fertility. Oocyte-derived paracrine factors were required for promoting cumulus cell expression of TGFB1. Therefore, oocyte-dependent and cumulus cell-derived TGFB1 promotes the expression of NPPC in oviductal ampulla, which is critical for sperm migration in the oviduct and subsequent fertilization., (© 2022. The Author(s).)

The current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight. While this approach maximizes the number of fertilized oocytes, on the other side it exposes them to high concentration of reactive oxygen species (ROS) generated by active and degenerating spermatozoa, and positively correlates with polyspermy. Here we set up to precisely define the time frame during which spermatozoa effectively penetrates and fertilizes the oocyte, in order to drastically reduce spermatozoa-oocyte interaction. To do that, in vitro matured sheep oocytes co-incubated with spermatozoa in IVF medium were sampled every 30 min (start of incubation time 0) to verify the presence of a fertilizing spermatozoon. Having defined the fertilization time frame (4 h, data from 105 oocytes), we next compared the standard IVF procedures overnight (about 16 h spermatozoa/oocyte exposure, group o/nIVF) with a short one (4 h, group shIVF). A lower polyspermic fertilization (> 2PN) was detected in shIVF (6.5%) compared to o/nIVF (17.8%), P < 0.05. The o/nIVF group resulted in a significantly lower 2-cell stage embryos, than shIVF [34.6% (81/234) vs 50.6% (122/241) respectively, P < 0.001]. Likewise, the development to blastocyst stage confirmed a better quality [29% (70/241) vs 23.5% (55/234), shIVF vs o/nIVF respectively] and an increased Total Cell Number (TCN) in shIVF embryos, compared with o/n ones. The data on ROS have confirmed that its generation is IVF time-dependent, with high levels in the o/nIVF group. Overall, the data suggest that a shorter oocyte-spermatozoa incubation results in an improved embryo production and a better embryo quality, very likely as a consequence of a shorter exposure to the free oxygen radicals and the ensuing oxidative stress imposed by overnight culture., (© 2021. The Author(s).)

Compelling evidence has shown that parental experiences and age at conception may potentially shape the future health of the next generation(s). Certain factors may affect both the female and, strikingly, the male gametes potentially causing the transmission of acquired traits, which was strongly defended by Jean-Baptiste Lamarck. Neurodevelopmental psychiatric disorders, trinucleotide repeat-associated diseases, cardiovascular pathologies, diabetes, obesity and cancer in the offspring, among others, have now been associated with events occurring at the preconception level. The potential implications of a (trans)generational inheritance of parental disease and exposure effects should be taken into account in counselling and public policy. Further research into how exactly gametes apparently deliver more than DNA to a new generation is warranted., (© 2021 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)

Propagation of paternal sperm-contributed mitochondrial genes, resulting in heteroplasmy, is seldom observed in mammals due to post-fertilization degradation of sperm mitochondria, referred to as sperm mitophagy. Whole organelle sperm mitochondrion degradation is thought to be mediated by the interplay between the ubiquitin-proteasome system (UPS) and the autophagic pathway (Song et al., Proc. Natl. Acad. Sci. USA, 2016). Both porcine and primate post-fertilization sperm mitophagy rely on the ubiquitin-binding autophagy receptor, sequestosome 1 (SQSTM1), and the proteasome-interacting ubiquitinated protein dislocase, valosin-containing protein (VCP). Consequently, we anticipated that sperm mitophagy could be reconstituted in a cell-free system consisting of permeabilized mammalian spermatozoa co-incubated with porcine oocyte extracts. We found that SQSTM1 was detected in the midpiece/mitochondrial sheath of the sperm tail after, but not before, co-incubation with oocyte extracts. VCP was prominent in the sperm mitochondrial sheath both before and after the extract co-incubation and was also detected in the acrosome and postacrosomal sheath and the subacrosomal layer of the spermatozoa co-incubated with extraction buffer as control. Such patterns are consistent with our previous observation of SQSTM1 and VCP associating with sperm mitochondria inside the porcine zygote. In addition, it was observed that sperm head expansion mimicked the early stages of paternal pronucleus development in a zygote during prolonged sperm-oocyte extract co-incubation. Treatment with anti-SQSTM1 antibody during extract co-incubation prevented ooplasmic SQSTM1 binding to sperm mitochondria. Even in an interspecific cellular environment encompassing bull spermatozoa and porcine oocyte extract, ooplasmic SQSTM1 was recruited to heterospecific sperm mitochondria. Complementary with the binding of SQSTM1 and VCP to sperm mitochondria, two sperm-borne pro-mitophagy proteins, parkin co-regulated gene product (PACRG) and spermatogenesis associated 18 (SPATA18), underwent localization changes after extract coincubation, which were consistent with their degradation observed inside fertilized porcine oocytes. These results demonstrate that the early developmental events of post-fertilization sperm mitophagy observed in porcine zygote can be reconstituted in a cell-free system, which could become a useful tool for identifying additional molecules that regulate mitochondrial inheritance in mammals.

Successful reproduction is very important for individuals and for society. Currently, the human health span and lifespan are the object of intense and productive investigation with great achievements, compared to the last century. However, reproduction span does not progress concomitantly with lifespan. Reproductive organs age, decreasing the levels of sexual hormones, which are protectors of health through their action on several organs of the body. Thus, this is the starting point of the organismal decay and infertility. This starting point is easily detected in women. In men, it goes under the surface, undetected, but it goes, nevertheless. Regarding fertility, aging alters the hormonal equilibrium, decreases the potential of reproductive organs, diminishes the quality of the gametes and worsen the reproductive outcomes. All these events happen at a different pace and affecting different organs in women and men. The question is what molecular pathways are involved in reproductive aging and if there is a possible halting or even reversion of the aging events. Answers to all these points will be explained in the present review., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Sperm-oocyte binding initiates an outside-in signaling event in the mouse oocyte that triggers recruitment and activation of the cytosolic protein kinase PTK2B in the cortex underlying the bound sperm. While not involved in gamete fusion, PTK2B activity promotes actin remodeling events important during sperm incorporation. However, the mechanism by which sperm-oocyte binding activates PTK2B is unknown, and the present study examined the possibility that sperm interaction with specific oocyte surface proteins plays an important role in PTK2B activation. Imaging studies revealed that as IZUMO1R and CD9 became concentrated at the sperm binding site, activated (phosphorylated) PTK2B accumulated in the cortex underlying the sperm head and in microvilli partially encircling the sperm head. In order to determine whether IZUMO1R and/or CD9 played a significant role in PTK2B recruitment and activation at the sperm binding site, the ability of oocytes null for Izumo1r or Cd9, to initiate an increase in PTK2B content and activation was tested. The results revealed that IZUMO1R played a minor role in PTK2B activation and had no effect on actin remodeling; however, CD9 played a very significant role in PTK2B activation and subsequent actin remodeling at the sperm binding site. These findings suggest the possibility that interaction of sperm surface proteins with CD9 or CD9-associated oocyte proteins triggers PTK2B activation at the sperm binding site., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

To trigger gamete fusion, spermatozoa need to activate the molecular machinery in which sperm IZUMO1 and oocyte JUNO (IZUMO1R) interaction plays a critical role in mammals. Although a set of factors involved in this process has recently been identified, no common factor that can function in both vertebrates and invertebrates has yet been reported. Here, we first demonstrate that the evolutionarily conserved factors dendrocyte expressed seven transmembrane protein domain-containing 1 (DCST1) and dendrocyte expressed seven transmembrane protein domain-containing 2 (DCST2) are essential for sperm-egg fusion in mice, as proven by gene disruption and complementation experiments. We also found that the protein stability of another gamete fusion-related sperm factor, SPACA6, is differently regulated by DCST1/2 and IZUMO1. Thus, we suggest that spermatozoa ensure proper fertilization in mammals by integrating various molecular pathways, including an evolutionarily conserved system that has developed as a result of nearly one billion years of evolution., Competing Interests: NI, YH, IW No competing interests declared, (© 2021, Inoue et al.)

Disorders affecting the sperm, oocyte, or embryo may cause a significant fraction of spontaneous miscarriages and cases of recurrent pregnancy loss (RPL). Altered chromosomal integrity of sperm and oocytes, which is highly dependent of the age of the mother, represents a major cause of miscarriage and in turn RPL. Avoiding transfers of abnormal embryos is possible with preimplantation genetic testing for aneuploidies. Chromosomal anomalies may also be caused by structural rearrangements of one or several chromosomes in either parents, a finding encountered in 12% of couples with RPL, including in those who have had one or several healthy babies. More than 40% of these chromosomal rearrangements are identifiable on regular karyotypes. When abnormal findings are made, preimplantation genetic testing for monogenic disorders allows selection of disease-free embryos. Finally, asymmetric inactivation of the X chromosome has been found more commonly in women with RPL, but no specific treatment is currently available., (Copyright © 2020. Published by Elsevier Inc.)

Purpose: Intracytoplasmic sperm injection (ICSI) is the most widely utilized assisted reproductive technique (ART) worldwide. In this feature, we review the early assisted fertilization attempts that eventually led to the development of ICSI, and discuss its current utilization in cases of male and non-male factor infertility., Methods: We researched the literature related to the development, indications, and current use of ICSI, such as sperm structural abnormalities, male genetic indications, surgically retrieved sperm, high sperm chromatin fragmentation, oocyte dysmorphism, and preimplantation genetic testing (PGT). We also describe the potential future applications of ICSI., Results: This review summarizes the early micromanipulation techniques that led to the inception of ICSI. We also explore its current indications, including non-male factor infertility, where its use is more controversial. Finally, we consider the benefits of future advancements in reproductive biology that may incorporate ICSI, such as in vitro spermatogenesis, neogametogenesis, and heritable genome editing., Conclusion: The versatility, consistency, and reliability of ICSI have made it the most prevalently utilized ART procedure worldwide.

The aim of this study was to evaluate the fertilizing capacity of frozen or vitrified stallion sperm after assessing different warming procedures. In Experiment 1, different warming procedures were compared after sperm vitrification: immersion in extender at 43 °C (C), or in a water bath at 37 °C/30 s (W37), 43 °C/10 s (W43) or 60 °C/5 s (W60). With the W60 treatment, there were greater values (P < 0.05) for VCL (83.93 ± 3.6 μm/s) and ALH (3.00 ± 0.2 μm) than freezing and with the C group, and greater values (P < 0.001) for PM (35.33 ± 2.5 %) than with the W43 treatment. In Experiment 2, the fertilizing capacity of vitrified and frozen sperm was assessed utilizing heterologous IVF procedures, using cattle oocytes. Vitrification resulted in greater values (P < 0.05) than freezing for the number of bound sperm (1.36 ± 0.3 and 0.69 ± 0.2, respectively). There were no differences between frozen or vitrified sperm in pronuclear formation (26 hours post-insemination - hpi; 14.08 ± 4.2 % and 22.78 ± 4.8 %, respectively) or cleavage rate (32.77 ± 4.3 % and 39.66 ± 4.6 %, respectively). In conclusion, vitrified stallion sperm warmed in a water bath at 60 ºC had the capacity to penetrate cattle oocytes, leading to pronuclear formation and hybrid embryo cleavage after heterologous IVF., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: To evaluate the impact of sperm morphology (SM) on laboratory and pregnancy outcomes in conventional intracytoplasmic sperm injection (c-ICSI) cycles, using the egg donation model to minimize female confounding variables., Materials and Methods: We retrospectively collected data of oocyte donation cycles from October 2016 to February 2020. Median seminal parameters, total (1-2-3PN) fertilization rate (FR), 2PN FR, cleavage rate (CR), implantation rate (IR), pregnancy rate (PR), miscarriage rate (MR), and live birth rate (LBR) were collected. The study population was divided into three groups: Group 1 with SM < 4%, Group 2 with SM between 4% and 6%, and Group 3 with SM > 6%., Results: Of 741 fresh ICSI cycles and 4507 warmed oocytes were included. Male age was 46.0 (31.0-72.0) years, and recipients' age was 44.0 (29.0-54.0) years. Normal SM was 5.0% (1.0%-15.0%). Male age was negatively correlated with normal SM (P = .002; Rho -0.113). Oocyte survival rate was 83.3% (16.7%-100.0%). Total FR was 75.0% (11.1%-100.0%), 2PN FR was 66.7% (11.1%-100.0%) %, and CR was 100% (0.0%-100%). Comparing samples with SM > 6% and those with SM < 4%, 2PN FR was significantly higher in the first group (P = .04). No significant associations were found among groups in terms of CR. IR was 27.7%, resulting significantly higher when normal SM was > 6% (P < .01). Clinical PR was 36.0%, MR was 23.9%, and LBR was 25.9%. PR and LBR were significantly higher in samples with normal SM > 6%, compared to other groups (P = .02 and P < .01, respectively)., Conclusions: Although c-ICSI technique allows the embryologist to select the best quality spermatozoa, male factor plays a key role in achieving successful assisted reproductive outcomes. Normal SM has been shown to have implications not only for laboratory outcomes, in terms of fertilization, but also for clinical findings, as regards implantation, pregnancy, and live birth., (© 2020 American Society of Andrology and European Academy of Andrology.)

The present study aimed to investigate and compare fitness-related traits and ploidy levels of purebreds and hybrids produced from sturgeon broodstock with both normal and abnormal ploidy levels. We used diploid Acipenser ruthenus and tetraploid A. baerii males and females to produce purebreds and reciprocal hybrids of normal ploidy levels. Likewise, we used diploid A. ruthenus and tetraploid A. baerii females mated to pentaploid and hexaploid A. baerii males to produce hybrids of abnormal ploidy levels. Fertilization of ova of A. ruthenus and A. baerii of normal ploidy with the sperm of pentaploid and hexaploid A. baerii produced fully viable progeny with ploidy levels that were intermediate between those of the parents as was also found in crosses of purebreds and reciprocal hybrids of normal ploidy levels. The A. ruthenus × pentaploid A. baerii and A. ruthenus × hexaploid A. baerii hybrids did not survive after 22 days post-hatch (dph). Mean body weight and cumulative survival were periodically checked at seven-time intervals. The recorded values of mean body weight were significantly higher in A. baerii × pentaploid A. baerii hybrids than other groups at three sampling points (160, 252 and 330 dph). In contrast, the highest cumulative survival was observed in A. baerii × A. ruthenus hybrids at all sampling points (14.47 ± 5.70 at 497 dph). Overall, most of the studied sturgeon hybrids displayed higher mean BW and cumulative survival compared to the purebreds. The utilization of sturgeon hybrids should be restricted to aquaculture purposes because they can pose a significant genetic threat to native populations through ecological interactions.

The interaction of sperm with the oocyte is pivotal during the process of mammalian fertilization. The limited numbers of sperm that reach the fallopian tube as well as anatomic restrictions indicate that human sperm-oocyte encounter is not a matter of chance but a directed process. Chemotaxis is the proposed mechanism for re-orientating sperm toward the source of a chemoattractant and hence to the oocyte. Chemokines represent a superfamily of small (8-11 kDa), cytokine-like proteins that have been shown to mediate chemotaxis and tissue-specific homing of leukocytes through binding to specific chemokine receptors such as CCRs. Here we show that CCR6 is abundantly expressed on human sperms and in human testes. Furthermore, radioligand-binding experiments showed that CCL20 bound human sperm in a specific manner. Conversely, granulosa cells of the oocyte-surrounding cumulus complex as well as human oocytes represent an abundant source of the CCR6-specific ligand CCL20. In human ovaries, CCL20 shows a cycle-dependent expression pattern with peak expression in the preovulatory phase and CCL20 protein induces chemotactic responses of human sperm. Neutralization of CCL20 in ovarian follicular fluid significantly impairs sperm migratory responses. Conversely, analyses in infertile men with inflammatory conditions of the reproductive organs demonstrate a significant increase of CCL20/CCR6 expression in testis and ejaculate. Taken together, findings of the present study suggest that CCR6-CCL20 interaction may represent an important factor in directing sperm-oocyte interaction., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: To evaluate reproductive outcomes of artificial insemination and IVF with donor sperm (AID or IVF-D) for male-factor couples with a history of unsuccessful ICSI attempt., Methods: This retrospective cohort includes couples with severe male-factor infertility who failed ICSI treatment, and subsequently underwent semen donation treatment. We report the following outcomes: (1) live birth rates in AID and IVF-D treatment for couples with severe male infertility factors and prior ICSI failures; (2) paternal impact on embryo development of the same oocyte cohort; (3) prognostic factors in obtaining a live birth with donor semen., Results: Of 92 women with failed ICSI cycles (26 with multiple attempts), 45 couples underwent AID treatment. Live birth rate per cycle of AID was 18.9%. Fifty-three patients underwent IVF-D including 6 couples who previously did not conceive with AID. Embryological outcomes including fertilization, viable cleavage embryos, and blastocyst formation rates were significantly lower in ICSI cycles with partner sperm compared with IVF-D (P < 0.01). Logistic regression analysis showed that female age and the severity of spermatogenetic disorder are prognostic factors in obtaining a live birth with donated sperm., Conclusion: Couples with severe male infertility factor (azoospermia or extreme oligoasthenospermia) and a history of unsuccessful ICSI cycles benefit from treating with donor sperm. ICSI fertilization, embryo viability, and progression of the embryo to the blastocyst stage are significantly deteriorated by semen parameters. The prognostic factors identified may help couples plan their treatment and prepare for their parenthood journey.

Objective: To investigate the applicability of phospholipase C zeta (PLCζ) analysis in assisting the clinical decision-making process when considering artificial oocyte activation (AOA) for infertile males in assisted reproductive technology., Design: Fifty-six males (43 infertile/13 fertile) were screened using our PLCζ assay., Setting: Fertility unit/university laboratory., Patient(s): Infertile males with abnormal sperm morphology or total fertilization failure, low fertilization rate (<50%), or repeated fertilization failure in assisted reproductive technology., Intervention(s): We analyzed PLCζ levels in sperm from fertile and infertile males. Eligible patients subsequently underwent intracytoplasmic sperm injection (ICSI)/artificial oocyte activation (AOA) with calcimycin (GM508)., Main Outcome Measure(s): PLCζ localization and level and the proportion of sperm expressing PLCζ. Thresholds of PLCζ deficiency, fertilization rates, pregnancy rates, and live birth rates of AOA and non-AOA cycles., Result(s): Compared with 13 fertile controls, 34 of the 43 infertile males had significantly lower levels of PLCζ and/or a significantly lower proportion of sperm exhibiting PLCζ. Of these 34 patients, 15 showed a significant PLCζ reduction in both parameters, which we termed "PLCζ deficiency." Five PLCζ-deficient patients opted for AOA; all five achieved fertilization, and four achieved clinical pregnancies and live births. The fertilization rate improved significantly from 18.6% (ICSI) to 56.8% (ICSI/AOA). The clinical pregnancy rate and live birth rate with AOA were both 40% per initiated cycle. Youden index analysis revealed that the cutoffs below which infertile males were likely to benefit from AOA were 71% for the proportion of sperm expressing PLCζ and 15.57 arbitrary units for mean PLCζ level., Conclusion(s): PLCζ analysis is a useful diagnostic tool to determine patient eligibility for subsequent AOA treatment., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Sperm-oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins. Sperm membrane protein IZUMO1 and its counterpart oocyte membrane protein JUNO make a protein complex allowing sperm to interact with the oocyte, and subsequent sperm-oocyte fusion. Oocyte tetraspanin protein CD9 also contributes to sperm-oocyte fusion. However, the fusion process cannot be explained solely by these three essential factors. In this study, we focused on analyzing a testis-specific gene 4930451I11Rik and generated mutant mice using the CRISPR/Cas9 system. Although IZUMO1 remained in 4930451I11Rik knockout (KO) spermatozoa, the KO spermatozoa were unable to fuse with oocytes and the KO males were severely subfertile. 4930451I11Rik encodes two isoforms: a transmembrane (TM) form and a secreted form. Both CRISPR/Cas9-mediated TM deletion and transgenic (Tg) rescue with the TM form revealed that only the TM form plays a critical role in sperm-oocyte fusion. Thus, we renamed this TM form Fertilization Influencing Membrane Protein (FIMP). The mCherry-tagged FIMP TM form was localized to the sperm equatorial segment where the sperm-oocyte fusion event occurs. Thus, FIMP is a sperm-specific transmembrane protein that is necessary for the sperm-oocyte fusion process., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The endogenous opioid peptides have been reported to be involved in the regulation of reproductive physiology. Many of the studies conclude with sentences around the harmful effect of opioids in male fertility but, actually, there is only one study regarding the real fertility potential of spermatozoa that have been exposed to mu specific opioids. The aim of the present study was to see if the modulation of delta (OPRD1) and kappa (OPRK1) opioid receptors in mouse sperm during capacitation was able to vary the embryo production after in vitro fertilization (IVF). The presence of OPRD1 and OPRK1 in mouse mature spermatozoa was analyzed by RT-PCR and immunofluorescence. Incubating the sperm with, on one hand, the delta specific agonist DPDPE and/or antagonist naltrindole, and, on the other hand, the kappa specific agonist U-50488 and antagonist nor-binaltorphimine, we analyzed the involvement of OPRD1 and OPRK1 on IVF and preimplantational embryo development. We verified the presence of OPRD1 and OPRK1 in mouse mature spermatozoa, not only at the mRNA level but also at protein level. Moreover, the sperm incubation with DPDPE, before the IVF, had an effect on the fertilization rate of sperm and reduced the number of reached blastocysts, which was reverted by naltrindole. Instead, the use of the kappa agonist U-50488 and the antagonist nor-binaltophimine did not have any effect on the amount and the quality of the achieved blastocysts. Although nowadays the pure delta or kappa opioid ligands are not used for the clinic, clinical trials are being conducted to be used in the near future, so it would be interesting to know if the modulation of these receptors in sperm would generate any consequence in relation to fertilization capacity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Sperm preparation in IVF cycles using density gradient centrifugation (DGC) in combination with swim-up (SU) has been widely adopted in reproductive centres worldwide. It is a fact that the sperm recovery rate following one DGC from poor semen samples (showing liquefaction defects/containing too many unresolvable clots or rare sperm) is relatively low. Our results showed that double DGC (DDGC) is effective at increasing the sperm recovery rate from poor semen samples. However, DDGC may increase the mechanical stress of sperm, thereby potentially impairing embryo development. Therefore, it is necessary to evaluate the safety of using sperm prepared by DDGC/SU for IVF cycles. In this study, we retrospectively analysed the data generated from a total of 529 IVF cycles (from June 2017 to June 2018), and these IVF cycles contributed 622 transfer cycles (from June 2017 to December 2018) in Changzhou Maternal and Child Health Care Hospital. Of them, 306 IVF cycles and the related 355 transfer cycles (normal semen samples prepared by DGC/SU) were set as the normal group, while 223 IVF cycles and the related 267 transfer cycles (poor semen prepared by DDGC/SU) were set as the observation group. The main outcome measures, including the normal fertilization rate, top D3 embryo formation rate, blastocyte formation rate, clinical pregnancy rate and live birth rate, birth weight and duration of pregnancy, were compared between the two groups. Compared to semen in the DGC/SU group, semen in the DDGC/SU group showed increased levels of the DNA fragmentation index (DFI) and reduced sperm concentration, percentage of progressive motility (PR) sperm, and percentage of normal morphology sperm. The indicators reflecting in vitro embryo development and clinical outcomes were similar in the DGC/SU group and DDGC/SU group, including the normal fertilization rate, top D3 embryo formation rate, blastocyte formation rate, pregnancy rate, implantation rate, spontaneous abortion rate, live birth rate, birth weight and duration of pregnancy. Furthermore, we found that the 1PN zygote formation rate was significantly lower in the DDGC/SU group than that in the DGC/SU group. We concluded that oocytes fertilized by sperm from poor semen samples separated by DDGC/SU achieved the same outcomes as oocytes fertilized by sperm from normal semen separated by DGC/SU, suggesting that DDGC/SU is an effective and safe method of sperm enrichment for poor semen samples in IVF. The main contribution of the present study is the verification of the effectiveness of DDGC/SU in improving sperm recovery from poor semen samples and the safety of using sperm prepared by DDGC/SU for IVF.

Cell-cell adhesion molecules have critically important roles in the early events of reproduction including gamete transport, sperm-oocyte interaction, embryonic development, and implantation. Major adhesion molecules involved in reproduction include cadherins, integrins, and disintegrin and metalloprotease domain-containing (ADAM) proteins. ADAMs on the surface of sperm adhere to integrins on the oocyte in the initial stages of sperm-oocyte interaction and fusion. Cadherins act in early embryos to organize the inner cell mass and trophectoderm. The trophoblast and uterine endometrial epithelium variously express cadherins, integrins, trophinin, and selectin, which achieve apposition and attachment between the elongating conceptus and uterine epithelium before implantation. An overview of the major cell-cell adhesion molecules is presented and this is followed by examples of how adhesion molecules help shape early reproductive events. The argument is made that a deeper understanding of adhesion molecules and reproduction will inform new strategies that improve embryo survival and increase the efficiency of natural mating and assisted breeding in cattle., (© 2020 Wiley-Liss, Inc., A Wiley Company.)

During mammalian fertilization, sperm is fused with the oocyte's membrane, triggering the resumption of meiosis from the metaphase II arrest, the extrusion of the second polar body, and the exocytosis of cortical granules; these events are collectively called 'oocyte activation.' In all species studied to date, the transient rise in the cytosolic level of calcium (in particular, the repeated calcium increases called 'calcium oscillations' in mammals) is required for these events. Researchers have focused on identifying the factor(s) that can induce calcium oscillations during fertilization. Sperm-specific phospholipase C, i.e., PLC zeta (PLCζ), is a strong candidate of the factor(s), and several research groups using different species obtained evidence that PLCζ is a sperm factor that can induce calcium oscillations during fertilization. However, postacrosomal sheath Tryptophan-Tryptophan (WW)-domain-binding protein (PAWP) was recently shown to have a pivotal role in inducing calcium oscillations in some species. In this review, we focus on PLCζ and PAWP as sperm factors, and we discuss this controversy: Which of these two molecules survives as a sperm factor?, (© 2020 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.)

Purpose: To investigate whether the ability of human spermatozoa to decondense in vitro in the presence of heparin (Hep) and glutathione (GSH) is related to assisted reproduction (ART) success., Methods: Cross-sectional pilot study involving male partners of 129 infertile couples undergoing ICSI with (45) or without (84) donor oocytes at two infertility clinics in CABA, Argentina, between October 2012 and December 2013. In vitro decondensation kinetics with Hep and GSH and DNA fragmentation (TUNEL) were determined on the same sample used for ICSI. The possible relationship of decondensation parameters (maximum decondensation and decondensation velocity) and TUNEL values with ART success was evaluated., Results: Embryo quality correlated positively with decondensation velocity (D60/D30) (Spearman's correlation, p < 0.05). According to D60/D30 values, patients were classified as slow decondensers (SlowD) (n = 68) or fast decondensers (FastD) (n = 61). Embryo quality was better in FastD (unpaired t test, p < 0.05). FastD and SlowD were subdivided according to use of donor oocytes. Among SlowD, biochemical and clinical pregnancy rates per transfer were significantly higher in donor (n = 19) vs. in non-donor (n = 31) cycles (Fisher's exact test, p < 0.05). TUNEL values were not related to embryo quality, but no clinical pregnancies or live births were achieved in TUNEL+ SlowD (n = 7)., Conclusion: Decondensation kinetics of human spermatozoa in vitro with Hep and GSH could be related to embryo quality and ART success.

The pig is an important livestock animal. Biotechnological interest in this species has increased due to its use, among others, in the generation of transgenic animals for use in biomedicine based on its greater physiological proximity to the human species than other large domestic animals. This development has paralleled an improvement in Assisted Reproduction Techniques (ART) used for this species. However, the ability to generate animals from embryos produced entirely in vitro is still limited and a wide margin for improvement remains. Here we review the procedures, additives, and devices used during pig in vitro fertilization (IVF), focusing on the main points of each step that have offered the best results in terms of increased efficiency of the system. The lack of standardized protocols and consensus on the parameters to be assessed makes it difficult to compare results across different studies, but some conclusions are drawn from the literature. We anticipate that new physiological protocols will advance the field of swine IVF, including induction of prefertilization ZP hardening with oviductal fluid, sperm preparation by swim-up method, increased viscosity through the addition of inert molecules or reproductive biofluids, and the incorporation of 3D devices. Here we also reflect on the need to expand the variables on which the efficiency of pig IVF is based, providing new parameters that should be considered to supply more objective and quantitative assessment of IVF additives and protocols., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The effect of extracellular sperm ubiquitination was examined from many aspects and the majority of existing studies negatively correlated the amount of highly ubiquitinated sperm cells in the sample with the ejaculate quality and the fertilization success rate. In the present study, we compared an early embryonic development up to blastocyst stage in the pig using two defined sperm cell populations sorted by flow cytometry (FACS) based on the rate of the extracellular ubiquitination. This novel approach allows studying the direct effect of extracellular ubiquitin (eUb), which is a marker for epididymal recognition and degradation of defective sperm cells. We further examined the hypothesis that eUb could be recognized directly in the ooplasm. In the porcine model, the significance of results might be seriously affected by a high variability among sperm cell doses from individual boars as well as by the variability among separate sample collections. To overcome this obstacle, we used cryopreserved sperm cells from a single dose. Comparison of an early embryonic development employing intracytoplasmic sperm cell injection (ICSI) with cryopreserved (frozen/thawed, F/T) and fresh sperm cells did not reveal significant difference regarding blastocyst formation rate. We also observed no difference in the male and female pronuclei formation and the first zygote cleavage after fertilization of oocytes with high or non-ubiquitinated sperm cells sorted by FACS. However, results of the early embryonic development to the blastocyst stage showed the difference between both experimental groups (16.67% of blastocysts in non-ubiquitinated group vs. 6.20% of blastocyst in the high-ubiquitinated group, P < 0.001). We further confirmed the negative effect of eUb by the masking of Ub epitopes with the appropriate primary antibody in fresh sperm cells prior to ICSI. This procedure improved the blastocyst formation rate from 14.19% in the untreated group to 24.03% concerning antibody masked sperms (P < 0.01). We conclude our results support a generally accepted hypothesis concerning the negative correlation of the presence of eUb on the sperm cell membrane and developmental competence of fertilized oocytes. However, experiments with masking Ub antibody indicate the direct negative effect of the membrane ubiquitin rather than sperm cell quality on the early embryonic development to the blastocyst stage, at least in the porcine model., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In spite of many attempts to establish an in vitro fertilization (IVF) technique in the equine, no efficient conventional IVF technique is available. The presence of oviductal fluid or oviductal cells during IVF helps to improve embryo production in vitro but is not sufficient to reach high fertilization rates. Thus, our aim was to perform equine IVF either after sperm pre-incubation with oviductal fluid or in the presence of oviductal cells, and to evaluate the effect of cumulus removal from the oocyte or sperm pre-incubation with progesterone. In experiments 1 and 2, IVF was performed in the presence of porcine oviduct epithelial cells. The removal of cumulus cells from equine oocytes after in vitro maturation tended to increase the percentage of fertilization when fresh sperm was used (1/33 vs. 4/31, p > 0.05) but had no effect when frozen sperm was used (1/32 vs. 1/32). Equine sperm pre-incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/14 vs. 2/18 for fresh, 1/29 vs. 1/25 for frozen). In experiments 3 and 4, IVF was performed after pre-incubation of sperm with porcine oviductal fluid. The removal of cumulus cells tended to increase the percentage of fertilization when fresh sperm was used (1/24 vs. 3/26, p > 0.05). Sperm pre-incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/39 vs. 2/36 for fresh, 2/37 vs. 1/46 for frozen), but two 3-4 cell stage zygotes were obtained with fresh sperm pre-incubated with progesterone. This is an encouraging result for the setting up of an efficient IVF procedure in equine., (© 2019 Blackwell Verlag GmbH.)

Intracytoplasmic sperm injection (ICSI) is a technique developed to help attain successful fertilisation for couples with severe male factor. However, a small percentage of couples confront low or failed fertilisation, mainly due to failed oocyte activation. Several studies have introduced phospholipase Cζ (PLCζ) as the main sperm factor inducing oocyte activation and thereby has the potential to act as a biomarker for the prediction of ICSI fertilisation outcome. On the other hand, researchers have focused on novel sperm selection procedures based on cellular characteristics of spermatozoa such as surface electrical charge (Zeta potential) to isolate normal sperm subpopulation with intact chromatin. Therefore, we aimed to compare PLCζ between Zeta method and routine sperm preparation procedure: density gradient centrifugation (DGC). Our results showed that number of PLCζ-positive spermatozoa was significantly low in the Zeta method, but the intensity of PLCζ protein in such spermatozoa was significantly higher than DGC procedure. Therefore, the combination of DGC with Zeta procedure may allow selecting the population of spermatozoa with a high percentage of PLCζ which may also contain a high amount of PLCζ and with intact chromatin. This sperm selection procedure can open a new approach for infertile men with previously failed fertilisation., (© 2019 Blackwell Verlag GmbH.)

Sex preselection has always generated great interest among livestock producers. Among the prevalent sperm sorting methods, there is much evidence that sex sorting has a negative effect on sperm quality with an altered pattern of sperm motility, ultimately reducing the period of cell viability. In this study, we have established a new approach for the preselected embryo production by using WholeMom®; a monoclonal antibody developed against bull sperm epitopes for simple and easy separation of X- and Y-sperm. There were no significant differences (P >  0.05) in the percentage of presumptive zygotes between the control and the X-sperm sorted group, but there was a difference in early cleaving embryos with there being 81.2 ± 1.4%, 78.3 ± 1.0%, and 66.7 ± 1.1% for the control, X-sperm sorted, and Y-sperm sorted groups, respectively. Similarly, the percentage of embryos that developed to the blastocyst stage (Day 7) were also greater (P <  0.05) in the control and X-sperm sorted group compared with the Y-sperm sorted group being 34.8 ± 1.0%, 32.1 ± 0.8%, and 23.7 ± 1.0% in the control, X-sperm sorted, and Y-sperm sorted groups, respectively. Furthermore, B-SRY F2 and B-SRY R2 gene expression data indicated there was a detection accuracy of 81.0% for the female embryos and 72.5% for the male embryos produced in vitro. In conclusion, in cattle in vitro derived embryo production using pre-selected sexed semen and subsequent embryo transfer can facilitate the mass production of individuals that are genetically superior., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We determined if microfluidic sorting (MF) of frozen-thawed stallion sperm improves sperm population characteristics and results in embryo development after intracytoplasmic sperm injection (ICSI). The efficiency and efficacy of MF sperm separation was evaluated by comparing pre- and post-separation sperm population variables. Procedural comparisons were performed after sorting with MF, single-layer colloidal centrifugation (SLC) or swim-up (SU), and cleavage and embryo development were evaluated after ICSI using MF-sorted sperm. In Experiment 1, when compared to the original sperm sample, MF sorting resulted in a sperm subpopulation with greater motility, morphology, viability, and membrane as well as DNA integrity. After sorting by MF, SLC and SU in Experiment 2, motility, viability, and membrane integrity were similar for sperm sorted using MF and SLC; however, morphology and DNA integrity were greater in sperm sorted using MF when compared with SLC. Swim-up was the least effective sorting method. In Experiment 3, sperm were processed using MF and SLC prior to ICSI. Motility, morphology and DNA integrity were similar for sperm subpopulations sorted using either method; but viability was greater for sperm sorted using MF than SLC. Sorting did not improve sperm membrane integrity. Sorting with MF prior to ICSI resulted in similar cleavage and blastocyst development rates as SLC. We concluded that MF separation of stallion sperm resulted in a subpopulation with improved sperm population parameters, comparable or better than SLC and SU. Embryos were produced after ICSI using MF sperm sorting., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Dog sperm cryopreservation is gaining importance both in breeding dogs for commercial purposes and for pet animals. Anyway, cryopreservation of mammalian spermatozoa, including dog ones, induces some negative effect on sperm fertility, leading to a lower use of this technique and limiting its widespread use. Therefore, studies to improve the quality of canine semen after cryopreservation could have a relevant impact on both the scientific advancement and the clinical practice. The aim of the present work was to investigate the putative ameliorative effect of Epigallochatechin-3-gallate (EGCG) addition to post thawing medium on dog sperm motility, mitochondrial activity, acrosome integrity and on zona-binding ability (zona binding assay). Spermatozoa were thawed in Tris-fructose-citrate medium supplemented with EGCG (0, 25 and 50 μM) and sperm motility, mitochondrial activity and acrosome integrity were assayed at 0.5, 1.5, 3 and 6 h after post thawing incubation at 37 °C. An aliquot of semen from each treatment group after 1.5 h post thawing incubation was washed and used to perform heterologous (using porcine oocytes) or homologous zona binding assay. The results obtained showed that no significant effect is exerted by EGCG on sperm parameters analysed neither at 0.5, 1.5, 3 or 6 h after thawing excepting for the reduction of the percentage of live cells with active mitochondria at the higher dose at 6 h; furthermore, both homologous or heterologous zona binding ability, was not influenced by EGCG. In conclusion, EGCG supplementation to thawing medium does not improve dog sperm quality or zona binding capacity., (Copyright © 2018 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

SummaryGerm plasm-related structures (GPRS) are known to accompany meiotic cell differentiation but their dynamics are still poorly understood. In this study, we analyzed the ultrastructural mechanisms of GPRS transformation during oogenesis and spermatogenesis of the bivalve mollusc Ruditapes philippinarum (Manila clam), exploring patterns of GPRS activity occurring at meiosis onset, sex-specific difference/similarity of such patterns, and the involvement of mitochondria during GPRS-assigned events. In the two sexes, the zygotene-pachytene stage of meiosis is anticipated by three shared steps. First, the dispersion of germ plasm granules containing the germ line determinant VASA occurs. Second, the VASA protein deriving from germ plasm granules enters neighbouring mitochondria and appears to induce mitochondrial matter release, as supported by cytochrome B localization outside the mitochondria. Third, intranuclear VASA entrance occurs and the protein appears involved in chromatin reorganization, as supported by VASA localization in synaptonemal complexes. In spermatogenesis, these three steps are sufficient for the normal course of meiosis. In oogenesis, these are followed by the action of 'germ plasm granule formation complex', a novel type of structure that appears alternative to the Balbiani body. The possibility of germ plasm involvement in reproductive technologies is also suggested.

Testis-specific PRSS55 is a highly conserved chymotrypsin-like serine protease among mammalian species. So far, the physiological function of PRSS55 remains unknown. Here, we show that PRSS55 is a GPI-anchored membrane protein, specifically expressed in adult mouse testis and mainly observed in the luminal side of seminiferous tubules and sperm acrosome. Mice deficient for Prss55 develop male infertile with normal reproduction-related parameters observed. Interestingly, in vivo fertilization rate of Prss55 -/- males is dramatically decreased, possibly due to incapable migration of Prss55 -/- sperm from uterus into oviduct. However, in vitro fertilization rate has no difference between two genotypes although Prss55 -/- sperm presents defective recognition/binding to zona-intact or zona-free oocytes. Further study reveals that mature ADAM3 is almost undetectable in Prss55 -/- sperm, while precursor ADAM3 remains unchanged in the testis. However, it is shown that ADAM3 has no interaction with PRSS55 by immunoprecipitation with anti-PRSS55 antibody. The expression levels of several proteins known to be related to the observed phenotypes remain comparable between wt and Prss55 -/- mice. Moreover, we found that Prss55 deficiency has no effect on PRSS37 or vice versa albeit two mutant males share almost the same phenotypes. Microarray analysis reveals a total of 72 differentially expressed genes in Prss55 -/- testis, most of which are associated with cellular membrane and organelle organization, protein transport and complex assembly, and response to stimulus and signaling. In conclusion, we have demonstrated that PRSS55 plays vital roles in regulating male fertility of mice, including in vivo sperm migration and in vitro sperm-egg interaction, possibly by affecting the maturation of ADAM3 in sperm and the expression of multiple genes in testis.

Polyspermy is the most commonly observed cause of embryonic abnormalities in fertilization, often resulting in death. In sterlet (Acipenser ruthenus), however, polyspermic embryos have high survival (similar to a control group) and morphological development is similar to monospermic larvae. Ploidy of these individuals is n/2n mosaic (whereas the normal state for A. ruthenus is a functional diploid). This study was undertaken to test whether sturgeon eggs can be fertilized by several spermatozoa from different species to produce viable offspring from three interspecific parents: A. ruthenus (2n), A. gueldenstaedtii (4n), and A. baerii (4n). Four trials were performed: (1) and (2) A. baerii eggs were fertilized with a mixture of A. ruthenus and A. gueldenstaedtii sperm; (3) A. gueldenstaedtii eggs were fertilized with a mixture of A. baerii and A. ruthenus sperm; and (4) A. gueldenstaedtii eggs were fertilized with a mixture of A. gueldenstaedtii and A. ruthenus sperm. Fertilized embryos with abnormal cleavage (3, 5, 6, 7, 9, and 10 cells) were collected and kept separately until 14 days post-fertilization. Ploidy level of 25 larvae (hatched from abnormal cleaved embryos) was evaluated by flow cytometry. Forty-four percent of observed hybrids had a mosaic 2n/3n ploidy. Five larva were processed further with microsatellite analysis and demonstrated that three specimens were heterospecific polyspermic larvae, containing alleles from three parents, and two specimens were conspesific polyspermic larvae from two parents. This astonishing phenomenon was emphasized by the fact that it was generated without any significant intervention.

Purpose: To investigate the effect of sperm concentration, motility and advanced paternal age on reproductive outcomes., Methods: A retrospective analysis of 1266 intracytoplasmic sperm injection (ICSI) cycles between 2013 and 2017. The cohort was divided into four groups according to semen concentration based on the WHO criteria (2010): group A (conc. <1 M/ml), group B (1 ≤ conc. <5 M/ml), group C (5 ≤ conc. < 15 M/ml) and the control group D (conc. ≥15 M/ml). The primary outcome investigated was the blastulation rate. Secondary outcomes were fertilization rate, top quality blastocyst formation rate and ongoing pregnancy rate., Results: After adjustment for maternal age and number of oocytes recovered, a significant difference was observed between group A and group D on the rate of fertilized oocytes [66.7 (40.0-80.0) vs 75.0 (57.1-90.2), adjusted p < 0.001] and the blastocyst formation rate [50.0 (33.3-66.3) vs 55.6 (40.0-75.0), adjusted p < 0.05]. However, the male factor did not affect the top quality blastocyst formation rate nor the ongoing pregnancy rate. Considering the age of the male partner as confounding factor, at the increase of each year of age, a reduction of 0.3% on the fertilization rate was observed but no other outcome was impacted. A negative correlation was also observed between sperm motility and fertilization rate in the group with a motility <5%., Conclusion: Male factor infertility and advanced paternal age may compromise fertilization and blastulation rates but not top quality blastocyst formation rate or the establishment of pregnancy in ICSI cycles.

Effect of sperm concentrations and cumulus cells (CCs) on porcine IVF was re-evaluated using current improved IVM and IVC system. Our results showed that both CCs and sperm concentration had significant effect on penetration rate, frequency of polyspermy and embryonic development. The best IVF results were obtained with oocytes with CCs fertilized with 0.5 × 10 5 sperm/ml. Such an IVP system works on both sow and gilt oocytes., (© 2018 Blackwell Verlag GmbH.)

The selection of the best dairy heifers is mainly driven by the genetic value of their parents. The phenotype analysis of cows and of the daughters of bulls has been used to identify the best genetic value for decades before being replaced by genomic selection of individuals that are not yet parents. Because it is possible to predict the future value of an individual by its genetic makeup, it becomes feasible to do it as early as the blastocyst stage and to decide which should be transferred or not. Because we know the genotype of an animal at birth, or even before, it is becoming desirable to reproduce this animal as soon as possible to reduce generation interval and improve selection speed. Nature provides constraints that can be overcome: a single oocyte per cycle and age at puberty. Indeed, it is now possible to super-stimulate the ovary at any age and to start collecting oocytes at 6 mo by trans-vaginal ultrasonography. The challenge becomes the production of good eggs and embryos capable of implanting and developing into healthy calves. Our understanding of ovarian follicular physiology has been instrumental in designing stimulation protocols that may be adjusted to any physiological context including age, and even the individual animal, to obtain a good response. Therefore, the combination of procedures developed in cows to optimize oocyte quality, for example, FSH coasting, in association with in vitro fertilization and optimal culture conditions can now result in the production of several female embryos twice a month from animals 6 to12 mo of age. The transcriptomic and epigenetic analyses of embryos produced from the same females at different ages were compared and few differences were noted in particular in relation to embryo metabolism. These embryos are as good as the ones obtained from adult animals and can be produced with sexed sperm of bulls 12 mo of age. This combination of these technical optimizations with blastocyst genotyping allows the selection of a second generation within a year.

This study tested the hypothesis that sperm sialic acid (Sia) is required to reach the site of fertilization, and that successful fertilization requires recognition of Sia from both the sperm and oocyte to occur. In addition, it has recently been reported that Siglecs (Sia-binding-immunoglobulin-like lectins) are present on the sperm surface. Thus, the possibility that the recognition of oocyte Sia was sperm-Siglec-mediated was also addressed. Sperm exposed to neuraminidase (NMase) exhibited lower overall and progressive motility, which translated to a decreased ability to swim through cervical mucus from cows in oestrus. In addition, when either sperm or cumulus-oocyte complexes (COCs) were treated with NMase, a decrease in cleavage and blastocyst rate was observed. However, incubation of sperm with increasing concentrations of anti-Siglec-2, -5, -6 and -10 antibodies prior to fertilization had no effect on their fertilizing ability. Interestingly, treatment with NMase increased the number of sperm bound to the ZP but also the rate of polyspermic fertilization. Flow cytometry analysis revealed no differences in the percentage of capacitated or acrosome-reacted sperm. These results suggest that Sia are required to reach the site of fertilization but need to be removed for sperm-oocyte interaction. However, fine regulation is needed to avoid abnormal fertilization which can lead to impaired embryo development., (© 2018 Society for Reproduction and Fertility.)

Experiments were conducted to investigate the development of in vitro matured camel oocytes after their intra-cytoplasmic sperm injection (ICSI) with epididymal sperm collected from slaughtered male camels. Ovaries and testes were collected from a local slaughterhouse in normal saline solution (NSS) at 37 °C and on ice (0-1 °C), respectively. Cumulus-oocyte complexes (COCs) aspirated from the follicles were randomly distributed to 4-well culture plates (20-25 COCs/well) containing 500 μL of maturation medium and cultured at 38.5 °C in an atmosphere of 5% CO 2 in air for about 30 h. Spermatozoa were collected from the cauda epididymites in syringes containing 2-3 mL of tris-tes egg yolk extender. They were cooled down slowly and stored at refrigeration (4 °C) temperature. On the day of use, spermatozoa were prepared by the swim up technique before use in ICSI. The injected oocytes were either activated by ionomycin and roscovitine or put into the culture without any chemical activation. In Experiment 1, presumptive zygotes were fixed and stained with Hoechst 33342 for evaluation of fertilization after 18 h of culture, while, in Experiment 2, they were cultured in 500 μL of the culture medium at 38.5 °C in an atmosphere of 5% CO 2, 5% O 2 and 90% N 2 in air for 7 days to evaluate their development. The proportion of oocytes activated when ICSI was followed by chemical activation was significantly higher (P < 0.05) when compared with non-activated ones. In experiment 2, a higher number of oocytes cleaved (59 vs. 35%) and developed to blastocysts (20 vs. 7%) in the group with post-ICSI activation when compared with the group without chemical activation, respectively. In conclusion, to the best of our knowledge, this is the first report where embryos were produced by ICSI in camels. Chemical activation of oocytes by ionomycin and roscovitine, post -ICSI, enhanced their cleavage and development to blastocyst stage., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: To investigate the effectiveness of cumulus oophorus complexes (COCs) in the physiologic selection of spermatozoa for intracytoplasmic sperm injection (ICSI)., Design: A prospective sibling oocytes study., Setting: Center of reproductive medicine., Patient(s): Couples undergoing ICSI during 2016, females aged ≤38 years, and at least six metaphase II (MII) oocytes retrieved. Sixty patients were included in the study. Of 857 MII oocytes, 429 were allocated to the study group and were injected with the sperm selected via COCs; 428 MII oocytes were allocated as controls (C) and fertilized by conventional ICSI., Intervention(s): In the study group, ICSI was performed with spermatozoa that traversed the COCs in vitro., Main Outcomes Measure(s): Blastocyst/top blastocyst formation rate, fertilization rate, and oocyte utilization rate., Result(s): Oocytes injected with COC-selected spermatozoa had a significantly higher fertilization rate than the conventional ICSI group (85.31% vs. 74.77%). There were no statistically differences in cleavage and top embryo rate on day 3 between the COC-ICSI and C-ICSI groups. However, with day 5 or 6 embryos, compared with conventional ICSI, COC-ICSI significantly improved blastocyst formation rate (64.90% vs. 53.50%), blastocyst formation rate at day 5 (46.52% vs. 38.85%), top blastocyst rate (38.72% vs. 24.20%), and the usable blastocysts formation rate (62.12% vs. 46.82%). The oocyte utilization rate was improved greatly in the COC-ICSI group compared with the C-ICSI group (51.98% vs. 34.35%). Furthermore, the fertilization rate, top embryo rate on day 3, usable blastocyst rate, top blastocyst rate, and day 5 usable blastocysts rate were similar between the conventional IVF and COC-ICSI groups. Single-blastocyst transfer was performed in 82 cycles, including 44 fresh cycles and 38 frozen-thawed cycles. The cumulative embryo implantation rate in the COC-ICSI group was 64.29%, slightly higher than in the C-ICSI group (53.85%), but there was no statistical difference., Conclusion(s): The use of COCs to select spermatozoa for ICSI appears to be effective and led to a statistically significant improvement in blastocyst development and quality., (Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Purpose: The aim of this study was to evaluate the differential effects of conventional and Piezo-driven ICSI on blastocyst development, and on sperm component remodeling and oocyte activation, in an equine model., Methods: In vitro-matured equine oocytes underwent conventional (Conv) or Piezo ICSI, the latter utilizing fluorocarbon ballast. Blastocyst development was compared between treatments to validate the model. Then, oocytes were fixed at 0, 6, or 18 h after injection, and stained for the sperm tail, acrosome, oocyte cortical granules, and chromatin. These parameters were compared between injection techniques and between sham-injected and sperm-injected oocytes among time periods., Results: Blastocyst rates were 39 and 40%. The nucleus number was lower, and the nuclear fragmentation rate was higher, in blastocysts produced by Conv. Cortical granule loss started at 0H after both sperm and sham injection. The acrosome was present at 0H in both ICSI treatments, and persisted to 18H in significantly more Conv than Piezo oocytes (72 vs. 21%). Sperm head area was unchanged at 6H in Conv but significantly increased at this time in Piezo; correspondingly, at 6H significantly more Conv than Piezo oocytes remained at MII (80 vs. 9.5%). Sham injection did not induce significant meiotic resumption., Conclusions: These data show that Piezo ICSI is associated with more rapid sperm component remodeling and oocyte meiotic resumption after sperm injection than is conventional ICSI, and with higher embryo quality at the blastocyst stage. This suggests that there is value in exploring the Piezo technique, utilized with a non-toxic fluorocarbon ballast, for use in clinical human ICSI.

Purpose: Partial thawing of a vial of cryopreserved sperm (shaving) is sometimes applied as a measure to preserve sperm for further use, particularly in cases of very restricted sperm quantity. However, mechanical violence may disrupt the sperm-wall and lead to impaired in-vitro fertilization (IVF) outcomes., Material and Methods: In a retrospective case-control study at a tertiary, university-affiliated medical center, we compared the IVF/intracytoplasmic sperm injection (ICSI) outcomes of patients who used donor sperm following partial thawing (shaving) of the vial of cryopreserved sperm (n = 99) to a control group consisting of patients for whom the vial of sperm was completely thawed before use (n = 99)., Results: While no differences were observed in the rates of oocyte fertilization, the mean number of top-quality embryos (TQE) was significantly lower in the shaving group than in the complete thawing group (1.33 ± 0.17 vs. 1.87 ± 0.17, p < 0.02). Experimental analysis of aliquots from the same donors revealed significantly reduced motility in sperm samples that were shaved vs. fully thawed (6.5 vs. 37.1%, p < 0.001)., Conclusions: In cases in which available cryopreserved sperm samples are limited, shaving of the vial without thawing can be used but with caution and only when absolutely necessary. Further, large prospective studies are needed to better clarify whether there is post-thawing sperm damage and to compare IVF outcomes after these two thawing methods.