Altered N6-methyladenosine methylation level in spermatozoa messenger RNA of the male partners is related to unexplained recurrent pregnancy loss.

BACKGROUND: Understanding the pathogenesis of unexplained recurrent pregnancy loss is paramount for advancing effective treatments. Various biological processes, including spermatogenesis and embryo development, are tightly regulated by N6-methyladenosine modifications. However, few studies have focused on the impact of sperm N6-methyladenosine modifications on embryonic development. Therefore, we aimed to study altered N6-methyladenosine-mediated messenger RNA methylation modifications in the spermatozoa of male partners from couples experiencing unexplained recurrent pregnancy loss, to identify potential diagnostic markers and explore their potential molecular mechanisms in pregnancy loss and embryogenesis. METHODS: Methylated RNA immunoprecipitation (MeRIP) sequencing and RNA sequencing were conducted on the spermatozoa of men from couples in the 'unexplained recurrent pregnancy loss' group (n = 6), and the fertility control group (n = 6). To identify the role of the detected key genes, zebrafish model embryos were studied, and multi-omics (transcriptomics, proteomics, and metabolomics) analyses helped to explore the molecular mechanism of abnormal embryogenesis. FINDINGS: Comparing unexplained recurrent pregnancy loss with the fertility control group, 217 N6-methyladenosine peaks were significantly upregulated, and 40 were downregulated in the spermatozoa. The combined analyses of spermatozoa-methylated RNA immunoprecipitation sequencing and RNA sequencing indicated that N6-methyladenosine methylation and the expression of SEMA5A, MT-ATP6, ZNF662, and KDM4C were significantly different. In zebrafish embryos, the altered expression of the four genes increased embryonic mortality and malformations by disturbing several key signaling pathways and zygotic genome activation. INTERPRETATION: This study highlights the paternal epigenome, which could be one of the reasons for faulty embryogenesis leading to pregnancy loss. The N6-methyladenosine modification, the most prevalent RNA modification, contributes to the exploration and understanding of the paternal epigenome in the maintenance of pregnancy and fetal growth and development. The four genes identified in this study may serve as potential diagnostic markers and elucidate novel molecular mechanisms of embryogenesis.

The Simulated Physiological Oocyte Maturation (SPOM) System Enhances Cytoplasmic Maturation and Oocyte Competence in Cattle.

In vitro embryo production is a widely applied technique that allows the expansion of genetics and accelerated breeding programs. However, in cattle, this technique still needs improvement in order to reach quality and pregnancy rates comparable to in vivo-derived embryos. One of the limitations of this technique is related to in vitro maturation, where a heterogeneous population of oocytes is harvested from follicles and cultured in vitro in the presence of gonadotropic hormones to induce maturation. As a result, oocytes with different degrees of competence are obtained, resulting in a decrease in the quality and quantity of embryos obtained. A novel system based on the use of cyclic adenosine monophosphate (cAMP) modulators was developed to enhance bovine oocyte competence, although controversial results were obtained depending on the in vitro embryo production (IVP) system used in each laboratory. Thus, in the present work, we employed a reported cAMP protocol named Simulated Physiological Oocyte Maturation (SPOM) under our IVP system and analysed its effect on cytoplasmic maturation by measuring levels of stress-related genes and evaluating the activity and distribution of mitochondria as a marker for cytoplasmic maturation Moreover, we studied the effect of the cAMP treatment on nuclear maturation, cleavage, and blastocyst formation. Finally, we assessed the embryo quality by determining the hatching rates, total cell number per blastocyst, cryopreservation tolerance, and embryo implantation. We found that maturing oocytes in the presence of cAMP modulators did not affect nuclear maturation, although they changed the dynamic pattern of mitochondrial activity along maturation. Additionally, we found that oocytes subjected to cAMP modulators significantly improved blastocyst formation (15.5% vs. 22.2%, p < 0.05). Blastocysts derived from cAMP-treated oocytes did not improve cryopreservation tolerance but showed an increased hatching rate, a higher total cell number per blastocyst and, when transferred to hormonally synchronised recipients, produced pregnancies. These results reflect that the use of cAMP modulators during IVM results in competent oocytes that, after fertilisation, can develop in more blastocysts with a better quality than standard IVM conditions.

Cellular mechanisms of monozygotic twinning: clues from assisted reproduction.

BACKGROUND: Monozygotic (MZ) twins are believed to arise from the fission of a single fertilized embryo at different stages. Monochorionic MZ twins, who share one chorion, originate from the splitting of the inner cell mass (ICM) within a single blastocyst. In the classic model for dichorionic MZ twins, the embryo splits before compaction, developing into two blastocysts. However, there are a growing number of ART cases where a single blastocyst transfer results in dichorionic MZ twins, indicating that embryo splitting may occur even after blastocyst formation. OBJECTIVE AND RATIONALE: For monochorionic MZ twins, we conducted a comprehensive analysis of the cellular mechanisms involved in ICM splitting, drawing from both ART cases and animal experiments. In addition, we critically re-examine the classic early splitting model for dichorionic MZ twins. We explore cellular mechanisms leading to two separated blastocysts in ART, potentially causing dichorionic MZ twins. SEARCH METHODS: Relevant studies including research articles, reviews, and conference papers were searched in the PubMed database. Cases of MZ twins from IVF clinics were found by using combinations of terms including 'monozygotic twins' with 'IVF case report', 'ART', 'single embryo transfer', or 'dichorionic'. The papers retrieved were categorized based on the implicated mechanisms or as those with unexplained mechanisms. Animal experiments relating to MZ twins were found using 'mouse embryo monozygotic twins', 'mouse 8-shaped hatching', 'zebrafish janus mutant', and 'nine-banded armadillo embryo', along with literature collected through day-to-day reading. The search was limited to articles in English, with no restrictions on publication date or species. OUTCOMES: For monochorionic MZ twins, ART cases and mouse experiments demonstrate evidence that a looser ICM in blastocysts has an increased chance of ICM separation. Physical forces facilitated by blastocoel formation or 8-shaped hatching are exerted on the ICM, resulting in monochorionic MZ twins. For dichorionic MZ twins, the classic model resembles artificial cloning of mouse embryos in vitro, requiring strictly controlled splitting forces, re-joining prevention, and proper aggregation, which allows the formation of two separate human blastocysts under physiological circumstances. In contrast, ART procedures involving the transfer of a single blastocysts after atypical hatching or vitrified-warmed cycles might lead to blastocyst separation. Differences in morphology, molecular mechanisms, and timing across various animal model systems for MZ twinning can impede this research field. As discussed in future directions, recent developments of innovative in vitro models of human embryos may offer promising avenues for providing fundamental novel insights into the cellular mechanisms of MZ twinning during human embryogenesis. WIDER IMPLICATIONS: Twin pregnancies pose high risks to both the fetuses and the mother. While single embryo transfer is commonly employed to prevent dizygotic twin pregnancies in ART, it cannot prevent the occurrence of MZ twins. Drawing from our understanding of the cellular mechanisms underlying monochorionic and dichorionic MZ twinning, along with insights into the genetic mechanisms, could enable improved prediction, prevention, and even intervention strategies during ART procedures. REGISTRAITON NUMBER: N/A.

Developmental perturbation in human embryos: Clinical and biological significance learned from time-lapse images.

Background: Time-lapse technology (TLT) has gained widespread adoption worldwide. In addition to facilitating the undisturbed culture of embryos, TLT offers the unique capability of continuously monitoring embryos to detect spatiotemporal changes. Although these observed phenomena play a role in optimal embryo selection/deselection, the clinical advantages of introducing TLT remain unclear. However, manual annotation of embryo perturbation could facilitate a comprehensive assessment of developmental competence. This process requires a thorough understanding of embryo observation and the biological significance associated with developmental dogma and variation. This review elucidates the typical behavior and variation of each phenomenon, exploring their clinical significance and research perspectives. Methods: The MEDLINE database was searched using PubMed for peer-reviewed English-language original articles concerning human embryo development. Main findings: TLT allows the observation of consecutive changes in embryo morphology, serving as potential biomarkers for embryo assessment. In assisted reproductive technology laboratories, several phenomena have not revealed their mechanism, posing difficulties such as fertilization deficiency and morula arrest. Conclusion: A profound understanding of the biological mechanisms and significance of each phenomenon is crucial. Further collaborative efforts between the clinical and molecular fields following translational studies are required to advance embryonic outcomes and assessment.

A prospective study of the proteome of equine pre-implantation embryo.

The present study was conducted to investigate the global proteome of 8-day-old equine blastocysts. Follicular dynamics of eight adult mares were monitored by ultrasonography and inseminated 24 h after the detection of a preovulatory follicle. Four expanded blastocysts were recovered, pooled, and subjected to protein extraction and mass spectrometry. Protein identification was conducted based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, and PepExplorer). Enrichment analysis was performed using g:Profiler, Panther, and String platforms. After the elimination of identification redundancies among search tools (at three levels, based on identifiers, peptides, and cross-database mapping), 1977 proteins were reliably identified in the samples of equine embryos. Proteomic analysis unveiled robust metabolic activity in the 8-day equine embryo, highlighted by an abundance of proteins engaged in key metabolic pathways like the TCA cycle, ATP biosynthesis, and glycolysis. The prevalence of chaperones among highly abundant proteins suggests that regulation of protein folding, and degradation is a key process during embryo development. These findings pave the way for developing new strategies to improve equine embryo media and optimize in vitro fertilization techniques.

Multi-omics analyses and machine learning prediction of oviductal responses in the presence of gametes and embryos.

The oviduct is the site of fertilization and preimplantation embryo development in mammals. Evidence suggests that gametes alter oviductal gene expression. To delineate the adaptive interactions between the oviduct and gamete/embryo, we performed a multi-omics characterization of oviductal tissues utilizing bulk RNA-sequencing (RNA-seq), single-cell RNA-sequencing (scRNA-seq), and proteomics collected from distal and proximal at various stages after mating in mice. We observed robust region-specific transcriptional signatures. Specifically, the presence of sperm induces genes involved in pro-inflammatory responses in the proximal region at 0.5 days post-coitus (dpc). Genes involved in inflammatory responses were produced specifically by secretory epithelial cells in the oviduct. At 1.5 and 2.5 dpc, genes involved in pyruvate and glycolysis were enriched in the proximal region, potentially providing metabolic support for developing embryos. Abundant proteins in the oviductal fluid were differentially observed between naturally fertilized and superovulated samples. RNA-seq data were used to identify transcription factors predicted to influence protein abundance in the proteomic data via a novel machine learning model based on transformers of integrating transcriptomics and proteomics data. The transformers identified influential transcription factors and correlated predictive protein expressions in alignment with the in vivo-derived data. In conclusion, our multi-omics characterization and subsequent in vivo confirmation of proteins/RNAs indicate that the oviduct is adaptive and responsive to the presence of sperm and embryos in a spatiotemporal manner.

The role of DNA damage response in human embryonic stem cells exposed to atmospheric oxygen tension: Implications for embryo development and differentiation.

Previous retrospective cohort studies have found that, compared with oxygen tension in the uterus and fallopian tubes (2 %-8 %), exposure of pre-implantation embryos to atmospheric oxygen tension (AtmO2, 20 %) during assisted reproductive technology(ART) can affect embryo quality, pregnancy outcomes and offspring health. However, current research on the effects and mechanisms of AtmO2 on the development of embryos and offspring is mainly limited to animal experiments. Human embryonic stem cells (hESCs) play a special and irreplaceable role in the study of early human embryonic development. In this study, we used hESCs as a model to elucidate the possible effects and mechanisms of AtmO2 exposure on human embryonic development. We found that exposure to AtmO2 can reduce cell viability, produce oxidative stress, increase DNA damage, initiate DNA repair, activate autophagy, and increase cell apoptosis. We also noticed that approximately 50 % of hESCs survived, adapted and proliferated through high expression of self-renewal and pluripotency regulatory factors, and affected embryoid body differentiation. These data indicate that hESCs experience oxidative stress, accumulation of DNA damage, and activate DNA damage response under the selective pressure of AtmO2.Some hESCs undergo cell death, whereas other hESCs adapt and proliferate through increased expression of self-renewal genes. The current findings provide in vitro evidence that exposure to AtmO2 during the early preimplantation stage negatively affects hESCs.

Hydrogen sulfide and its role in female reproduction.

Hydrogen sulfide (H2S) is a gaseous signaling molecule produced in the body by three enzymes: cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST). H2S is crucial in various physiological processes associated with female mammalian reproduction. These include estrus cycle, oocyte maturation, oocyte aging, ovulation, embryo transport and early embryo development, the development of the placenta and fetal membranes, pregnancy, and the initiation of labor. Despite the confirmed presence of H2S-producing enzymes in all female reproductive tissues, as described in this review, the exact mechanisms of H2S action in these tissues remain in most cases unclear. Therefore, this review aims to summarize the knowledge about the presence and effects of H2S in these tissues and outline possible signaling pathways that mediate these effects. Understanding these pathways may lead to the development of new therapeutic strategies in the field of women's health and perinatal medicine.

In Vitro Culture of Mammalian Embryos: Is There Room for Improvement?

Preimplantation embryo culture, pivotal in assisted reproductive technology (ART), has lagged in innovation compared to embryo selection advancements. This review examines the persisting gap between in vivo and in vitro embryo development, emphasizing the need for improved culture conditions. While in humans this gap is hardly estimated, animal models, particularly bovines, reveal clear disparities in developmental competence, cryotolerance, pregnancy and live birth rates between in vitro-produced (IVP) and in vivo-derived (IVD) embryos. Molecular analyses unveil distinct differences in morphology, metabolism, and genomic stability, underscoring the need for refining culture conditions for better ART outcomes. To this end, a deeper comprehension of oviduct physiology and embryo transport is crucial for grasping embryo-maternal interactions' mechanisms. Research on autocrine and paracrine factors, and extracellular vesicles in embryo-maternal tract interactions, elucidates vital communication networks for successful implantation and pregnancy. In vitro, confinement, and embryo density are key factors to boost embryo development. Advanced dynamic culture systems mimicking fluid mechanical stimulation in the oviduct, through vibration, tilting, and microfluidic methods, and the use of innovative softer substrates, hold promise for optimizing in vitro embryo development.

Embryos from Prepubertal Hyperglycemic Female Mice Respond Differentially to Oxygen Tension In Vitro.

Reduced oxygen during embryo culture in human ART prevents embryo oxidative stress. Oxidative stress is also the major mechanism by which maternal diabetes impairs embryonic development. This study employed induced hyperglycemia prepubertal mice to mimic childhood diabetes to understand the effects of varying oxygen tension during in vitro embryonic development. The oocytes were fertilized and cultured at low (≈5%) oxygen (LOT) or atmospheric (≈20%) oxygen tension (HOT) for up to 96 h. Embryo development, apoptosis in blastocysts, inner cell mass (ICM) outgrowth proliferation, and Hif1α expression were assessed. Though the oocyte quality and meiotic spindle were not affected, the fertilization rate (94.86 ± 1.18 vs. 85.17 ± 2.81), blastocyst rate (80.92 ± 2.92 vs. 69.32 ± 2.54), and ICM proliferation ability (51.04 ± 9.22 vs. 17.08 ± 3.05) of the hyperglycemic embryos were significantly higher in the LOT compared to the HOT group. On the other hand, blastocysts from the hyperglycemic group, cultured at HOT, had a 1.5-fold increase in apoptotic cells compared to the control and lower Hif1α transcripts in ICM outgrowths compared to the LOT. Increased susceptibility of embryos from hyperglycemic mice to higher oxygen tension warrants the need to individualize the conditions for embryo culture systems in ART clinics, particularly when an endogenous maternal pathology affects the ovarian environment.

Metabolic regulation of preimplantation embryo development in vivo and in vitro: Molecular mechanisms and insights.

Understanding of embryonic development has led to the clinical application of Assisted Reproductive technologies (ART), with the resulting birth of millions of children. Recent developments in metabolomics, proteomics, and transcriptomics have brought to light new insights into embryonic growth dynamics, with implications spanning reproductive medicine, stem cell research, and regenerative medicine. The review explores the key metabolic processes and molecular pathways active during preimplantation embryo development, including PI3K-Akt, mTOR, AMPK, Wnt/ß-catenin, TGF-ß, Notch and Jak-Stat signaling pathways. We focused on analyzing the differences occurring in vitro as opposed to in vivo development and we discussed significant physiological and clinical implications.

High-fat diet-negative impact on female fertility: from mechanisms to protective actions of antioxidant matrices.

Introduction: Excessive calorie intake poses a significant threat to female fertility, leading to hormonal imbalances and reproductive challenges. Overconsumption of unhealthy fats exacerbates ovarian dysfunction, with an overproduction of reactive oxygen species causing oxidative stress, impairing ovarian follicle development and leading to irregular ovulation and premature ovarian failure. Interest in biological matrices with high antioxidant properties to combat diet-related oxidative stress has grown, as they contain various bioactive factors crucial for neutralizing free radicals potentially preventing female reproductive health. This systematic review evaluates the female reproductive impact of biological matrices in mitigating oxidative damages induced by over calory habits and, in particular, high fat diets. Methods: A comparative approach among mammalian models was utilized to interpret literature available data. This approach specifically investigates the antioxidant mechanisms of biological matrices on early and late ovarian folliculogenesis, under physiological and hormone-induced female reproductive cycle. Adhering to the PRISMA 2020 guidelines, only English-language publications from peer-reviewed international indexes were considered. Results: The analysis of 121 publications meeting the inclusion criteria facilitated the identification of crucial components of biological matrices. These components, including carbocyclic sugars, phytonutrients, organosulfur compounds, and vitamins, were evaluated for their impact on ovarian follicle resilience, oocyte quality, and reproductive lifespan. The detrimental effects of oxidative stress on female fertility, particularly exacerbated by high saturated fat diets, are well-documented. In vivo studies across mammalian preclinical models have underscored the potential of antioxidants derived from biological matrices to mitigate diet-induced conditions. These antioxidants enhance steroidogenesis and ovarian follicle development, thereby improving oocyte quality. Additionally, discussions within these publications emphasized the clinical significance of these biological matrices, translating research findings into practical applications for female health. Conclusion: Further research is essential to fully exploit the potential of these matrices in enhancing female reproduction and mitigating the effects of diets rich in fatty acids. This requires intensified in vitro studies and comprehensive collection of in vivo data before clinical trials. The promotion of ovarian resilience offers promising avenues for enhancing understanding and advancing female reproductive health world-wide.

Toxic Effects of Methamphetamine on Reproductive Systems, Embryo Development, and Newborns: An Evidence-Based Review.

Methamphetamine (METH), an amphetamine-type stimulant, has been extensively abused globally in the past decades. METH use causes great harm to the major systems of the human body. Specifically, METH has a negative impact on the hypothalamic- pituitary-testicular axis, testicular structure, sperm function, ovarian folliculogenesis, oocyte quality, embryo development, and newborns. However, the mechanisms underlying these toxic effects have not yet been fully described. This study reviews the evidence concerning the impact of METH on male and female reproduction in the context of the testis, sperm, ovaries, oocytes, reproductive hormones, embryo development, and newborns, discussing the potential pathophysiological mechanisms in the reproductive toxicity induced by METH.

Effect of cytoplasmic fragmentation on embryo development, quality, and pregnancy outcome: a systematic review of the literature.

The role of cytoplasmic fragmentation in human embryo development and reproductive potential is widely recognized, albeit without standard definition nor agreed upon implication. While fragmentation is best understood to be a natural process across species, the origin of fragmentation remains incompletely understood and likely multifactorial. Several factors including embryo culture condition, gamete quality, aneuploidy, and abnormal cytokinesis seem to have important role in the etiology of cytoplasmic fragmentation. Fragmentation reduces the volume of cytoplasm and depletes embryo of essential organelles and regulatory proteins, compromising the developmental potential of the embryo. While it has been shown that degree of fragmentation and embryo implantation potential are inversely proportional, the degree, pattern, and distribution of fragmentation as it relates to pregnancy outcome is debated in the literature. This review highlights some of the challenges in analysis of fragmentation, while revealing trends in our evolving knowledge of how fragmentation may relate to functional development of the human embryos, implantation, and pregnancy outcome.

Spatio-temporal expression patterns of glycine-rich beta proteins and cysteine-rich beta proteins in setae development of Gekko japonicus.

BACKGROUND: Setae on the pad lamellae of the Japanese gecko Gekko japonicus (Schlegel, 1836), a vital epidermal derivative, are primarily composed of cornified beta-proteins (CBPs) and play a pivotal role in adhesion and climbing. The amino acid composition of CBPs might be a determining factor influencing their functional properties. However, the molecular mechanisms governed by CBP genes with diverse amino acid compositions in setae development remain unexplored. RESULTS: Based on RNA-seq analyses, this study confirmed that all G. japonicus CBPs (GjCBPs) are involved in setae formation. Cysteine-rich CBPs encoding genes (ge-cprp-17 to ge-cprp-26) and glycine-rich CBPs encoding genes (ge-gprp-17 to ge-gprp-22) were haphazardly selected, with quantitative real-time PCR revealing their expression patterns in embryonic pad lamellae and dorsal epidermis. It is inferred that glycine-rich CBPs are integral to the formation of both dorsal scales and lamellar setae, cysteine-rich CBPs are primarily associated with setae development. Additionally, fluorescence in situ hybridization revealed spatiotemporal differences in the expression of a glycine-rich CBP encoding gene (ge-gprp-19) and a cysteine-rich CBP encoding gene (ge-cprp-17) during dorsal scales and/or lamellar development. CONCLUSIONS: All 66 CBPs are involved in the formation of setae. Glycine-rich CBPs hold a significant role in the development of dorsal scales and lamellar setae, whereas most cysteine-rich CBPs appear to be essential components of G. japonicus setae. Even GjCBPs with similar amino acid compositions may play diverse functions. The clear spatio-temporal expression differences between the glycine-rich and cysteine-rich CBP encoding genes during epidermal scale and/or setae formation were observed. Embryonic developmental stages 39 to 42 emerged as crucial phases for setae development. These findings lay the groundwork for deeper investigation into the function of GjCBPs in the development of G. japonicus setae.

MtPIN4 plays critical roles in amino acid biosynthesis and metabolism of seed in Medicago truncatula.

The regulation of seed development is critical for determining crop yield. Auxins are vital phytohormones that play roles in various aspects of plant growth and development. However, its role in amino acid biosynthesis and metabolism in seeds is not fully understood. In this study, we identified a mutant with small seeds through forward genetic screening in Medicago truncatula. The mutated gene encodes MtPIN4, an ortholog of PIN1. Using molecular approaches and integrative omics analyses, we discovered that auxin and amino acid content significantly decreased in mtpin4 seeds, highlighting the role of MtPIN4-mediated auxin distribution in amino acid biosynthesis and metabolism. Furthermore, genetic analysis revealed that the three orthologs of PIN1 have specific and overlapping functions in various developmental processes in M. truncatula. Our findings emphasize the significance of MtPIN4 in seed development and offer insights into the molecular mechanisms governing the regulation of seed size in crops. This knowledge could be applied to enhance crop quality by targeted manipulation of seed protein regulatory pathways.

MCT1-mediated transport of valeric acid promotes porcine preimplantation embryo development by improving mitochondrial function and inhibiting the autophagic AMPK-ULK1 pathway.

Oocytes and embryos are highly sensitive to environmental stress in vivo and in vitro. During in vitro culture, many stressful conditions can affect embryo quality and viability, leading to adverse clinical outcomes such as abortion and congenital abnormalities. In this study, we found that valeric acid (VA) increased the mitochondrial membrane potential and ATP content, decreased the level of reactive oxygen species that the mitochondria generate, and thus improved mitochondrial function during early embryonic development in pigs. VA decreased expression of the autophagy-related factors LC3B and BECLIN1. Interestingly, VA inhibited expression of autophagy-associated phosphorylation-adenosine monophosphate-activated protein kinase (p-AMPK), phosphorylation-UNC-51-like autophagy-activated kinase 1 (p-ULK1, Ser555), and ATG13, which reduced apoptosis. Short-chain fatty acids (SCFAs) can signal through G-protein-coupled receptors on the cell membrane or enter the cell directly through transporters. We further show that the monocarboxylate transporter 1 (MCT1) was necessary for the effects of VA on embryo quality, which provides a new molecular perspective of the pathway by which SCFAs affect embryos. Importantly, VA significantly inhibited the AMPK-ULK1 autophagic signaling pathway through MCT1, decreased apoptosis, increased expression of embryonic pluripotency genes, and improved embryo quality.

A longer period of epididymal sperm interaction with extender components during cryopreservation improves sperm quality, decreases the size of sperm distal cytoplasmic droplets, and changes the number of nanoparticles in the extender.

While cryopreservation of cauda epididymal sperm (SpCau) allows the preservation of post-mortem bulls' gametes, the process triggers sperm damage. Although improving post-thaw sperm quality, using egg yolk extenders (EY) raises biosafety concerns which forces the use of EY-free extenders (EYFE). Since EYFE are less efficient in preserving post-thaw sperm quality, a strategy for ejaculated sperm (SpEj) frozen with EYFE is to add an Equilibrium Time (ET) step period to the cryopreservation process. However, the ET effect on the quality of SpCau cryopreserved in EYFE remains unknown. Distinct from SpEJ, SpCau physiologically displays cytoplasmic droplets (CDs) in the flagellum that may benefit cell exchange during ET. We hypothesized that using ET in SpCau cryopreserved with EYFE impacts sperm morphofunctional features, CD area, and in vitro fertility ability. Extender nanoparticles were also assessed. Following collection from the cauda epididymis of six Nellore bulls by retrograde flow, SpCau were cryopreserved in EYFE BoviFree® (Minitube, Germany) using three ET protocols: ET0 (no-ET); ET2.5 (2.5 h-ET); and ET5 (5 h-ET). SpCau from ET2.5 and ET5 showed a higher (P ≤ 0.05) percentage of motility and integrity of plasma and acrosome membranes and a smaller (P ≤ 0.05) distal CD area. There are no differences in sperm abnormalities, oxidative stress, capacitation-like events, and in vitro fertility ability. However, a better sperm recovery was found after Percoll® selection for ET2.5 and ET5. Interestingly, the number of nanoparticles in the extender decreased in post-thawed samples. In conclusion, an ET of 2.5 or 5 h is required for an efficient SpCau cryopreservation using an EYFE.

Determinants of thermal homeostasis in the preimplantation embryo: a role for the embryo's central heating system?

A number of factors may impinge on thermal homeostasis in the early embryo. The most obvious is the ambient temperature in which development occurs. Physiologically, the temperature in the lumen of the female tract is typically lower than the core body temperature, yet rises at ovulation in the human, while in an IVF setting, embryos are usually maintained at core body temperature. However, internal cellular developmental processes may modulate thermal control within the embryo itself, especially those occurring in the mitochondria which generate intracellular heat through proton leak and provide the embryo with its own 'central heating system'. Moreover, mitochondrial movements may serve to buffer high local intracellular temperatures. It is also notable that the preimplantation stages of development would generate proportionally little heat within their mitochondria until the blastocyst stage as mitochondrial metabolism is comparatively low during the cleavage stages. Despite these data, the specific notion of thermal control of preimplantation development has received remarkably scant consideration. This opinion paper illustrates the lack of reliable quantitative data on these markers and identifies a major research agenda which needs to be addressed with urgency in view of laboratory conditions in which embryos are maintained as well as climate change-derived heat stress which has a negative effect on numerous clinical markers of early human embryo development.

Impact of preovulatory follicle maturity on oocyte metabolism and embryo development.

Improved oocyte competence for embryo development and pregnancy was observed following ovulation of preovulatory follicles with greater physiological maturity, as indicated by estradiol production, prior to the gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) surge. It was hypothesized that follicular fluid from preovulatory follicles of greater maturity better supports the maturing oocyte's metabolic requirements and improves embryo development. The objective was to determine if differences in preovulatory follicular fluid due to follicle maturity influence oocyte metabolism during in vitro maturation (IVM) and affect embryo development. Bovine preovulatory follicular fluid was collected 18 h after a GnRH-induced LH surge. Serum estradiol concentration at GnRH administration categorized follicles as greater or lesser maturity. Immature bovine oocytes were submitted to 24 h IVM in medium supplemented with 20% follicular fluid from preovulatory follicles of greater or lesser maturity. Embryo development was recorded. Oocyte maturation media and media conditioned by developing embryos were submitted for metabolomics. A randomized block design was utilized to determine differences in embryo development and media metabolites (P ≤ 0.05). Blastocysts from oocytes matured in greater vs. lesser maturity follicular fluid had a more moderate rate of development (P = 0.01). At the conclusion of 24 h IVM, abundance of 66 metabolites differed between greater and lesser follicle maturity treatments. Nine metabolites differed in media conditioned by developing embryos. Metabolome results suggest improved amino acid, purine, and glucose metabolism, followed by a more efficient rate of embryo development, in oocytes matured in greater vs lesser maturity follicular fluid.