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Assisted reproductive technologies are an emerging field in equine reproduction, with species dependent peculiarities, such as the low success rate of conventional in vitro fertilisation. Here, the "cumulome" was related to the developmental capacity of its corresponding oocyte. Cumulus oocyte complexes (COCs) collected from slaughterhouse ovaries were individually matured, fertilised by intracytoplasmic sperm injection (ICSI), and cultured. After maturation, the cumulus was collected for proteomics analysis using label-free mass spectrometry (MS) based protein profiling by nano-HPLC MS/MS and metabolomics analysis by UPLC-nanoESI MS. Overall, a total of 1671 proteins and 612 metabolites were included in the quantifiable "cumulome". According to the development of the corresponding oocytes, three groups were compared with each other: not matured (NM; n = 18), cleaved (CV; n = 15) and blastocyst (BL; n = 19) groups. CV and BL were also analysed together as the matured group (M; n = 34). The dataset revealed a closer connection within the two M groups and a more distinct separation from the NM group. Over-representation analysis detected enrichments related to energy metabolism as well as vesicular transport in the M group. Functional enrichment analysis found only the KEGG pathway of oxidative phosphorylation as significantly enriched in NM group. A compound attributed to ATP was observed with significantly higher concentrations in the BL group compared with the NM group. Finally, in the NM group, proteins related to degradation of glycosaminoglycans were lower and components of cumulus extracellular matrix were higher compared to the other groups. In summary, the study revealed novel pathways associated with the maturational and developmental competence of oocytes.

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Extracellular vesicles, also known as exosomes, influence numerous cellular functions by regulating different signaling pathways. However, their role in animal reproduction remains understudied. This study aimed to evaluate the effects of porcine follicular fluid-derived exosomes (pff-Exos) on porcine oocyte in vitro maturation and parthenogenetic embryo development. We obtained pff-Exos through mixed-method ultracentrifugation and size-exclusion chromatography. Transmission electron microscopy revealed an increase in the expression of exosome markers in the first four of thirteen fractions. The number of pff-Exo was 2.2 × 106 particles per microliter. The highest maturation rate of porcine oocytes treated with pff-Exo was observed with 1.1 × 107 particles of pff-Exo in the absence of porcine follicular fluid (pFF) culture conditions. Moreover, increased expression of Gdf9 and Bmp15 was observed. The developmental rate was the highest upon treatment with 1.1 × 107 particles of pff-Exo, which increased the total cell number in blastocysts. Embryonic development to the 2-cell stage was similar between the control and pff-Exo groups; however, development to the 4-cell stage and blastocyst was significantly increased in the pff-Exo group (61.6 ± 6.08 % and 29.72 ± 1.41 %, respectively; P < 0.05) compared with that in the control group (42.0 ± 5.19 % and 18.14 ± 1.78 %, respectively). The expression levels of Oct4, Sox2, Bcl2, Elf4, and Gcn5 significantly increased at the pff-Exo 2-cell stage, whereas those of Bax, Hdac1, Hdac6, and Sirt6 decreased. Specifically, the Oct4, Sox2, Elf4, Gcn5, and Hdac6 levels remained stable in pff-Exo 4-cell embryos, whereas those of p53 and Hat1 were reduced and increased, respectively. Treatment with pffExos significantly increased H3K9 and H3K14 acetylation levels. These results demonstrate that pff-Exo affects the in vitro maturation of porcine oocytes and early embryonic development by regulating gene expression.

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BACKGROUND: Within the follicular fluid, extracellular vesicles (EVs) guide oocyte growth through their cargo microRNAs (miRNAs). Here, we investigated the role of EVs and their cargo miRNAs by linking the miRNAs found in EVs, derived from the fluid of an individual follicle, to the ability of its oocyte to become a blastocyst (competent) or not (non-competent). METHODS: Bovine antral follicles were dissected, categorized as small (2-4 mm) or large (5-8 mm) and the corresponding oocytes were subjected to individual maturation, fertilization and embryo culture to the blastocyst stage. Follicular fluid was pooled in 4 groups (4 replicates) based on follicle size and competence of the corresponding oocyte to produce a blastocyst. Follicular fluid-derived EVs were isolated, characterized, and subjected to miRNA-sequencing (Illumina Miseq) to assess differential expression (DE) in the 4 groups. Functional validation of the effect of miR-34c on embryo development was performed by supplementation of mimics and inhibitors during in vitro maturation (IVM). RESULTS: We identified 16 DE miRNAs linked to oocyte competence when follicular size was not considered. Within the large and small follicles, 46 DE miRNAs were driving blastocyst formation in each group. Comparison of EVs from competent small and large follicles revealed 90 DE miRNAs. Cell regulation, cell differentiation, cell cycle, and metabolic process regulation were the most enriched pathways targeted by the DE miRNAs from competent oocytes. We identified bta-miR-34c as the most abundant in follicular fluid containing competent oocytes. Supplementation of miR-34c mimic and inhibitor during IVM did not affect embryo development. However, blastocyst quality, as evidenced by higher cell numbers, was significantly improved following oocyte IVM in the presence of miR-34c mimics, while miR-34c inhibitors resulted in the opposite effect. CONCLUSION: This study demonstrates the regulatory effect of miRNAs from follicular fluid-derived EVs on oocyte competence acquisition, providing a further basis for understanding the significance of miRNAs in oocyte maturation and embryonic development. Up-regulation of miR-34c in EVs from follicular fluid containing competent oocytes and the positive impact of miR-34c mimics added during IVM on the resulting blastocysts indicate its pivotal role in oocyte competence.

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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.

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Success of animal cloning is limited by oocyte quality, which is closely linked to reprogramming ability. The number of layers of cumulus cells is typically used to assess the quality of oocyte; a minimum of one-third of collected cumulus-oocyte complexes (COCs) are discarded as inferior oocytes because they have less cumulus cells. Melatonin, which has been recognised for its ability to sequester free radicals and perform multiple functions, has emerged as a potentially effective candidate for enhancing inferior oocytes quality and, consequently, embryo development competency. The current study investigates to improve the quality of inferior oocytes by supplementation of melatonin (10-9 M) during in vitro maturation (IVM) and subsequent cloned embryo production and its mechanism. The results indicate that melatonin supplementation significantly (p<0.05) enhances inferior oocytes maturation, reduces oxidative stress by reducing ROS levels, and improves mitochondrial function by boosting GSH levels. The melatonin treatment (10-9 M) enhances the expression of SOD, GPx1, GDF 9, BMP 15, ATPase 6, and ATPase 8 in inferior oocytes. Furthermore, melatonin treatment increases the total cell number in the treated groups, promoting cloned blastocyst formation rates derived from inferior oocytes. Furthermore, compared to the control, 10-9 M melatonin supplementation enhances H3K9ac acetylation and lowers H3K27me3 methylation in cloned blastocysts derived from inferior oocytes. In conclusion, 10-9 M melatonin supplementation during IVM increased inferior oocyte maturation and promoted cloned buffalo embryo development by lowering oxidative stress and promoting epigenetic alterations. These studies show that melatonin may improve the quality of poor oocytes and buffalo cloning.

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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.

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The role of cumulus cells (CCs) in the acquisition of oocyte developmental competence is not yet fully understood. In a previous study, we matured cumulus-denuded fully-grown mouse oocytes to metaphase II (MII) on a feeder layer of CCs (FL-CCs) isolated from developmentally competent (FL-SN-CCs) or incompetent (FL-NSN-CCs) SN (surrounded nucleolus) or NSN (not surrounding nucleolus) oocytes, respectively. We observed that oocytes cultured on the former could develop into blastocysts, while those matured on the latter arrested at the 2-cell stage. To investigate the CC factors contributing to oocyte developmental competence, here we focused on the CCs' release into the medium of extracellular vesicles (EVs) and on their miRNA content. We found that, during the 15-h transition to MII, both FL-SN-CCs and FL-NSN-CCs release EVs that can be detected, by confocal microscopy, inside the zona pellucida (ZP) or the ooplasm. The majority of EVs are <200 nm in size, which is compatible with their ability to cross the ZP. Next-generation sequencing of the miRNome of FL-SN-CC versus FL-NSN-CC EVs highlighted 74 differentially expressed miRNAs, with 43 up- and 31 down-regulated. Although most of these miRNAs do not have known roles in the ovary, in silico functional analysis showed that seven of these miRNAs regulate 71 target genes with specific roles in meiosis resumption (N = 24), follicle growth (N = 23), fertilization (N = 1), and the acquisition of oocyte developmental competence (N = 23). Overall, our results indicate CC EVs as emerging candidates of the CC-to-oocyte communication axis and uncover a group of miRNAs as potential regulatory factors.

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The possibility of detecting the developmental competence of individually cultured embryos through analysis of spent media is a major current trend in an ART setting. However, individual embryo culture is detrimental compared with high-density group culture due to the reduced concentration of putative embryotropins. The main aim of this study was to identify an individual culture system that is not detrimental over high-density group culture in the bovine model. Blastocyst rates and competence were investigated in a conventional (GC) group, semi-confined group (MG), and individual culture (MS) in a commercial microwell device. Main findings showed that: (1) individual embryos can be continuously cultured for 7 days in ~70 nL microwells (MS) without detrimental effects compared with the GC and MG; (2) MS and MG blastocysts had a reduced number of TUNEL-positive cells compared to GC blastocysts; (3) though blastocyst mean cell numbers, mitochondrial activity, and lipid content were not different among the three culture conditions, MS blastocysts had a higher frequency of small-sized lipid droplets and a reduced mean droplet diameter compared with GC and MG blastocysts. Overall, findings open the way to optimize the development and competence of single embryos in an ART setting.

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Mammalian oocytes develop and mature in a mutually dependent relationship with surrounding cumulus cells. The oocyte actively regulates cumulus cell differentiation and function by secreting soluble paracrine oocyte-secreted factors (OSFs). We characterized the molecular mechanisms by which two model OSFs, cumulin and BMP15, regulate oocyte maturation and cumulus-oocyte cooperativity. Exposure to these OSFs during mouse oocyte maturation in vitro altered the proteomic and multispectral autofluorescence profiles of both the oocyte and cumulus cells. In oocytes, cumulin significantly upregulated proteins involved in nuclear function. In cumulus cells, both OSFs elicited marked upregulation of a variety of metabolic processes (mostly anabolic), including lipid, nucleotide, and carbohydrate metabolism, whereas mitochondrial metabolic processes were downregulated. The mitochondrial changes were validated by functional assays confirming altered mitochondrial morphology, respiration, and content while maintaining ATP homeostasis. Collectively, these data demonstrate that cumulin and BMP15 remodel cumulus cell metabolism, instructing them to upregulate their anabolic metabolic processes, while routine cellular functions are minimized in the oocyte during maturation, in preparation for ensuing embryonic development.NEW & NOTEWORTHY Oocyte-secreted factors (OSFs) promote oocyte and cumulus cell cooperativity by altering the molecular composition of both cell types. OSFs downregulate protein catabolic processes and upregulate processes associated with DNA binding, translation, and ribosome assembly in oocytes. In cumulus cells, OSFs alter mitochondrial number, morphology, and function, and enhance metabolic plasticity by upregulating anabolic pathways. Hence, the oocyte via OSFs, instructs cumulus cells to increase metabolic processes on its behalf, thereby subduing oocyte metabolism.

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BACKGROUND: While oocyte IVM is practiced sporadically it has not achieved widespread clinical practice globally. However, recently there have been some seminal advances in our understanding of basic aspects of oocyte biology and ovulation from animal studies that have led to novel approaches to IVM. A significant recent advance in IVM technology is the use of biphasic IVM approaches. These involve the collection of immature oocytes from small antral follicles from minimally stimulated patients/animals (without hCG-priming) and an ∼24 h pre-culture of oocytes in an advanced culture system ('pre-IVM') prior to IVM, followed by routine IVF procedures. If safe and efficacious, this novel procedure may stand to make a significant impact on human ART practices. OBJECTIVE AND RATIONALE: The objectives of this review are to examine the major scientific advances in ovarian biology with a unique focus on the development of pre-IVM methodologies, to provide an insight into biphasic IVM procedures, and to report on outcomes from animal and clinical human data, including safety data. The potential future impact of biphasic IVM on ART practice is discussed. SEARCH METHODS: Peer review original and review articles were selected from PubMed and Web of Science searches for this narrative review. Searches were performed using the following keywords: oocyte IVM, pre-IVM, biphasic IVM, CAPA-IVM, hCG-triggered/primed IVM, natural cycle IVF/M, ex-vivo IVM, OTO-IVM, oocyte maturation, meiotic competence, oocyte developmental competence, oocyte capacitation, follicle size, cumulus cell (CC), granulosa cell, COC, gap-junction communication, trans-zonal process, cAMP and IVM, cGMP and IVM, CNP and IVM, EGF-like peptide and IVM, minimal stimulation ART, PCOS. OUTCOMES: Minimizing gonadotrophin use means IVM oocytes will be collected from small antral (pre-dominant) follicles containing oocytes that are still developing. Standard IVM yields suboptimal clinical outcomes using such oocytes, whereas pre-IVM aims to continue the oocyte's development ex vivo, prior to IVM. Pre-IVM achieves this by eliciting profound cellular changes in the oocyte's CCs, which continue to meet the oocyte's developmental needs during the pre-IVM phase. The literature contains 25 years of animal research on various pre-IVM and biphasic IVM procedures, which serves as a large knowledge base for new approaches to human IVM. A pre-IVM procedure based on c-type natriuretic peptide (named 'capacitation-IVM' (CAPA-IVM)) has undergone pre-clinical human safety and efficacy trials and its adoption into clinical practice resulted in healthy live birth rates not different from conventional IVF. WIDER IMPLICATIONS: Over many decades, improvements in clinical IVM have been gradual and incremental but there has likely been a turning of the tide in the past few years, with landmark discoveries in animal oocyte biology finally making their way into clinical practice leading to improved outcomes for patients. Demonstration of favorable clinical results with CAPA-IVM, as the first clinically tested biphasic IVM system, has led to renewed interest in IVM as an alternative, low-intervention, low-cost, safe, patient-friendly ART approach, and especially for patients with PCOS. The same new approach is being used as part of fertility preservation in patients with cancer and holds promise for social oocyte freezing.

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Objective To investigate the protective effects of melatonin(MT)on early embryo in vitro development of mice after exposure to benzophenone-3(BP-3).Methods Fertilized mouse oocytes at the synge-neic stage were cultured in KSOM culture medium,0.8 μmol/L BP-3 culture medium,and 1×10-7 mol/L MT + 0.8 μmol/L BP-3 mixed culture medium,respectively.The rescue effect of MT on the early embryos developmental potential of BP-3-exposed mice in vitro was explored by detecting the blastocyst rate,gene transcription level,protein expression level,and the degree of DNA damage in the three groups of embryos.Results MT improved the developmental potential of mouse embryos exposed to BP-3 in vitro.Compared with the control group,MT treatment significantly increased the protein expression of ATP5A and ATP5B and decreased the DNA damage(P<0.05).Furthermore,the transcription levels of antioxidant gene Gpx1 and pluripotency related genes Pou5f1 and Cdx2 were significantly up-regulated in MT-treated blastocysts,and the expression of pro-apoptotic gene Bax was decreased.Compared with the control group,BP-3 treatment enhanced the signal intensity of γ-H2AX in blastocysts(P<0.05),while adding MT could effectively alleviate DSBs(P<0.05).Conclusion The physiological concentration of BP-3 exposure has reproductive toxicity,but the addition of appropriate con-centration of MT could significantly improve the in vitro developmental potential and quality of BP-3-exposed early embryos.

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During the transition period and early lactation of ruminants with higher production, the reproductive organs are exposed to various stressors, like inflammation stimulators such as lipopolysaccharides (LPS), as a consequence of high concentrate consumption. In this study, we aimed to determine the probable potential of α-linolenic acid (ALA) in alleviating LPS-induced effects in ovine oocytes in vitro as well as the underlying controlling mechanisms. Different concentrations of LPS (0, 0.01, 0.1, 1, and 10 µg/mL) were added to the oocyte maturation medium to evaluate its effect on oocyte developmental competence. Likewise, different concentrations of ALA (0, 10, 50, 100, and 200 µM/mL) were added to the maturation medium to define its effects on oocyte developmental competence. Accordingly, a combination of ALA and LPS in a dose-dependent manner was added to the maturation medium to elucidate their effect on oocyte developmental competence and uncover any possible potential of ALA to alleviate the detrimental effect induced by the presence of LPS. The expressions of candidate genes were measured in mature oocytes treated either with ALA, LPS, or ALA plus LPS. Adding LPS to the maturation medium decreased the cleavage rate of the treated oocytes, and those oocytes reached the blastocyst stage at a lower rate. Adding ALA to the maturation medium in the presence of LPS alleviated the detrimental effects of LPS in a dose-dependent manner, which ultimately led to higher cleavage and blastocyst formation. A higher expression of Trim26, GRHPR, NDUFA, PGC-1α, SOD, CS, SDH, p53, and CAT was observed in LPS-treated oocytes compared with the ALA and control groups. Additionally, CS and CAT transcripts were down-regulated in oocytes in LPS plus ALA-treated group compared to that of the LPS-treated group. These findings revealed that ALA has the potential to alleviate the detrimental effects induced by LPS on in ovine oocytes during maturation in vitro. Thus, LPS-detrimental effect and ALA-preventing mechanisms seem to be regulated through the expression of genes involved in mitochondrial biogenesis and function, oxidative stress, and antioxidant systems.

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Since the developmental stage of oocyte is a challenging issue in the success of vitrification, this study investigated the effects of vitrification, before and after in vitro maturation, on the survival and maturation rates, developmental competence and the expression levels of genes involved in apoptosis, oxidative stress and epigenetic modifications. Mouse germinal vesicle (GV) oocytes were divided into four groups: fresh in vitro matured oocytes without vitrification (fIVM), in vitro matured oocytes after vitrification (vIVM), in vitro matured oocytes before vitrification (IVMv). In addition, in vivo matured oocytes (MII) were used as control. After oocytes collection, maturation and survival rates as well as the intracellular reactive oxygen species (ROS) level were evaluated. Also, the expression level of various genes was analyzed by qRT-PCR. In addition, following artificial activation (parthenogenesis), the developmental competence of oocytes to the blastocyst stage was evaluated. A significant decrease in maturation rate and survival of vIVM oocytes was observed compared to fIVM and IVMv oocytes. Intracellular ROS levels were significantly increased in both vitrified groups compared to the fIVM group, and no significant difference between vitrified groups. Pro-apoptotic genes; BAX and Bcl2 as well as genes related to oxidative stress response Hsp1a, Hsp1b and SOD1were significantly increased in the vIVM group compared to the IVMv group. Interestingly, epigenetic regulators genes DNMT1, DNMT3a and DNMT3b were highly expressed in IVMv oocytes along with a decrease in the artificial activation rate compared to the vIVM oocytes. Our results indicated that despite observing more negative effects of vitrification before IVM on the survival rate and maturation as well as apoptosis status, less epigenetic changes in vIVM oocytes can make this process a better option in the treatment of infertility than IVM of oocytes followed by vitrification, a hypothesis that needs to be investigation in human oocytes.

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The establishment and maintenance of a pregnancy that goes to term is sine qua non for the long-term sustainability of dairy and beef cattle operations. The oocyte plays a critical role in providing the factors necessary for preimplantation embryonic development. Furthermore, the female, or maternal, environment where oocytes and embryos develop is crucial for the establishment and maintenance of a pregnancy to term. During folliculogenesis, the oocyte must sequentially acquire meiotic and developmental competence, which are the results of a series of molecular events preparing the highly specialized gamete to return to totipotency after fertilization. Given that folliculogenesis is a lengthy process in the cow, the occurrence of disease, metabolic imbalances, heat stress, or other adverse events can make it challenging to maintain oocyte quality. Following fertilization, the newly formed embryo must execute a tightly planned program that includes global DNA remodeling, activation of the embryonic genome, and cell fate decisions to form a blastocyst within a few days and cell divisions. The increasing use of assisted reproductive technologies creates an additional layer of complexity to ensure the highest oocyte and embryo quality given that in vitro systems do not faithfully recreate the physiological maternal environment. In this review, we discuss cellular and molecular factors and events known to be crucial for proper oocyte development and maturation, as well as adverse events that may negatively affect the oocyte; and the importance of the uterine environment, including signaling proteins in the maternal-embryonic interactions that ensure proper embryo development. We also discuss the impact of assisted reproductive technologies in oocyte and embryo quality and developmental potential, and considerations when looking into the prospects for developing systems that allow for in vitro gametogenesis as a tool for assisted reproduction in cattle.

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The oocyte donor plays a pivotal role in bovine in vitro embryo production (IVP) success. The individual factor affects blastocyst/oocyte ratio and determine the existence of outstanding performing animals. The aim of this study was to assess the extent of individual factor effect to IVP efficiency, in a population of Gir oocyte donors. Extreme (high or low IVP efficiency based on blastocyst/oocyte ratio) animals were selected out of a population of 250 oocyte donors (1,734 observations) to form high (>0.48, n = 40), average (0.17-0.48, n = 168), and low (<0.17, n = 42) efficiency donor groups. Cumulus-oocyte complex indicators (total number, IVF-grade number, and IVF-grade/total ratio) were lower (p < 0.05) in high efficiency donors. The number of blastocysts per OPU was analyzed for highest performing bull, and an increase (p < 0.05) in high efficiency donors and a decrease (p < 0.05) in low efficiency donors were noticed, compared to average efficiency donors. The number of pregnancies obtained per OPU was affected (p = 0.017) by donor's efficiency (low: 0.60 ± 0.09 $$ 0.60\pm 0.09 $$ , average: 1.17 ± 0.07 $$ 1.17\pm 0.07 $$ , high: 2.57 ± 0.26 $$ 2.57\pm 0.26 $$ ), being 4.3-fold higher in high than in low efficiency donors. We conclude that producing embryos from high efficiency blastocyst/oocyte ratio donors increases blastocyst and pregnancy numbers by OPU, being an important indicator for donor selection in IVP programs.

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Throughout the reproductive life of women, cumulus cells (CC) protect the dormant oocyte from damage, act as sensors of the follicular microenvironment, and act as a gatekeeper for oocyte developmental potential. One such mechanism relies on the hypoxia-tolerance response, which, with age, decreases systematically, including in the ovary. We aimed to evaluate the association between gene expression related to hypoxia and aging in CC and reproductive results in in vitro fertilization cycles. We recruited 94 women undergoing controlled ovarian stimulation. Total RNA was extracted from pooled CCs collected after oocyte pick-up (OPU) and reverse-transcribed to complementary DNA using random hexamers to test 14 genes related to hypoxia response via HIF1α activation, oxidative stress, and angiogenic responses. The expression of CLU, NOS2, and TXNIP had a positive correlation with age (rs = 0.25, rs = 0.24, and rs = 0.35, respectively). Additionally, NOS2 and HMOX1 expression correlated positively with the retrieval of immature oocytes (rs = 0.22 and rs = 0.40, respectively). Moreover, VEGFC levels decreased overall with increasing fertilization rate, independently of age (rs = -0.29). We found that the fertilization potential of a cohort of oocytes is related to the ability of CC to respond to oxidative stress and hypoxia with age, pointing at NOS2, HMOX1, and VEGFC expression as markers for oocyte maturation and fertilization success.

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Introduction: Acquisition of germinal vesicle (GV) stage oocytes for fertility preservation (FP) offers several benefits over in vivo matured oocyte cryopreservation following ovarian stimulation, particularly for cancer patients necessitating immediate treatment. Two FP approaches for GV oocytes are available: vitrification before in vitro maturation (IVM) at the GV stage (GV-VI) or post-IVM at the metaphase II (MII) stage (MII-VI). The optimal method remains to be determined. Methods: In this study, mouse oocytes were collected without hormonal stimulation and vitrified either at the GV stage or the MII stage following IVM; non-vitrified in vitro matured MII oocytes served as the control (CON). The oocyte quality and developmental competence were assessed to obtain a better method for immediate FP. Results: No significant differences in IVM and survival rates were observed among the three groups. Nevertheless, GV-VI oocytes exhibited inferior quality, including abnormal spindle arrangement, mitochondrial dysfunction, and early apoptosis, compared to MII-VI and CON oocytes. Oocyte vitrification at the GV stage impacted maternal mRNA degradation during IVM. In addition, the GV-VI group demonstrated significantly lower embryonic developmental competence relative to the MII-VI group. RNA sequencing of 2-cell stage embryos revealed abnormal minor zygotic genome activation in the GV-VI group. Conclusion: Vitrification at the GV stage compromised oocyte quality and reduced developmental competence. Consequently, compared to the GV stage, oocyte vitrification at the MII stage after IVM is more suitable for patients who require immediate FP.

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The vertebrate head mesoderm provides the heart, the great vessels, some smooth and most head skeletal muscle, in addition to parts of the skull. It has been speculated that the ability to generate cardiac and smooth muscle is the evolutionary ground-state of the tissue. However, whether indeed the entire head mesoderm has generic cardiac competence, how long this may last, and what happens as cardiac competence fades, is not clear. Bone morphogenetic proteins (Bmps) are known to promote cardiogenesis. Using 41 different marker genes in the chicken embryo, we show that the paraxial head mesoderm that normally does not engage in cardiogenesis has the ability to respond to Bmp for a long time. However, Bmp signals are interpreted differently at different time points. Up to early head fold stages, the paraxial head mesoderm is able to read Bmps as signal to engage in the cardiac programme; the ability to upregulate smooth muscle markers is retained slightly longer. Notably, as cardiac competence fades, Bmp promotes the head skeletal muscle programme instead. The switch from cardiac to skeletal muscle competence is Wnt-independent as Wnt caudalises the head mesoderm and also suppresses Msc-inducing Bmp provided by the prechordal plate, thus suppressing both the cardiac and the head skeletal muscle programmes. Our study for the first time suggests a specific transition state in the embryo when cardiac competence is replaced by skeletal muscle competence. It sets the stage to unravel the cardiac-skeletal muscle antagonism that is known to partially collapse in heart failure.

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Melatonin, an antioxidant hormone secreted by the pineal gland, has been recognized as a regulator for numerous biological events. The deleterious effects of juglone, a polyphenolic extract of walnut trees, on embryo development has been previously reported. In the current study, we aimed to display the impact of melatonin administrated during in vitro oocyte maturation (IVM) on juglone-treated oocytes. Thus, in vitro matured oocytes were collected after 24 h post incubation with juglone in the presence or absence of melatonin. Reactive oxygen species (ROS), glutathione (GSH) content, mitochondrial distribution, and the relative abundance of mRNA transcription levels were assessed in oocytes, in addition, oocytes were in vitro fertilized to check the competency levels of oocytes to generate embryos. We found that administration of melatonin during the maturation of oocytes under juglone stress significantly improved the cleavage rate, 8-16 cell-stage embryos and day-8 blastocysts when compared to the sole juglone treatment. In addition, the fluorescence intensity of ROS increased, whereas the GSH decreased in juglone-treated oocytes compared to melatonin-juglone co-treated and untreated ones. Additionally, a significant increase in the mitochondrial aberrant pattern, the pattern that was normalized following melatonin supplementation, was observed following juglone administration. The mRNA analysis using RT-qPCR revealed a significant upregulation of autophagy and oxidative-stress-specific markers in the juglone-treated group compared to the co-treatment and control. In conclusion, the study reveals, for the first time, a protective effect of melatonin against the oxidative stress initiated following juglone treatment during the in vitro maturation of oocytes.