Application of amphiregulin in IVM culture of immature human oocytes and pre-insemination culture for COCs in IVF cycles.

Background: Amphiregulin (AR) is a growth factor that resembles the epidermal growth factor (EGF) and serves various functions in different cells. However, no systematic studies or reports on the role of AR in human oocytes have currently been performed or reported. This study aimed to explore the role of AR in human immature oocytes during in vitro maturation (IVM) and in vitro fertilization (IVF) in achieving better embryonic development and to provide a basis for the development of a pre-insemination culture medium specific for cumulus oocyte complexes (COCs). Methods: First, we examined the concentration of AR in the follicular fluid (FF) of patients who underwent routine IVF and explored the correlation between AR levels and oocyte maturation and subsequent embryonic development. Second, AR was added to the IVM medium to culture immature oocytes and investigate whether AR could improve the effects of IVM. Finally, we pioneered the use of a fertilization medium supplemented with AR for the pre-insemination culture of COCs to explore whether the involvement of AR can promote the maturation and fertilization of IVF oocytes, as well as subsequent embryonic development. Results: A total of 609 FF samples were examined, and a positive correlation between AR levels and blastocyst formation was observed. In our IVM study, the development potential and IVM rate of immature oocytes, as well as the fertilization rate of IVM oocytes in the AR-added groups, were ameliorated significantly compared to the control group (All P < 0.05). Only the IVM-50 group had a significantly higher blastocyst formation rate than the control group (P < 0.05). In the final IVF study, the maturation, fertilization, high-quality embryo, blastocyst formation, and high-quality blastocyst rates of the AR-added group were significantly higher than those of the control group (All P < 0.05). Conclusion: AR levels in the FF positively correlated with blastocyst formation, and AR involvement in pre-insemination cultures of COCs can effectively improve laboratory outcomes in IVF. Furthermore, AR can directly promote the in vitro maturation and developmental potential of human immature oocytes at an optimal concentration of 50 ng/ml.

Oocyte maturation, fertilization, and embryo development in vitro by green and chemical iron oxide nanoparticles: a comparative study.

Oxidative stress is considered one of the main challenges for in vitro maturation (IVM) and makes assisted reproductive technology (ART), including IVF and embryonic development less effective. Reducing free radicals via biocompatible nanoparticles (NPs) is one of the most promising approaches for developing IVM. We investigated the comparative effect of green and chemically synthesized iron oxide nanoparticles (IONPs) with an aqueous extract of date palm pollen (DPP) on oocyte parameters related to the IVM process. To this end, IONPs were synthesized by chemical (Ch-IONPs) and green methods (G-IONPs using DPP) and characterized. The mature oocyte quality of the Ch-IONPs and G-IONPs groups was evaluated by JC1 and Hoechst staining, Annexin V-FITC-Propidium Iodide, 2', 7'-dichlorofluorescein diacetate, and dihydroethidium staining compared to the control group. Eventually, the mature oocytes were fertilized, promoted to blastocysts (BL), and evaluated in vitro. Compared with the control and G-IONPs groups, the Ch-IONPs-treated group produced more hydrogen peroxide and oxygen radicals. Compared with the Ch-IONPs group, the fertilization rate in the G-IONPs and control groups increased significantly. Finally, the G-IONPs and control groups exhibited a significant increase in the 2PN, 2-cell, 4-cell, 8-cell, compacted morula (CM), and BL rates compared with the Ch-IONPs group. Green synthesis of IONPs can reduce the toxicity of chemical IONPs during the IVM process. It can be concluded that G-IONPs encased with DPP compounds have the potential to protect against exogenous reactive oxygen species (ROS) production in an IVM medium, which can have a crucial effect on oocyte maturation and fertilization efficiency.

Effects of limonin on oxidative stress and early apoptosis in oocytes during in vitro maturation.

To investigate the effects of limonin (Lim) on oxidative stress and early apoptosis in bovine oocytes during in vitro maturation (IVM), different concentrations of Lim (0, 10, 20, 50 µmol/L) were added to bovine IVM medium. Oocyte maturation rates and development 24 h after in vitro fertilization (IVF) were examined to determine the optimal Lim concentration. The optimal Lim concentration was added to the IVM medium, and 0 µmol/L Lim was used as the control. Immunofluorescence staining was used to detect the abnormal rate of spindle assembly, reactive oxygen species (ROS), glutathione (GSH), mitochondrial membrane potential (MMP) levels, mitochondrial distribution, and the fluorescence intensity of cathepsin B (CB)-active LC3 protein. RT‒qPCR was used to detect the mRNA expression levels of antioxidant-, apoptosis- and autophagy-related genes in oocytes. The total number of blastocysts and the proportion of apoptotic cells among blastocysts were detected. The results showed that the PBI ejection rate, cleavage rate and blastocyst rate of bovine oocytes in the 20 µmol/L Lim group were significantly higher than those in the control group (P < 0.05). Compared with those in the control group, ROS levels, abnormal mitochondrial distribution, the proportion of abnormal spindle assembly, CB activity and LC3 protein fluorescence intensity of oocytes in the 20 µmol/L Lim group were significantly decreased (P < 0.05), and GSH and MMP levels were significantly increased (P < 0.05). The expression of antioxidant genes (Prdx3, Prdx6, Sirt1) and antiapoptotic genes (Bcl-xl, Survivin) were significantly upregulated (P < 0.05), and the expression levels of proapoptotic genes (Caspase-4, BAX) and autophagy-related genes (LC3) were significantly downregulated (P < 0.05). The total number of cells among in vitro fertilized embryos was significantly increased (P < 0.05), and the apoptosis rate of blastocysts was significantly decreased (P < 0.05). Here, we show that Lim exerts positive effects on bovine oocyte IVM by regulating REDOX homeostasis, reducing spindle damage and enhancing mitochondrial function during IVM, thereby inhibiting oocyte apoptosis and autophagy.

Antioxidant sericin averts the disruption of oocyte-follicular cell communication triggered by oxidative stress.

Antioxidants are free radical scavengers that increase oocyte quality and improve female fertility by suppressing oxidative stress. However, the related mechanisms remain unclear. The present study was designed to examine whether a reduction of oxidative stress from using the antioxidant sericin led to expanded cumulus cell (CC)-oocyte communication and oocyte developmental acquisition in a bovine model. We found that cumulus-oocyte complexes (COCs) matured in the presence of sericin showed a significantly increased oocyte meiotic maturation rate (P < 0.01) and accelerated subsequent blastocyst formation, as more blastocysts were found at the hatched stage (P < 0.05) compared to that in the control group. In contrast to the control group, sericin suppressed H2O2 levels in COCs, resulting in a markedly enhanced CC-oocyte gap junction communication index and number of transzonal projections, which were preserved until 18 h of oocyte maturation. These findings indicate that sericin reduces disruption of oocyte-follicular cell communication induced by oxidative stress. Sericin consistently increased intra-oocyte glutathione (GSH) levels and reduced oocyte H2O2 levels (P < 0.05), both of which were ablated when GSH synthesis was inhibited by buthionine sulfoximide (an inhibitor of GSH synthesis). Furthermore, the inhibition of GSH synthesis counteracted the positive effects of sericin on subsequent embryo developmental competence (P < 0.01). Intra-oocyte GSH levels were positively associated with blastocyst development and quality. These outcomes demonstrate new perspectives for the improvement of oocyte quality in assisted reproductive technology and may contribute to developing treatment strategies for infertility and cancer.

A fresh start for IVM: capacitating the oocyte for development using pre-IVM.

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.

Cathepsin L regulates oocyte meiosis and preimplantation embryo development.

Early embryonic loss, caused by reduced embryo developmental competence, is the major cause of subfertility in humans and animals. This embryo developmental competence is determined during oocyte maturation and the first embryo divisions. Therefore, it is essential to identify the underlying molecules regulating these critical developmental stages. Cathepsin L (CTSL), a lysosomal cysteine protease, is involved in regulating cell cycle progression, proliferation and invasion of different cell types. However, CTSL role in mammalian embryo development is unknown. Using bovine in vitro maturation and culture systems, we show that CTSL is a key regulator for embryo developmental competence. We employed a specific CTSL detection assay in live cells to show that CTSL activity correlates with meiotic progression and early embryo development. Inhibiting CTSL activity during oocyte maturation or early embryo development significantly impaired oocyte and embryo developmental competence as evidenced by lower cleavage, blastocyst and hatched blastocyst rates. Moreover, enhancing CTSL activity, using recombinant CTSL (rCTSL), during oocyte maturation or early embryo development significantly improved oocyte and embryo developmental competence. Importantly, rCTSL supplementation during oocyte maturation and early embryo development significantly improved the developmental competence of heat-shocked oocytes/embryos which are notoriously known for reduced quality. Altogether, these results provide novel evidence that CTSL plays a pivotal role in regulating oocyte meiosis and early embryonic development.

Three-dimensional glass scaffolds improve the In Vitro maturation of porcine cumulus-oocyte complexes and subsequent embryonic development after parthenogenetic activation.

In vitro maturation (IVM) methods for porcine oocytes are still deficient in achieving full developmental capacity, as the currently available oocyte in vitro culture systems still have limitations. In vitro embryo production must also improve the porcine oocyte IVM system to acquire oocytes with good developmental potential. Herein, we tested a three-dimensional (3D) glass scaffold culture system for porcine oocyte maturation. After 42 h, we matured porcine cumulus-oocyte complexes (COCs) on either two-dimensional glass dishes (2D-B), two-dimensional microdrops (2D-W), or 3D glass scaffolds. The 3D glass scaffolds were tested for porcine oocyte maturation and embryonic development. Among these culture methods, the extended morphology of the 3D group maintained a 3D structure better than the 2D-B and 2D-W groups, which had flat COCs that grew close to the bottom of the culture vessel. The COCs of the 3D group had a higher cumulus expansion index and higher first polar body extrusion rate, cleavage rate, and blastocyst rate of parthenogenetic embryos than the 2D-B group. In the 3D group, the cumulus-expansion-related gene HAS2 and anti-apoptotic gene Bcl-2 were significantly upregulated (p < 0.05), while the pro-apoptotic gene Caspase3 was significantly downregulated (p < 0.05). The blastocysts of the 3D group had a higher relative expression of Bcl-2, Oct4, and Nanog than the other two groups (p < 0.05). The 3D group also had a more uniform distribution of mitochondrial membrane potential and mitochondria (p < 0.05), and its cytoplasmic active oxygen species content was much lower than that in the 2D-B group (p < 0.05). These results show that 3D glass scaffolds dramatically increased porcine oocyte maturation and embryonic development after parthenogenetic activation, providing a suitable culture model for porcine oocytes.

In vitro reduction of bovine oocyte ATP production with oligomycin affects embryo epigenome.

In brief: Epigenetic programming is a crucial process during early embryo development that can have a significant impact on the results of assisted reproductive technology (ART) and offspring health. Here we show evidence using a bovine in vitro experiment that embryo epigenetic programing is dependent on oocyte mitochondrial bioenergetic activity during maturation. Abstract: This study investigated if oocyte and early embryo epigenetic programming are dependent on oocyte mitochondrial ATP production. A bovine in vitro experiment was performed in which oocyte mitochondrial ATP production was reduced using 5 nmol/L oligomycin A (OM; ATP synthase inhibitor) during in vitro maturation (IVM) compared to control (CONT). OM exposure significantly reduced mitochondrial ATP production rate in MII oocytes (34.6% reduction, P = 0.018) and significantly decreased embryo cleavage rate at 48 h post insemination (7.6% reduction, P = 0.031). Compared to CONT, global DNA methylation (5mC) levels were decreased in OM-exposed MII oocytes (9.8% reduction, P = 0.019) while global histone methylation (H3K9me2) was increased (9.4% increase, P = 0.024). In zygotes, OM exposure during IVM increased 5mC (22.3% increase, P < 0.001) and histone acetylation (H3K9ac, 17.3% increase, P = 0.023) levels, while H3K9me2 levels were not affected. In morulae, 5mC levels were increased (10.3% increase, P = 0.041) after OM exposure compared to CONT, while there was no significant difference in H3K9ac and H3K9me2 levels. These epigenetic alterations were not associated with any persistent effects on embryo mitochondrial ATP production rate or mitochondrial membrane potential (assessed at the four-cell stage). Also, epigenetic regulatory genes were not differentially expressed in OM-exposed zygotes or morulae. Finally, apoptotic cell index in blastocysts was increased after OM exposure during oocyte maturation (41.1% increase, P < 0.001). We conclude that oocyte and early embryo epigenetic programming are dependent on mitochondrial ATP production during IVM.

Cyclophosphamide reduces gene transcriptional activity and embryo in vitro development by inhibiting NF-κB expression through decreasing AcH4K12.

Cyclophosphamide (CTX), a widely used chemotherapeutic agent for cancer treatment, has been associated with long-term toxicity and detrimental effects on oocytes and ovaries, resulting in female reproductive dysfunction. This study aimed to investigate the potential impact of CTX on in vitro maturation (IVM) injury of porcine oocytes and subsequent embryonic development, as well as its effects on epigenetic modification and gene activation during early embryonic development. The results demonstrated that CTX treatment caused aberrant spindle structure and mitochondrial dysfunction during oocyte maturation, inducing DNA damage and early apoptosis, which consequently disrupted meiotic maturation. Indeed, CTX significantly reduced the in vitro developmental capacity of porcine embryos, and induced DNA damage and apoptosis in in vitro fertilization (IVF) blastocysts. Importantly, CTX induced abnormal histone modification of AcH4K12 in early porcine embryos. Moreover, addition of LBH589 before zygotic genome activation (ZGA) effectively increased AcH4K12 levels and restored the protein expression of NF-κB, which can effectively enhance the in vitro developmental potential of IVF embryos. The DNA damage and apoptosis induced by CTX compromised the quality of the blastocysts, which were recovered by supplementation with LBH589. This restoration was accompanied by down-regulation of BAX mRNA expression and up-regulation of BCL2, POU5F1, SOX2 and SOD1 mRNA expression. These findings indicated that CTX caused abnormal histone modification of AcH4K12 in early porcine embryos and reduced the protein expression of NF-κB, a key regulator of early embryo development, which may block subsequent ZGA processes.

Ellagic acid treatment during in vitro maturation of porcine oocytes improves development competence after parthenogenetic activation and somatic cell nuclear transfer.

Ellagic acid (EA) is a natural polyphenol and a free radical scavenger with antioxidant properties. This study investigated the protective effects of EA during in vitro maturation (IVM) of porcine oocytes. To determine the optimal concentration, IVM medium was supplemented with various concentrations of EA. Treatment with 10 µM EA (10 EA) resulted in the highest cleavage rate, blastocyst formation rate, and total cell number per blastocyst and the lowest percentage of apoptotic cell in parthenogenetic blastocysts. In the 10 EA group, abnormal spindle and chromosome misalignment were rescued and the ratio of phosphorylated p44/42 to total p44/42 was increased. Furthermore, the reactive oxygen species and glutathione levels were significantly decreased and increased, respectively, and antioxidant genes (Nrf2, HO-1, CAT, and SOD1) were significantly upregulated in the 10 EA group. mRNA expression of developmental-related (CDX2, POU5F1, and SOX2) and anti-apoptotic (BCL2L1) genes was significantly upregulated in the 10 EA group, while mRNA expression of pro-apoptotic genes (BAK, FAS, and CASP3) was significantly downregulated. Ultimately, following somatic cell nuclear transfer, the blastocyst formation rate was significantly increased and the percentage of apoptotic cell in blastocysts was significantly decreased in the 10 EA group. In conclusion, addition of 10 EA to IVM medium improved oocyte maturation and the subsequent embryo development capacity through antioxidant mechanisms. These findings suggest that EA can enhance the efficiencies of assisted reproductive technologies.

In vitro maturation (IVM) of human immature oocytes: is it still relevant?

In vitro maturation (IVM) of human immature oocytes has been shown to be a viable option for patients at risk of ovarian hyperstimulation syndrome (OHSS), those seeking urgent fertility preservation and in circumstances where controlled ovarian stimulation is not feasible. Moreover, IVM techniques can be combined with ovarian tissue cryobanking to increase the chances of conception in cancer survivors. The clinical applications of IVM in the field of reproductive medicine are rapidly expanding and the technique is now classified as non-experimental. In contrast to conventional IVF (in vitro fertilization), IVM offers several advantages, such as reduced gonadotropin stimulation, minimal risk of ovarian hyperstimulation syndrome (OHSS), reduced treatment times and lower costs. However, the technical expertise involved in performing IVM and its lower success rates compared to traditional IVF cycles, still pose significant challenges. Despite recent advances, such as innovative biphasic IVM systems, IVM is still an evolving technique and research is ongoing to refine protocols and identify techniques to improve its efficiency and effectiveness. A comprehensive understanding of the distinct mechanisms of oocyte maturation is crucial for obtaining more viable oocytes through in vitro methods, which will in turn lead to significantly improved success rates. In this review, the present state of human IVM programs and future research directions will be discussed, aiming to promote a better understanding of IVM and identify potential strategies to improve the overall efficiency and success rates of IVM programs, which will in turn lead to better clinical outcomes.

The study of the efficiency of in vitro maturation of ovarian tissue oocytes in pediatric patients.

PURPOSE: Although recent in vitro maturation (IVM) studies in pediatric patients have demonstrated successful retrieval and maturation of oocytes, the studies included only a small number of premenarchal patients. In the present study, we examined the potential use of oocyte retrieval and maturation for pediatric patients who undergo ovarian tissue cryopreservation (OTC). METHODS: We retrospectively examined the clinical records of pediatric patients who underwent OTC at our institution between October 2015 and December 2022. Data on the age, primary disease, menstrual history, pre-procedure chemotherapy, anti-Müllerian hormone (AMH) level, number of oocytes collected ex vivo from ovarian tissue, and number of mature oocytes from IVM were examined. RESULTS: Data of 60 pediatric patients (aged 1 to 17 years) were included for analysis. Oocytes were retrieved from 36 patients; the oocytes of 18 of these patients could be cryopreserved. The IVM rate was significantly lower in the premenarchal patients than in the postmenarchal patients. The number of mature oocytes retrieved from IVM was higher in the no-chemotherapy group than in the chemotherapy group. A significant positive correlation was observed between the AMH level and the IVM outcomes. CONCLUSION: Oocyte retrieval and maturation in pediatric patients undergoing OTC is particularly useful in those not receiving chemotherapy. In patients receiving chemotherapy, the AMH level may be useful for predicting the IVM outcome. Activation of the oocyte maturation process in vivo in pediatric patients and better understanding of the major regulators of oocyte maturation are necessary to improve the utility of the IVM procedure.

Attenuation of Oxidative Stress and Regulation of AKT Signaling by Vanillic Acid during Bovine Pre-Implantation Embryo Development.

Vanillic acid (VA) has shown antioxidant and anti-inflammatory activities in different cell types, but its biological effects in the context of early embryo development have not yet been clarified. In the current study, the impact of VA supplementation during in vitro maturation (IVM) and/or post-fertilization (in vitro culture; IVC) on redox homeostasis, mitochondrial function, AKT signaling, developmental competence, and the quality of bovine pre-implantation embryos was investigated. The results showed that dual exposure to VA during IVM and late embryo culture (IVC3) significantly improved the blastocyst development rate, reduced oxidative stress, and promoted fatty acid oxidation as well as mitochondrial activity. Additionally, the total numbers of cells and trophectoderm cells per blastocyst were higher in the VA-treated group compared to control (p < 0.05). The RT-qPCR results showed down-regulation of the mRNA of the apoptosis-specific markers and up-regulation of AKT2 and the redox homeostasis-related gene TXN in the treated group. Additionally, the immunofluorescence analysis showed high levels of pAKT-Ser473 and the fatty acid metabolism marker CPT1A in embryos developed following VA treatment. In conclusion, the study reports, for the first time, the embryotrophic effects of VA, and the potential linkage to AKT signaling pathway that could be used as an efficacious protocol in assisted reproductive technologies (ART) to improve human fertility.

Establishment and optimization of an in vitro guinea pig oocyte maturation system.

Guinea pigs are a valuable animal model for studying various diseases, including reproductive diseases. However, techniques for generating embryos via embryo engineering in guinea pigs are limited; for instance, in vitro maturation (IVM) technique is preliminary for guinea pig oocytes. In this study, we aimed to establish and optimize an IVM method for guinea pig oocytes by investigating various factors, such as superovulation induced by different hormones, culture supplementation (e.g., amino acids, hormone, and inhibitors), culture conditions (e.g., oocyte type, culture medium type, and treatment time), and in vivo hCG stimulation. We found that oocytes collected from guinea pigs with superovulation induced by hMG have a higher IVM rate compared to those collected from natural cycling individuals. Moreover, we found that addition of L-cysteine, cystine, and ROS in the culture medium can increase the IVM rate. In addition, we demonstrated that in vivo stimulation with hCG for 3-8 h can further increase the IVM rate. As a result, the overall IVM rate of guinea pig oocytes under our optimized conditions can reach ~69%, and the mature oocytes have high GSH levels and normal morphology. In summary, we established an effective IVM method for guinea pig oocytes by optimizing various factors and conditions, which provides a basis for embryo engineering using guinea pigs as a model.

The combination of rolipram and cilostamide improved the developmental competence of cloned porcine embryos.

In vitro maturation of porcine oocytes is characterized by asynchronous cytoplasmic and nuclear maturation, leading to less competent oocytes supporting embryo development. The purpose of this study was to evaluate the combined effect of rolipram and cilostamide as cyclic Adenine monophosphate (cAMP) modulators to find the maximum cAMP levels that temporarily arrest meiosis. We determined the optimal time to maintain functional gap junction communication during pre-in vitro maturation to be four hours. Oocyte competence was evaluated by the level of glutathione, reactive oxygen species, meiotic progression, and gene expression. We evaluated embryonic developmental competence after parthenogenetic activation and somatic cell nuclear transfer. The combined treatment group showed significantly higher glutathione and lower reactive oxygen species levels and a higher maturation rate than the control and single treatment groups. Cleavage and blastocyst formation rates in parthenogenetic activation and somatic cell nuclear transfer embryos were higher in two-phase in vitro maturation than in the other groups. The relative levels of BMP15and GDF9 expression were increased in two-phase in vitro maturation. Somatic cell nuclear transfer blastocysts from two-phase in vitro maturation oocytes showed a lower level of expression of apoptotic genes than the control, indicating better pre-implantation developmental competence. The combination of rolipram and cilostamide resulted in optimal synchrony of cytoplasmic and nuclear maturation in porcine in vitro matured oocytes and there by enhanced the developmental competence of pre-implantation embryos.

Characterization of ovarian tissue oocytes from transgender men reveals poor calcium release and embryo development, which might be overcome by spindle transfer.

STUDY QUESTION: Can spindle transfer (ST) overcome inferior embryonic development of in vitro matured ovarian tissue oocytes (OTO-IVM) originating from testosterone-treated transgender men? SUMMARY ANSWER: ST shows some potential to overcome the embryo developmental arrest observed in OTO-IVM oocytes from transgender men. WHAT IS KNOWN ALREADY: OTO-IVM is being applied as a complementary approach to increase the number of oocytes/embryos available for fertility preservation during ovarian tissue cryopreservation in cancer patients. OTO-IVM has also been proposed for transgender men, although the potential of their oocytes remains poorly investigated. Currently, only one study has examined the ability of OTO-IVM oocytes originating from transgender men to support embryo development, and that study has shown that they exhibit poor potential. STUDY DESIGN, SIZE, DURATION: Both ovaries from 18 transgender men undergoing oophorectomy were collected for the purposes of this study, from November 2020 to September 2022. The patients did not wish to cryopreserve their tissue for fertility preservation and donated their ovaries for research. All patients were having testosterone treatment at the time of oophorectomy and some of them were also having menses inhibition treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sibling ovaries were collected in either cold or warm medium, to identify the most optimal collection temperature. Cumulus oocyte complexes (COCs) from each condition were isolated from the ovarian tissue and matured in vitro for 48 h. The quality of OTO-IVM oocytes was assessed by calcium pattern releasing ability, embryo developmental competence following ICSI, and staining for mitochondrial membrane potential. In vitro matured metaphase I (MI) oocytes, germinal vesicle (GV) oocytes, and in vivo matured oocytes with aggregates of smooth endoplasmic reticulum (SERa) were donated from ovarian stimulated women undergoing infertility treatment and these served as Control oocytes for the study groups. ST was applied to overcome poor oocyte quality. Specifically, enucleated mature Control oocytes served as cytoplasmic recipients of the OTO-IVM spindles from the transgender men. Embryos derived from the different groups were scored and analysed by shallow whole genome sequencing for copy number variations (CNVs). MAIN RESULTS AND THE ROLE OF CHANCE: In total, 331 COCs were collected in the cold condition (OTO-Cold) and 282 were collected in the warm condition (OTO-Warm) from transgender men. The maturation rate was close to 54% for OTO-Cold and 57% for OTO-Warm oocytes. Control oocytes showed a calcium releasing ability of 2.30 AU (n = 39), significantly higher than OTO-Cold (1.47 AU, P = 0.046) oocytes (n = 33) and OTO-Warm (1.03 AU, P = 0.036) oocytes (n = 31); both values of calcium release were similar between the two collection temperatures. Mitochondrial membrane potential did not reveal major differences between Control, OTO-Warm, and OTO-Cold oocytes (P = 0.417). Following ICSI, 59/70 (84.2%) of Control oocytes were fertilized, which was significantly higher compared to 19/47 (40.4%) of OTO-Cold (P < 0.01) and 24/48 (50%) of OTO-Warm oocytes (P < 0.01). In total, 15/59 (25.4%) blastocysts were formed on Day 5 in the Control group, significantly higher than 0/19 (0%) from the OTO-Cold (P = 0.014) and 1/24 (4.1%) in OTO-Warm oocytes (P = 0.026). Application of ST rescued the poor embryo development, by increasing the Day 5 blastocyst rate from 0% (0/19) to 20.6% (6/29) (P = 0.034), similar to that in the ICSI-Control group (25.4%, 15/59). A normal genetic profile was observed in 72.7% (8/11) of OTO-Cold, 72.7% (8/11) of OTO-Warm and 64.7% (11/17) of Control Day 3-Day 5 embryos. After ST was applied for OTO-IVM oocytes, 41.1% (7/17) of the embryos displayed normal genetic patterns, compared to 57.1% (4/7) among ST-Control Day 3-Day 5 embryos. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Due to the limited access to human oocytes and ovarian tissue, our results should be interpreted with some caution, as only a limited number of human oocytes and embryos could be investigated. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study, clearly indicate that OTO-IVM oocytes originating from transgender patients are of inferior quality, which questions their use for fertility preservation. The poor quality is likely to be related to cytoplasmic factors, supported by the increased blastocyst numbers following application of ST. Future research on OTO-IVM from transgender men should focus on the cytoplasmic content of oocytes or supplementation of media with factors that promote cytoplasmic maturation. A more detailed study on the effect of the length of testosterone treatment is also currently missing for more concrete guidelines and guidance on the fertility options of transgender men. Furthermore, our study suggests a potentially beneficial role of experimental ST in overcoming poor embryo development related to cytoplasmic quality. STUDY FUNDING/COMPETING INTEREST(S): A.C. is a holder of FWO grants (1S80220N and 1S80222N). A.B. is a holder of an FWO grant (1298722N). B.H. and A.V.S. have been awarded with a special BOF (Bijzonder Onderzoeksfonds), GOA (Geconcerteerde onderzoeksacties) and 2018000504 (GOA030-18 BOF) funding. B.H. has additional grants from FWO-Vlaanderen (Flemish Fund for Scientific Research, G051516N and G1507816N) and Ghent University Special Research Fund (Bijzonder Onderzoeksfonds, BOF funding (BOF/STA/202109/005)), and has been receiving unrestricted educational funding from Ferring Pharmaceuticals (Aalst, Belgium). The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Dendrobine enhances bovine oocyte maturation and subsequent embryonic development and quality.

Strategies for improving the quality of oocytes have important theoretical and practical significance for increasing the efficiency of livestock breeding. In this respect, the accumulation of reactive oxygen species (ROS) is a major factor affecting the development of oocytes and embryos. This study investigated the effects of Dendrobium nobile extract (DNE) on the in vitro maturation of bovine oocytes and embryonic development after IVF. DNE is an extract from Dendrobium rhizomes that contains alkaloids with anti-inflammatory, anti-cancer and anti-ageing functions. Various concentrations of DNE (0, 5, 10, 20 and 50 µmol/L) were added during oocyte maturation in vitro, and we found that 10 µmol/L of DNE remarkably increased the oocyte maturation rate, the subsequent blastocyst formation rate and embryo quality. Further, we found that DNE treatment decreased the frequency of spindle/chromosome defects and ROS and increased the oocyte glutathione and mitochondrial membrane potential in oocytes. Moreover, DNE upregulated the expression of oxidative stress-related genes (Sirt1, Sirt2, Sirt3 and Sod1) in oocytes and apoptosis-related genes (Caspase-3, Caspase-4, Bax, Bcl-xl and Survivin) in blastocysts. These results suggest that DNE supplementation can promote oocyte maturation and subsequent embryonic development by regulating redox reactions and inhibiting embryonic apoptosis.

Therapeutic effects of L-arginine, L-carnitine, and mesenchymal stem cell-conditioned medium on endometriosis-induced oocyte poor quality in an experimental mouse model.

AIM: The present study aimed to explore the potential ameliorative effects of L-arginine (LA), L-carnitine (LC), and bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on endometriosis (EMS) model in vivo and in vitro. METHODS: The animals were divided into two main groups, normal and EMS-induced mice. Normal and EMS-induced groups were injected with or without LA (250 mg/kg), LC (250 mg/kg), and BMSC-CM (a final volume of 100 µL of CM/mouse). At the end of the study, the level of total antioxidant capacity (TAC), nitric oxide (NO), and total oxidative status (TOS) were measured in plasma. Furthermore, immature oocytes were collected from two groups and cultured in a maturation medium. Subsequently, the rates of in vitro maturation, in vitro fertilization (IVF), and in vitro embryonic development were evaluated. RESULTS: The results revealed that administration of LA, LC, and BMSC-CM ameliorated the oxidative status through maintaining TAC and alleviating TOS and NO levels. More importantly, the maturation and fertilization rates, blastocyst development, and total blastocyst cell numbers significantly increased in LA, LC, and BMSC-CM-administrated groups compared to the control group. In both the normal and EMS groups, the highest IVF, cleavage, and blastocyst percentages were associated with BMSC-CM treatment (p < 0.05). CONCLUSION: Altogether, LA, LC, and BMSC-CM have therapeutic effects on impaired oocyte quality and promote subsequent development in vitro, probably through normalization of nitro-oxidative stress, thus offering potential alternatives to conventional therapies during assisted reproductive technologies for patients with EMS-associated sub/infertility.

Antioxidants supplementation improves the quality of in vitro produced ovine embryos with amendments in key development gene expressions.

The present study assessed the effects of supplementation of different antioxidants on oocyte maturation, embryo production, reactive oxygen species (ROS) production and expression of key developmental genes. In this study, using ovine as an animal model, we tested the hypothesis that antioxidant supplementation enhanced the developmental competence of oocytes. Ovine oocytes aspirated from local abattoir-derived ovaries were subjected to IVM with different concentrations of antioxidants [(Melatonin, Ascorbic acid (Vit C), alpha-tocopherol (Vit E), Sodium selenite (SS)]. Oocytes matured without any antioxidant supplementation were used as controls. The oocytes were assessed for maturation rates and ROS levels. Further, embryo production rates in terms of cleavage, blastocysts and total cell numbers were evaluated after performing in vitro fertilization. Real-Time PCR analysis was used to evaluate the expression of stress related gene (SOD-1), growth related (GDF-9, BMP-15), and apoptosis-related genes (BCL-2 and BAX). We observed that maturation rates were significantly higher in alpha-tocopherol (100 µM; 92.4%) groups followed by melatonin (30 µM; 89.1%) group. However, blastocyst rates in ascorbic acid (100 µM; 19.5%), melatonin (30 µM; 18.4%), alpha-tocopherol (100 µM; 18.2%), and sodium selenite (20 µM; 16.9%) groups were significantly higher (P 0.05) than that observed in the control groups. Total cell numbers in blastocysts in the melatonin, ascorbic acid and alpha-tocopherol groups were significantly higher than those observed in sodium selenite and control groups. ROS production was reduced in groups treated with melatonin (30 µM), vitamin C (100 µM), sodium selenite (20 µM) and α-tocopherol (200 µM) compared with that observed in the control group. Supplementation of antioxidants caused the alterations in mRNA expression of growth, stress, and apoptosis related gene expression in matured oocytes. The results recommend that antioxidants alpha-tocopherol (200 µM), sodium selenite (40 µM), melatonin (30 µM) and ascorbic acid (100 µM) during IVM reduced the oxidative stress by decreasing ROS levels in oocytes, thus improving embryo quantity and quality.

Beneficial Effect of Polysaccharide Gel Made of Xanthan Gum and Locust Bean Gum on Bovine Oocytes.

The present study examined the effect of polysaccharides gels made of xanthan gum and locust bean gum (gel culture system) on oocyte maturation and explored the molecules causing the beneficial effect of the gel culture system. Oocytes and cumulus cells complexes were collected from slaughterhouse-derived ovaries and cultured on a plastic plate or gel. The gel culture system improved the rate of development to the blastocyst stage. The oocytes that matured on the gel contained high lipid contents and F-actin formation, and the resultant 8-cell stage embryos had low DNA methylation levels compared to their plate counterparts. RNA sequencing of the oocytes and embryos revealed the differentially expressed genes between the gel and plate culture systems, and upstream regulator analysis revealed estradiol and TGFB1 as top activated upstream molecules. The medium of the gel culture system contained higher concentrations of estradiol and TGFB1 than that of the plate cultures system. Supplementation of the maturation medium with either estradiol or TGFB1 resulted in high lipid content in oocytes. In addition, TGFB1 improved the developmental ability of the oocytes and increased F-actin content while reducing DNA methylation levels in the 8-cell stage embryos. In conclusion, the gel culture system is useful for embryo production, potentially through the upregulation of TGFB1.