Vitrification of porcine immature oocytes at the germinal vesicle (GV) stage reduces subsequent embryo yield and changes at the molecular level may occur during embryonic development. Therefore, the present study used porcine parthenogenetic embryos as a model to investigate the effect of GV oocyte vitrification on the transcriptional profiles of the resultant embryos at the 4-cell and blastocyst stages using the Smart-seq2 RNA-seq technique. We identified 743 (420 up-regulated and 323 down-regulated) and 994 (554 up-regulated and 440 down-regulated) differentially expressed genes (DEGs) from 4-cell embryos and blastocysts derived from vitrified GV oocytes, respectively. Functional enrichment analysis of DEGs in 4-cell embryos showed that vitrification of GV oocytes influenced regulatory mechanisms related to transcription regulation, apoptotic process, metabolism and key pathways such as the MAPK signaling pathway. Moreover, DEGs in blastocysts produced from vitrified GV oocytes were enriched in critical biological functions including cell adhesion, cell migration, AMPK signaling pathway, GnRH signaling pathway and so on. In addition, the transcriptomic analysis and quantitative real-time PCR results were consistent. In summary, the present study revealed that the vitrification of porcine GV oocytes could alter gene expression patterns during subsequent embryonic developmental stages, potentially affecting their developmental competence.

The growth and development of early mammalian embryos mainly take place in the fallopian tube, which not only provides nutrients for embryonic growth and development but also offers suitable mechanical conditions. The embryo culture system established in assisted reproductive technology mainly simulates the environment in which oocytes and embryos grow and develop in vivo. However, current in vitro embryo culture is mainly static and cannot completely mimic the mechanical environment in which embryos grow and develop in vivo. Therefore, to more accurately simulate the mechanical environment of embryos in the fallopian tube, we have developed a dynamic culture device to investigate the effects of mechanical stimulation on the in vitro maturation of immature oocytes and their parthenogenetic developmental potential. Immature mice oocytes were subjected to in vitro maturation by static culture and vibration (3 Hz, 6 Hz) with tilting for 15∼16 hours. The maturation of oocytes was observed after the culture period. The mature oocytes were activated by parthenogenesis and the rate of embryo compaction and formation of parthenogenetic blastocysts was analyzed. The results showed that using 3 Hz vibration and tilting can significantly improve the parthenogenetic development potential of immature mice oocytes.

BACKGROUND: The influence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on assisted reproductive technology (ART) has received increasing attention. It has been reported that the SARS-CoV-2 RiboNucleic Acid (RNA) cannot be detected in follicular fluid and granulosa cells. However, the detection rate of SARS-CoV-2 RNA in immature oocytes and blastocysts has still unknown. Moreover, the effect of SARS-CoV-2 infection on embryological outcomes in ART during the Omicron epidemic is limited.
METHODS: A prospective study was performed to explore the detection rate of viral RNA in biological specimens from patients who tested positive for SARS-CoV-2 RNA and the effects of SARS-CoV-2 infection on embryological outcomes. A total of 211 patients underwent transvaginal oocyte retrieval at the Third Affiliated Hospital of Guangzhou Medical University between December 13, 2022 and December 30, 2022. Prior to transvaginal oocyte retrieval, 61 individuals tested positive for SARS-CoV-2 RNA within 24 h. Follicular fluid was preserved during oocyte retrieval. Granular cells were collected after degranulation (Intracytoplasmic sperm injection only). Immature oocytes were collected at the end of the ICSI. Unavailable blastocysts were collected on day 6 (D6). The TIANLONG SARS-CoV-2 RT-PCR-Kit was used to detect SARS-CoV-2 RNA in all samples. The COVID-19 and Non COVID-19 groups were contrasted in the following areas: fertilization rate, 2PN rate, Day 3 (D3) available embryos rate, D3 good-quality embryos rate, blastocyst formation rate, good-quality blastocyst formation rate.
RESULTS: All samples were negative except for an immature oocytes sample that was positive for SARS-CoV-2 viral RNA with a detection rate of 6.67%. Whether in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), the rate of fertilization, 2PN, D3 available embryos, D3 good-quality embryos, blastocyst formation, good-quality blastocyst formation was not significantly negative different between the COVID-19 and the Non COVID-19 groups. Our findings were validated by an overview of the embryological outcome from the cycles before SARS- Cov-2 infection from the same patient.
CONCLUSIONS: Except for immature oocytes, none of the follicular fluid, granulosa cells, or blastocysts samples contained viral RNA. In addition, SARS-CoV-2 infection had no detrimental effects on the embryological outcomes of ART.

Some studies have been conducted to explore the influence of growth hormone (GH) on oocytes in in vitro maturation (IVM); however, previous studies reporting showed different results, and the specific mechanisms were not clear. In the present study, GH supplementation improved oocyte maturation rate. The rate of germinal vesicle breakdown (GVBD) in the GH group was 83.9%, which was significantly higher than that (72.1%) in the control group (p = 0.001). The maturation rate of the GH group (79.2%) was significantly higher than that (65.4%) of the control group (p = 0.000). The fertilization (68.6 vs. 59.3%) and blastocyst (30 vs. 25.3%) rates showed an increasing trend in the GH group compared to those in controls. The dynamic parameters of nuclear maturation of oocytes were recorded by time-lapse monitoring system; oocytes in the GH group completed nuclear maturation earlier than did those in the control group. GH reduced cAMP levels to promote oocyte maturation. Single-cell RNA sequencing analysis revealed that the majority of differentially expressed genes (DEGs) involved in mitochondrial oxidative phosphorylation was upregulated in the GH group. Furthermore, the mitochondrial membrane potential of oocytes significantly increased, and the levels of intracellular reactive oxygen species (ROS) and Ca2+ largely decreased in the GH group. Finally, single-oocyte transcriptome analysis indicated that GH decreased the expression of apoptosis-related genes in oocytes. GH treatment reduced the expression of γH2AX and caspase-3. Therefore, GH improves the developmental potential of immature oocytes by reducing cAMP levels more rapidly within 0.5 h, protecting mitochondrial function, and reducing DNA damage and apoptosis.

This research aims to study the effects of continuous microvibration stimulation on the parthenogenetic development of human germinal vesicle oocytes.
Ninety-five discarded germinal vesicle oocytes from intracytoplasmic sperm injection treatment (ICSI) cycles performed at Amcare Women\'s & Children\'s Hospital between January and December 2021 were used for conventional static culture as well as 10 Hz microvibration culture. We investigated the differences between the two groups in terms of oocyte maturation rate, parthenogenetic activation rate, and parthenogenetic blastocyst formation rate.
The static culture and 10 Hz microvibration culture of 95 oocytes showed that the parthenogenetic blastocyst formation rate in the microvibration culture group was significantly higher than those in the traditional static culture group.
A continuous microvibration stimulation can significantly improve the parthenogenetic developmental potential of human immature oocytes.

As the immature oocytes are submitted to cryopreservation, their surrounding cumulus cells (CCs) will inevitably suffer, which may have some adverse effects on subsequent oocyte maturation and development. So far, little is known about the molecular differences in CCs of immature oocytes after vitrification. The aim of this study therefore was to analyze the protein profile of CCs derived from vitrified porcine immature oocytes following in vitro maturation, using TMT-based quantitative proteomic approach. A total of 5910 proteins were identified, and 88 of them presented significant difference, with 46 up-regulated and 42 down-regulated proteins. Gene Ontology enrichment analysis revealed that cell cycle phase transition, mitotic cell cycle phase transition, positive regulation of cell differentiation and regulation of oogenesis were significantly down-regulated within the biological process. After Kyoto Encyclopedia of Genes and Genomes pathway analysis, some up-regulated proteins were significantly enriched in TGF-beta signaling pathway and 4 pathways related to steroid hormones. Furthermore, 10 selected proteins were quantified and verified by a parallel reaction monitoring technique, indicating a high reliability of the TMT results. In conclusion, vitrification affects protein profile of CCs as well as their biological functions, which will offer a new perspective to understand the reasons for decline in maturation quality of vitrified immature oocytes.

OBJECTIVE: Previous reports have demonstrated that melatonin exists in multiple extrapineal sites, and higher amounts of melatonin are present in human follicular fluid than in serum, which indicates that it might play key roles in human oocyte maturation and subsequent embryonic development. Melatonin has been shown to be a potent antioxidant and might be beneficial to human oocytes during in vitro maturation (IVM). However, the underlying mechanisms of melatonin action during IVM have not been thoroughly investigated.
METHODS: Immunofluorescence staining, western blotting, and ELISA were applied to investigate whether melatoninergic components are expressed in the cultured human ovarian cumulus cells. TMRE staining and Fluo-4 AM staining were performed to detect the mitochondrial membrane potential and intracellular Ca2+ levels of immature human oocytes respectively.
RESULTS: First, cultured human ovary cumulus cells synthesized melatonin in vitro, and it expressed serotonin (the precursor of melatonin) and the two key enzymes, i.e. N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). Additionally, the results suggest that melatonin maintains the mitochondrial membrane potential and decrease excessive Ca2+ levels in immature human oocytes during IVM.
CONCLUSIONS: In conclusion, we provide evidence that the melatoninergic components were expressed in cultured human ovarian cumulus cells, and melatonin might reduce oxidative stress of human oocytes by ameliorating mitochondrial function. In view of the significant clinical value that immature human oocytes have in assisted reproductive technology (ART), our findings highlight a potential treatment strategy of using melatonin to improve mitochondrial function and to enhance the quality of human oocytes during IVM.

It is essential to enhance the in vitro maturation (IVM) condition for immature oocytes after cryopreservation, particularly if limited numbers of oocytes collected from specific donors. The objective of this study was to determine if quality of vitrified porcine immature oocytes was enhanced by coculturing with fresh oocytes during IVM. To distinguish fresh versus vitrified oocytes, we used two types of coculture systems: (a) transwell two-chamber coculture; (b) labeling and tracing fresh oocytes with CellTracker™ Green CMFDA during conventional culture. Coculture systems significantly accelerated meiotic progression of vitrified oocytes and significantly increased blastocyst formation rates following parthenogenetic activation and somatic cell nuclear transfer. Reactive oxygen species generation in vitrified oocytes was ameliorated by the coculture conditions, with no significant difference between fresh and vitrified oocytes for intracellular glutathione level. Both coculture systems significantly increased rate of normal mitochondrial distribution in vitrified oocytes, but did not affect fluorescence intensity of mitochondria. The percentage of oocytes with normal endoplasmic reticulum (ER) distribution and ER fluorescence intensity were significantly higher in vitrified oocytes cocultured with fresh oocytes. After 20 hr of IVM, mRNA expression of COX2, HAS2, PTX3, and TNFAIP6 remained significantly higher in cumulus cells derived from vitrified oocytes and coculture systems significantly decreased the expression of these genes. Additionally, coculture methods prevented the reduction of mRNA expression for BMP15, ZAR1, POU5F1, and DNMT3A in vitrified oocytes. In conclusion, oocyte quality and subsequent embryo development of vitrified porcine immature oocytes were significantly improved by fresh oocyte coculture during IVM.

The objectives of this study were to compare the effectiveness of liquid helium (LHe) and liquid nitrogen (LN2) as cryogenic liquid for vitrification of bovine immature oocytes with open-pulled straw (OPS) system and determine the optimal cryoprotectant concentration of LHe vitrification. Cumulus oocyte complexes were divided into three groups, namely, untreated group (control), LN2 vitrified with OPS group, and LHe vitrified with OPS group. Oocyte survival was assessed by morphology, nuclear maturation, and developmental capability. Results indicated that the rates of normal morphology, maturation, cleavage, and blastocyst (89.3%, 52.8%, 42.7%, and 10.1%, respectively) in the LHe-vitrified group were all higher than those (79.3%, 43.4%, 34.1%, and 4.7%) in the LN2-vitrified group (P < 0.05) although the corresponding rates in both treated groups decreased compared with the control group (100%, 75.0%, 64.9%, and 40.8%; P < 0.05). Normal calves were obtained after the transfer of blastocysts derived from LHe- and LN2-vitrified oocytes. The effects of the different vitrification solutions (EDS30, EDS35, EDS40, EDS45, and EDS50) in LHe vitrification for bovine immature oocytes vitrification were examined. No difference was found in the rates of morphologically normal oocytes among the EDS30 (87.9%), EDS35 (90.1%), EDS40 (89.4%), and EDS45 (87.2%) groups (P > 0.05). The maturation rate of the EDS35 group (65.0%) was higher than those of the EDS30 (51.3%), EDS40 (50.1%), EDS45 (52.1%), and EDS50 groups (36.9%; P < 0.05). No significant differences were observed in the cleavage and blastocyst rates between the EDS35 (49.0% and 12.1%) and EDS40 (41.7% and 10.2%) groups. However, the cleavage and blastocyst rates in the EDS35 group were higher (P < 0.05) than those of the EDS30 (36.2% and 6.8%), EDS45 (35.9% and 5.8%), and EDS50 (16.6% and 2.2%) groups. In conclusion, LHe can be used as a cryogenic liquid for vitrification of bovine immature oocytes, and it is more efficient than LN2-vitrified oocytes in terms of blastocyst production. EDS35 was the optimal cryoprotectant agent combination for LHe vitrification in this study.