Metaphase II Research Articles

Transcriptome analyses reveal transcriptional profiles of horse oocytes before and after in vitro maturation.

Oocyte in vitro maturation is necessary for the study and application of animal-assisted reproduction technology in animal reproduction and breeding. The comprehensive transcriptional profile of equine oocyte maturated in vitro has not been fully mined yet, which makes many key transcriptional events still unidentified. Here, Smart-seq2 was performed to analyse the gene expression pattern and the underlying regulatory mechanism of horse germinal vesicle (GV) and in vitro metaphase II (MII) oocytes. The results showed that 6402 genes (2640 up-regulated and 3762 down-regulated in MII samples compared to GV) and 4021 lncRNA transcripts (1210 up-regulated and 2811 down-regulated in MII samples compared to GV) were differentially expressed in GV and MII oocytes. Further, GO and KEGG analysis found that differentially expressed mRNAs and lncRNAs were mainly enriched in the pathways related to energy and lipid metabolism. In addition, LGALS3 was found a key gene in mediating the regulation of oocyte meiosis recovery and fertilization ability. This study provides novel knowledge about gene expression and energy metabolism during equine oocyte maturation and a reference for the further study and application of assisted reproductive technology in horse reproduction and breeding.

Extracellular Vesicle-Derived Cord Blood Plasma and Photobiomodulation Therapy Down-Regulated Caspase 3, LC3 and Beclin 1 Markers in the PCOS Oocyte: An In Vitro Study.

Introduction: Polycystic ovary syndrome (PCOS) is the communal endocrine illness in women and the most common cause of infertility due to lack of ovulation. The exact cause of PCOS is still unknown. Affected women may have difficulty getting pregnant due to ovulation problems. Various methods have not been effective in the treatment of PCOS due to the positive role of photobiomodulation therapy (PBMT) and extracellular vesicles (ECV) obtained from cord blood plasma in the treatment of various diseases. The aim of this study was to study the role of ECV and PBMT in maturation and improvement of infertility in women with PCOS. Methods: In this research, a number of oocytes were obtained after ovarian stimulation from women who had been referred to the hospital for infertility treatment after obtaining personal consent, and they were divided into three groups: control, ECV and PBMT. Subsequently, in vitro maturation (IVM) was assessed, then some oocytes were cultured with a routine medium and others were treated with ECV and PBMT. Real-time PCR was used to evaluate BCL-2, BAX, caspase-3, and autophagy gene (ATG5, LC3, Beclin 1). Oocyte glutathione (GSH), oxidised gluathione (GSSG), and reactive oxygen species (ROS) were measured. Results: The metaphase II (MII) oocyte ratio formation significantly increased in the ECV and PBMT groups (P<0.05). The expression of the BCL-2 gene was significantly up-regulated in the ECV and PBMT groups, but the expression of BAX and caspase-3 significantly decreased (P<0.05). The expression of the ATG5, LC3, BECLIN-2 genes significantly decreased in the ECV and PBMT groups (P<0.05). ROS, GSSG decreased in ECV and PBMT groups but GSH increased (P<0.05). Conclusion: The use of ECV and PBMT can increase the rate of fertilization and maturation of an oocyte and cause a decrease in apoptosis, autophagy, and ROS in a PCOS oocyte.

The Effect of The Conditioned Medium from Human Embryonic Stem Cells on Mouse Oocytes In Vitro Maturation.

Some reports have indicated that conditioned medium from growing mouse embryonic stem cells (ESCs) provides a supportive condition for small follicles growing, oocyte maturation, and following embryo growth. The aim of this study is assessing in vitro maturation (IVM) and consequent in vitro fertilization (IVF) outcome of immature mouse oocytes using human embryonic stem cells conditioned medium (HESCM). In this experimental study, 240 germinal vesicle (GV) oocytes were took from NMRI female mice, aged 4-6 weeks, 48 hours before injection of 5 IU pregnant mare serum gonadotropin (PMSG). 120 GV oocytes without cumulus cells were cultured in each of the groups. 120 GV were cultured in HESCM as test groups and also 120 GV cultured in human embryonic stem cells medium (HESM) as control groups. After evaluating the metaphase II (MII) oocyte maturation rate at 8, 16 and 24 hours, the MII oocytes subsequently were fertilized in vitro and the two-cell embryo development rate was recorded at days 1, 2, and 3. Statistical analysis was performed by using the generalized estimating equations (GEE) method that calculated their rate ratio. Our data indicated there are significant differences between the maturation rates in HESCM and HESM (P=0.004), also the two-cell embryo development was significant between two culture media (P=0.00). Similar to some other studies, the secretome of the HESCM showed a significant impact on the IVM outcomes in mice.

Predicting nuclear maturation speed of oocytes from Japanese Black beef heifers through non-invasive observations during IVM: An attempt using machine learning algorithms

Nuclear maturation is an essential process in which oocytes acquire the competence to develop further. However, the time required for nuclear maturation during IVM varies among oocytes. Therefore, predicting nuclear maturation speed (NMS) could help identify the optimal timing for IVF and maximize the developmental competence of each oocyte. This study aimed to establish machine learning-based prediction models for NMS using non-invasive indicators during the individual IVM of Japanese Black (JB) beef heifer oocytes. We collected ovaries from abattoirs and aspirated cumulus-oocyte complexes (COCs) from follicles with diameters ranging between 2 and 8 mm. The COCs were matured individually for 18 h, and photographs of each COC were taken at the beginning and every 3 h from 12 h to the end of maturation. After IVM culture, we denuded COCs and stained oocytes to confirm the progression of meiosis. Only oocytes that reached the metaphase II (MII) stage were considered to have a fast NMS. Morphological features, including COC area, cumulus expansion ratio, expansion rate per hour, and expansion pattern, were extracted from the recorded photos and applied to develop prediction models for NMS using machine learning algorithms. The MII rates of oocytes with fast- and slow-predicted NMS differed when the decision tree (DT) and random forest (RF) models were employed (P < 0.05). To evaluate the relationship between predicted NMS by DT and RF models and fertilization dynamics during individual IVF, sperm penetration and pronuclear formation were evaluated at 3, 6, 9, and 12 h after IVF start, following 24 h of IVM. The association between predicted NMS and embryo development was investigated by performing IVC for seven days using microwell culture dishes after 24 h of IVM and 6 h of IVF. Predicted NMS did not show a significant association with fertilization dynamics. However, oocytes with fast-predicted NMS by the RF model exhibited a tendency towards a higher cleavage rate 48 h after IVF start (P = 0.08); no other relationship was found between predicted NMS and embryo development. These findings demonstrate the feasibility of using non-invasive indicators during IVM to develop prediction models for NMS of JB beef heifer oocytes. Although the effect of predicted NMS on embryo development remains unclear, customized treatments based on NMS predictions have the potential to improve the efficiency of in vitro embryo production following individual IVM culture.

Effects of meiotic stage-specific oocyte vitrification on mouse oocyte quality and developmental competence.

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. 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. 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. 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.

Luteolin supplementation during porcine oocyte maturation improves the developmental competence of parthenogenetic activation and cloned embryos.

Luteolin (Lut), a polyphenolic compound that belongs to the flavone subclass of flavonoids, possesses anti-inflammatory, cytoprotective, and antioxidant activities. However, little is known regarding its role in mammalian oocyte maturation. This study examined the effect of Lut supplementation during in vitro maturation (IVM) on oocyte maturation and subsequent developmental competence after somatic cell nuclear transfer (SCNT) in pigs. Lut supplementation significantly increased the proportions of complete cumulus cell expansion and metaphase II (MII) oocytes, compared with control oocytes. After parthenogenetic activation or SCNT, the developmental competence of Lut-supplemented MII oocytes was significantly enhanced, as indicated by higher rates of cleavage, blastocyst formation, expanded or hatching blastocysts, and cell survival, as well as increased cell numbers. Lut-supplemented MII oocytes exhibited significantly lower levels of reactive oxygen species and higher levels of glutathione than control MII oocytes. Lut supplementation also activated lipid metabolism, assessed according to the levels of lipid droplets, fatty acids, and ATP. The active mitochondria content and mitochondrial membrane potential were significantly increased, whereas cytochrome c and cleaved caspase-3 levels were significantly decreased, by Lut supplementation. These results suggest that Lut supplementation during IVM improves porcine oocyte maturation through the reduction of oxidative stress and mitochondria-mediated apoptosis.

P-249 Exploring the possible impact of single-step culture media refreshment on embryo ploidy: A prospective sibling oocyte study

Abstract Study question Does refreshing the media on day 3 of development improve rates of blastocyst utilization and euploidy rate? Summary answer Refreshing single-step culture medium on day 3 of embryo development does not improve blastocyst euploidy rate, neither does it improve embryo utilization rate. What is known already The impact of culture conditions on blastocyst development and aneuploidy is not fully understood. Single-step culture media provide embryos with all the nutrients required throughout the development. The debated practice till today is whether refreshing media during the seven days of culture is required to compensate for possible, even subtle, osmolarity changes, and to replenish nutrients and exchange metabolites, alleviating embryos from a possible toxic environment. Up to date, no study has investigated the effect of using the same type of culture media, refreshing it or not, in a sibling oocyte manner, on blastocyst developmental quality and embryo ploidy. Study design, size, duration A prospective sibling oocyte study, conducted between July 2021 and December 2022 in a tertiary IVF center. A total of 1549 metaphase II (MII) oocytes from 93 PGT-A cycles were randomized between the 2 groups. Outcomes were defined as euploid rate, mosaicism rate and blastocyst utilization rate. Participants/materials, setting, methods Patients undergoing ICSI and PGT-A with females aged 18-40yrs and BMI&amp;lt;35kg/m2 who signed the informed consent. MII oocytes were randomized between Group 1(GI): embryos were moved into a newly calibrated culture media only on day 3, 67-69 hours post ICSI (hpi), and Group 2(GII): only on day 5, 114-118hpi. ICSI was performed by the same operator for both groups. Fertilization check was done 18-20hpi and no further assessment was done till day 5. Main results and the role of chance Patients and cycle characteristics presented as median (IQR) were female age 31yrs (28-34yrs), BMI 26.5 kg/m2 (23-28.6 kg/m2), AMH 3.4ng/ml (2.1-5.9ng/ml), cumulus-oocyte complex (COC) 21 (14-26) and oocyte maturation rate 0.8 (0.7-0.9). In GI, 775 MIIs and in GII, 774 MIIs were inseminated by ICSI. Fertilization rates were similar in both groups (77.2% vs 78.7%, mean difference: -1.5%, 95% CI: -5.9% to 2.9%, GI vs GII, respectively, P = 0.499). No significant differences were observed in day 5 blastulation rates between groups (66.3% vs 63.9%, mean difference: 2.4%, 95% CI: -3.7% to 8.5%, G1 vs G2, respectively, P = 0.436). Blastocyst utilization rate (blastocysts biopsied per 2PN) were similar between the groups (52.6% vs 50.7%, mean difference: 1.8%, 95% CI: -4.4% to 8.0%, G1 vs G2, respectively, P = 0.567). PGT-A outcomes were defined as euploid, aneuploid or mosaic aneuploid. Overall euploid rates were similar between GI and GII (57.86% vs 59.5%, p = 0.68, respectively), as well as mosaicism rates (7.7% vs 6.8%, p = 0.67, respectively). The rate of blastocysts biopsied on day 5,6 or 7 was comparable between both GI and GII (p = 0.62, 0.83 and 0.59, respectively). Limitations, reasons for caution While the outcome of this study did not show improvement in euploid rate or blastocyst utilization rate by refreshing the culture medium on day 3, the results may not be reproducible in other laboratories applying different protocols since culture conditions and protocols may differ among laboratories. Wider implications of the findings The fact that there are no significant differences in blastulation and euploid rates between the groups, supports the stability of culture conditions without the need for media refreshment and allows for reducing embryo manipulation, workload and costs adhered by culture medium refreshment on day 3. Trial registration number NCT04969575

O-184 Validation of a model capable of estimating the minimum number of mature oocytes needed to obtain at least one euploid blastocyst regarding female age

Abstract Study question Which is the minimum number of mature oocytes needed to obtain at least one euploid blastocyst regarding female age in In Vitro Fertilization (IVF) treatments? Summary answer The validated model estimates with a 74% accuracy the probability of having one euploid blastocyst regarding the number of mature oocytes and female age. What is known already Female age is significantly and directly related to embryo aneuploidy rates, thus lowering the chances of success in IVF treatments. The current delay in motherhood has led to a large proportion of women of advanced maternal age seeking infertility treatment. The aim of the present study is to determine the number of metaphase II (MII) oocytes needed to obtain at least one euploid blastocyst regarding female age. This information will help to decide the best strategy for each patient, taking also into account other variables such as ovarian reserve, semen quality and oocyte quality. Study design, size, duration Retrospective analysis of IVF cycles with pre-implantational genetic testing for aneuploidies (PGT-A) performed over the last 5 years in an infertility clinic in Spain, from January 2017 to March 2022. Participants/materials, setting, methods Patients undergoing an IVF cycle in an infertility clinic with own or donated oocytes, regardless semen origin. Only trophoectoderm biopsies performed on day 5 or 6 of development and analyzed using Next Generation Sequencing (NGS) were included. PGT-A was used for reasons such as advanced maternal age, implantation failure and recurrent miscarriage. PGT-A cycles due to a known abnormal karyotype were excluded. Endpoints were analized using binary logistic regression models. Main results and the role of chance A total of 3840 IVF-PGT-A cycles meeting the inclusion criteria were performed in the study period. Of them, 939 cycles were discarded due to the absence of any biopsied/analyzed embryo (final sample size=2901). A model for the probability of having at least one euploid blastocyst (pEB) regarding female age and the number of MII oocytes retrieved was created with 80% of the sample (n = 2320) and validated in the remaining 20% (n = 581). The validation of this model showed that it was capable of estimate with an accuracy of 73.88%. The pEB was directly related to the number of MII oocytes retrieved (odds ratio (OR) 1.130, confidence interval (IC) 95% (1.110-1.150); p &amp;lt; 0.001), but indirectly related to female age (OR 0.751, IC95% (0.725-0.778); p &amp;lt; 0.001). The ROC curve showed a significant predictive value of the number of MII oocytes (area under the curve (AUC):0.8041 (0.7882-0.82)) for this model. A mathematical formula was created for the calculation of pEB using this model. The number of MII oocytes needed for a pEB of 75% regarding female age (female age:number MII) was 35:6, 36:9, 37:12, 38:15, 39:18, 40:21, 41:24, 42:27, 43:30, 44:32, 45:35, 46:38, 47:41, 48:44, 49:47, 50:50. Limitations, reasons for caution The main limitation of this study is its retrospective design. In addition, it’s important to keep in mind that many other variables should be taken into consideration along with maternal age in the assessment of the number of oocytes needed to obtain at least one euploid blastocyst in IVF treatments. Wider implications of the findings Results from this study may constitute a useful tool for both clinical and patient. The clinician may be able to better decide the best strategy for each patient, while the patient will understand more easily this information, helping her morally with the treatment. Trial registration number 2204-VLC-040-CR

O-159 Impact of open and closed methods of vitrification on oocyte mitochondria function and RNAseq transcriptome profile: a preclinical screening study

Abstract Study question Do current methods of open vitrification (OV) and closed vitrification (CV) impact on oocyte mitochondria function and transcriptome? Summary answer Oocyte vitrification confers unique mitochondria distribution patterns and alters the expression of stress response genes and mitochondria function relative to control (fresh) oocytes. What is known already Mitochondria are essential for maintenance of metabolic function and developmental competence at physiological conditions. Conversely, open and closed methods of oocyte vitrification are associated with physical, chemical and non-physiological changes, and exposures to cryoprotecting agents which could be harmful at extremely high and low temperatures. Study design, size, duration Ninety-four unfertilised Metaphase II (MII) oocytes were donated to research between May 2018 and August 2021. Fifty-seven oocytes were randomly allocated to the different study groups. Similarly, thirty-seven oocytes were allocated to the different study control groups. Participants/materials, setting, methods Fourteen women underdoing IVF/ICSI treatment cycles were involved in the study. cDNA was obtained for qPCR and the Smart-Seq2 protocol and single-cell RNA-Seq was performed (illumina NextSeq2000). Mitochondrial staining on live-oocytes was performed using Mito-tracker Deep Red stain, and images evaluated using fluorescence microscopy. Differentially expressed genes were identified using the R package edgeR and functional gene ontology was performed using an over-representation analysis via WebGestalt. Correction for multiple testing was calculated using Benjamini-Hochberg method Main results and the role of chance Mean mitochondrial intensity/density showed similarities between open (OV) and closed vitrification (CV) and control (fresh oocytes FO): 25.8 (12.7-44.9, standard deviation [SD] 10.2) for FO oocytes (n = 15), 30.3 (16.2-44.8, SD 10.1) for OV oocytes (n = 11), 26.6 (13.3-64.6, SD 15.3) for CV oocytes (n = 12) (p = 0.617). Distinct mitochondria distribution patterns (cytoplasmic and peripheral) were associated with OV and CV oocytes. Twice as many peripheral to cytoplasmic patterns were observed in FO and OV oocytes in contrast to CV oocytes (p = 037). Mean expression of the gene SDHB, which is associated with the TCA cycle and the mitochondrial electron transport chain was reduced in vitrified oocytes (n = 13) (0.0879 ± 0.0216 vs 0.0593 ± 0.0479), as was the anti-apoptotic gene BCL2 (p = 0.0061), relative to control oocytes (n = 13), (p = 0.01). Conversely, genes associated with apoptosis and stress response (BAX (p = 0.002) and BCL2L1 (p = 0.016)) were significantly enriched relative to control oocytes. RNAseq transcriptome analysis revealed a total of 71 differentially expressed genes following vitrification relative to control oocytes. Genes associated with critical metabolic processes (death domain binding, BCL-2 homology 3 (BH3) domains, and exopolyphosphatatse activities) were enriched in vitrified oocytes relative to the control group (FDR&amp;gt;0.05). Limitations, reasons for caution The use of unfertilised metaphase II (MII) oocytes on day 1 as a preclinical application screening model for vitrification may not reflect the molecular status and integrity of clinical grade MII oocytes on day 0. Wider implications of the findings This study emphasizes the inherent impact of vitrification on oocyte mitochondria function and transcriptome relative to control oocytes. Importantly, unintended perturbations may be detrimental to oocyte health and developmental competence, suggesting the need for a continuous safety screening and monitoring of ART methods of cryopreservation and offspring long-term health. Trial registration number National Institute for Health Research (NIHR). Grant Reference: ICA-CDRF-2015-01-068

O-105 Chromatin accessibility of oocytes contributes to PCOS transgenerational inheritance

Abstract Study question What is the underlying mechanism contributing to the transgenerational defects of oocytes and embryos of polycystic ovary syndrome (PCOS)? Summary answer The transgenerational inheritance of abnormal chromatin accessibility in the PCOS oocytes is responsible for the defect of oocytes and embryos in transgeneration. What is known already Studies have provided evidence of compromised oocyte quality and related adverse impacts. Observed PCOS animal-model oocyte defects include meiotic abnormalities, mitochondrial dysfunction, and increased oxidative stress. Elevated androgen levels are associated with reduced fertilization rates and lower cleavage rates during in vitro fertilization (IVF) treatment. PCOS could be transgenerationally transmitted, and unique altered gene expression has been observed in metaphase II (MII) oocytes from all offspring in PCOS mice. Study design, size, duration We profiled the transcriptome and chromatin accessibility of oocytes and preimplantation embryos in F0-F2 offspring of PCOS mice, to explored whether the inheritance of chromatin accessibility defects in PCOS oocytes was responsible for the abnormalities of oocytes and embryos in PCOS offspring. Besides, oocytes from 13 healthy women and 8 women with PCOS, 24 blood samples from 24 women with/without PCOS were analyzed to validate the molecular mechanisms of PCOS inheritance. Duration: 2019.5∼2022.12. Participants/materials, setting, methods PCOS mice models were established by means of DHEA-exposure. Smart-seq2 and DNase-seq were utilized to investigate the transcriptome and chromatin accessibility of oocytes and preimplantation embryos in F0-F2 offspring of PCOS mice. Bioinformatic analysis was applied to explore the differential gene expression and chromatin accessibility of oocytes and embryos in PCOS mice and offspring comparing to controls. Common gene signatures in the tissues of PCOS women and PCOS daughters are analyzed compared to control samples. Main results and the role of chance In PCOS mice, we observed transcriptional alteration in oocytes and 2-cell embryos compared to normal mice. There are 4229 and 1197 differentially expressed genes (DEGs) in PCOS oocytes and 2-cell embryos. The DEGs includes both maternal factors and zygotic genome activation (ZGA) genes. Interestingly, in PCOS 2-cell embryos, we observed hundreds of pre-activated genes which are silence in normal 2-cell embryo but expressed in PCOS 2-cell embryos. Notably, many DEGs in the oocytes of PCOS mice were also differentially expressed in the oocytes of F2 PCOS mice compared to control mice, supporting the transgenerational inheritance of transcriptional alteration in PCOS oocytes. Mechanismly, the changes of chromatin accessibility were associated with the DEGs in PCOS oocytes and embryos. In particular, chromatin accessibility of the pre-activated gene promoters is elevated in PCOS oocytes compared to that in normal oocytes. Furthermore, many of the abnormal chromatin accessibility signals detected in F0 PCOS oocytes were also observed in F2 PCOS oocytes. Finally, our data showed that some DEGs in the oocytes of PCOS mice were also differentially expressed in human PCOS oocytes compared to the control oocytes. Common genes identified in PCOS offspring may be used as potential predictors of PCOS phenotype. Limitations, reasons for caution Which factors contribute to the abnormalities of chromatin accessibility in PCOS oocytes are still unknown. Wider implications of the findings The candidate genes may be used as potential predictors of PCOS phenotype and provide the clues for PCOS offspring . Trial registration number not applicable

O-244 Mitochondrial fission factor (MFF) is required for female fertility and oocyte development

Abstract Study question To explore the role of Mitochondrial fission factor (MFF) in female fertility and oocyte development using a Mff knockout mouse model. Summary answer Mff deficiency influences mitochondrial dynamics in oocytes at germinal vesicle stage (GV), and results in premature ovarian failure (POF) and female infertility. What is known already Mff regulates mitochondrial quality control (MQC) by influencing mitochondrial dynamics. MQC has critical effects on oocyte development and POF. However, little is known about the role of Mff in oocyte development and female fertility. Study design, size, duration The Mff knockout (Mff-/-) mice were generated via the CRISPR-Cas9 system. Adult Mff-/- female mice were compared to wildtype (WT) female mice. Female mice at 2-month old from each group (Mff-/- or WT, n = 5) were used to assess the fecundity for 10 months. The follicle development (n = 4 mice), the number of GV oocytes (n = 5 mice) and metaphase II (MII) oocytes (n = 5 mice) were determined. Participants/materials, setting, methods Female mice from each group were mated with adult WT males to assess the fecundity. Follicle development was evaluated in ovaries after fixation, paraffin embedding, and sectioning, followed by hematoxylin and eosin staining. MFF and DRP1 protein expression was evaluated by western blot. Oocyte mitochondria were stained with the Mito-Tracker Red CMXRos. Main results and the role of chance The Mff deficient ovaries had significantly lower levels of MFF (0.056 vs. 1, p &amp;lt; 0.01) than the WT ovaries. Mff-/- female mice were infertile (0 vs. 39, p &amp;lt; 0.05). Mature (8-week-old) Mff deficient ovaries had significantly lower weight (0.0043 vs. 0.011, p &amp;lt; 0.05) compared to the WT. The 2-month-old female Mff-/- mice had a lower number of primordial (220 vs. 775, p&amp;lt; 0.05), primary (128 vs. 580, p&amp;lt; 0.05), secondary (29 vs. 82, p&amp;lt; 0.05) and antral follicles (5 vs. 23, p&amp;lt; 0.05). The numbers of GV oocytes (18 vs. 37, p&amp;lt; 0.05) and MII oocytes (2 vs. 16, p&amp;lt; 0.001) were lower in the Mff-/- mice than the WT mice. In addition, the Mff-/- ovaries expressed significantly less mitochondrial fission key regulator DRP1 (0.54 vs. 1, p&amp;lt; 0.05). Mff-/- oocytes contained elongated and aggregated mitochondria compared to the discrete and evenly distributed mitochondria in the ooplasm of WT oocytes. Herein, we found that the Mff played a concerted role in oocyte and follicle development. Limitations, reasons for caution The present observations in mice may not be applicable to humans. The results are derived global knockout of Mff. It is not known which kind of cell plays a vital role in fertility. Further investigation is needed to explore the regulatory mechanism of Mff in oocyte development and fertility regulation. Wider implications of the findings This study provides important insights into the relationship between mitochondrial dynamics and female fertility. Trial registration number not applicable

P-217 Artificial intelligence (AI)-supported MAGENTA oocyte assessments shown to prospectively correlate with utilizable blastocyst development in patients, and for the first time in oocyte donors

Abstract Study question Can MAGENTA, an AI oocyte assessment tool, prospectively assess the quality of oocytes retrieved from patients and donors as it relates to reproductive outcomes? Summary answer MAGENTA AI oocyte assessments provide insights on the quality of mature oocytes retrieved from both patients and young donors that correlate with utilizable blastocyst development. What is known already Oocyte donation has increasingly become a treatment path for many patients struggling with infertility. With specific criteria, oocyte donation aims to provide higher quality oocytes to increase the chances of success for recipients. However, not all oocytes from even a young donor will be of the same quality or have the same chances of reproductive success. MAGENTA is a non-invasive oocyte AI image analysis tool that provides an assessment of oocyte quality through a score on a scale of 0-10. MAGENTA scores in patient oocytes have previously been shown to correlate with subsequent development of a blastocyst, and its quality. Study design, size, duration A prospective study was conducted from April-November 2022 by Equipo Médico Crespo (Valencia, Spain) utilizing MAGENTA to assess the oocyte quality of patient and donor oocytes by assigning a score on a scale of 0-10. MAGENTA scores of denuded metaphase II (MII) oocytes were assessed for correlation to reproductive outcomes (fertilization and blastocyst development). 1,313 fresh MII oocytes retrieved from 126 subjects (68 patients, 59 donors) between the ages of 19-47 were included for analysis. Participants/materials, setting, methods Prior to ICSI, a non-invasive light microscopic image was taken of denuded MII oocytes utilizing specialized image capture software. Images were uploaded and analyzed by MAGENTA to provide a score, which remained blinded for the duration of the study. Reproductive outcomes regarding each mature oocyte were collected over the fertility cycle. Blastocyst development was defined by a Gardner grade by Day 5/6 post-ICSI. A utilizable blastocyst was considered a Gardner grade of 2CC or greater. Main results and the role of chance The following comparisons were assessed by Welch’s Two Sample t-tests. Overall, successfully fertilized oocytes had higher mean MAGENTA scores (4.5) than those that did not (3.9) (p &amp;lt; 0.01); furthermore, those oocytes that successfully developed into a blastocyst had higher mean MAGENTA scores (4.8) than those that did not (4.0) (p &amp;lt; 0.01). Patient oocytes were found significantly older (mean age: 38.5) compared to donated oocytes (mean age: 25) (p &amp;lt; 0.01). Patient oocytes that successfully developed into a blastocyst had a higher mean MAGENTA score (4.7) than those that did not (4.0) (p &amp;lt; 0.01); similarly, donated oocytes that successfully developed into a blastocyst had a higher mean MAGENTA score (4.9) than those that did not (4.1) (p &amp;lt; 0.01). The MAGENTA scale was divided into 4 groups: A: 0-2.5 (346 oocytes); B: 2.6-5 (454 oocytes); C: 5.1-7.5 (358 oocytes); D: 7.6-10 (155 oocytes), over the whole dataset. There was an overall increasing proportion of oocytes that developed into not only blastocysts, but more impressively, utilizable blastocysts from the lowest to highest MAGENTA score groups with significant differences between the patient oocytes scored in group B(38%) and C(50%) (p &amp;lt; 0.05), and the donated oocytes scored in group A(39%) and B(52%), and group C(54%) and D(66%) (p &amp;lt; 0.05) by Two Proportions Z-tests. Limitations, reasons for caution This study was conducted in fresh oocyte ICSI cycles. The use of frozen patient and donor oocytes should be assessed by VIOLET prior to vitrification, which takes into consideration freezing and thawing statistics. Further data is required to determine any correlation of MAGENTA with embryo ploidy or implantation status. Wider implications of the findings MAGENTA’s a valuable tool for oocyte quality assessments correlating with utilizable blastocyst development amongst both autologous and donated oocytes. Although young donors are assumed to have high quality oocytes, this is not always true. MAGENTA assessments for donor oocytes could be essential to provide insights when these cycles unexpectedly fail. Trial registration number not applicable

P-640 No impact of Progestin Primed Ovarian Stimulation (PPOS) on euploid blastocyst rate per cohort of metaphase-II oocytes during PGT-A cycles: a case-control study

Abstract Study question Can progestins be used to prevent spontaneous LH surge during ovarian stimulation (OS) without affecting the euploid blastocyst rate (EBR) per metaphase-II (MII) oocytes? Summary answer Progestin Primed Ovarian Stimulation (PPOS) (conducted with norethisterone acetate) and conventional-OS with GnRH-antagonist show similar EBR per MII-oocytes What is known already PPOS is a novel OS protocol based exogenous progesterone to inhibit LH surge and spontaneous ovulation as an alternative to GnRH analogues. The rationale of its use is that multiple follicular waves arise during the ovarian cycle and no spontaneous ovulation occurs during the luteal phase. PPOS effectiveness and safety have been supported lately in the literature. A recent study reported similar number of euploid blastocysts after PPOS (conducted with medroxyprogesterone acetate) versus conventional-OS with GnRH-antagonist. Study design, size, duration Case-control study involving advanced-maternal-age women undergoing ICSI with PGT-A at two private IVF centers (May-2017 to February-2020). 80 PPOS women were matched with 160 control based on maternal-age and ovarian reserve markers. Patients with poor-ovarian-reserve (AFC&amp;lt;3), premature-ovarian-failure, III-IV stage endometriosis, ovarian cyst, severe-male-factor, PGT-SR or PGT-M were excluded. EBR per MII-oocytes was the primary outcome. All other embryological and clinical outcomes were reported. Participants/materials, setting, methods Both groups underwent recombinant-FSH OS with GnRH-agonist ovulation trigger; the only difference was that norethisterone acetate 10mg/day was administered orally to PPOS patients starting from the second day of the menstrual cycle until trigger. The control group, instead, underwent conventional antagonist protocol. The laboratory procedures were similar in both groups: ICSI, blastocyst culture, trophectoderm biopsy, comprehensive-chromosome-testing to report non-mosaic aneuploidies and vitrified-warmed euploid single-embryo-transfers (SETs). Main results and the role of chance The mean fertilization rate per MII-oocytes and blastulation rate per 2PN-zygotes were similar among PPOS and control (75.5%±21.8% versus 71.7±20.7%, p = 0.1; and 56.5%±27.7% versus 53.2%±27.5%, p = 0.4, respectively). No difference was reported for the EBR per MII-oocytes among PPOS and control (15.4%±19.8% versus 14.8%±18.2%, p = 0.9). No difference was reported among the biopsied blastocysts in terms of morphological quality and day of development. A total of 47 and 86 SETs were conducted in the two groups. There was no difference in the live-birth-rates (LBR) per SET in the two groups (N = 19/47, 40.4%, 95%CI 26.7-55.7 versus N = 35/86, 40.7%, 95%CI 30.4-51.8, p = 0.9), nor in the miscarriage rate per clinical pregnancy (N = 1/20, 5%, 95%CI 0.3-26.9 versus N = 3/36, 8.3%, 95%CI 2.2-25.6, p = 0.9). At last, the cumulative-LBR among concluded cycles (i.e., LB achieved or no transferable/all transferred euploid blastocysts) was also similar (N = 19/68, 27.9%, 95%CI 18.1-40.3 versus N = 31/132, 22.0%, 95%CI 15.4-30.2, p = 0.4). Limitations, reasons for caution The main limitation is the retrospective and non-randomized design. More data are required, especially for follicle recruitment, oocyte yield, gestational and perinatal outcomes. Wider implications of the findings Norethisterone acetate-based PPOS protocol has no impact on oocyte competence when compared to conventional GnRH-antagonist OS. These promising data support further investigation on PPOS, especially in view of its reduced cost, and putative increase in patient compliance and decrease in discomfort. Trial registration number none

O-182 The clinical value of rescued MI-oocytes. Retrospective analysis of 625 MI-oocytes versus 2124 sibling MII-oocytes obtained in 285 fresh donor ICSI cycles

Abstract Study question What are the developmental competence and clinical value in ICSI cycles of retrieved metaphase-I oocytes matured in vitro (rescued-MI) to the metaphase-II (MII) stage? Summary answer Rescued MI-oocytes showed lower developmental but similar post-implantation competence than their sibling MII-oocytes, thereby contributing to a + 9.5% relative-increase in the cumulative-live-birth rate per completed-cycle. What is known already MI-oocytes are typically excluded from ICSI. Nevertheless, some authors reported their use in cancer or poor responder patients. On average, 15-20% of the oocytes retrieved in IVF are immature, but 45% of them may mature within a few hours of culture. These rescued-oocytes showed lower developmental, chromosomal and reproductive competence. Regardless, they might be valuable for some patients. To date, most of the studies were conducted including patients using own oocytes, thus implicitly involving the bias of infertility and poor prognosis. Here we aimed at outlining the clinical value of rescued-MI oocytes in the context of egg donation cycles. Study design, size, duration Observational study conducted at a private IVF center including 284 fresh oocyte donation cycles (Jan-2020 to Dec-2021). Two hours after oocyte-retrieval, all MI-oocytes identified after denudation were cultured for 1-2 additional hour(s). If reaching the MII-stage, they were inseminated via ICSI with ejaculated sperm, as for sibling MII-oocytes. Single culture in continuous media was conducted. MI- and MII-derived embryonic cohorts were monitored and compared. One-hundred-fifty-three cycles in the same period had no MI-oocyte available. Participants/materials, setting, methods Two-hundred-eighty-four cycles were conducted from 272 recipients (42.1±4.0 years) with fresh oocytes retrieved from 215 donors (25.5±5.0 years). All sperm samples were ejaculated (42.3% fresh; 15% oligoasthenoteratozoospermic). The number of MI- and MII-oocytes after denudation was 2.2±1.3 (range:1-7) and 7.5±1.6 per cycle, respectively. 1.7±1.2 (0-6) MI-oocytes matured in vitro (rescue-rate: 78.1±33.4%) and were inseminated. Embryological data were compared between sibling MI- and MII-derived cohorts. Clinical data and relative contributions of MI-oocytes were also reported. Main results and the role of chance The mean fertilization rate per cohort of inseminated MI- and MII-oocytes were 54.3±43.0% and 74.4±18.1%, respectively (paired t-test&amp;lt;0.01). The mean blastulation rate per cohort of MI- and MII-derived zygotes were 53.1±44.4% and 59.1±25.9%, respectively (paired t-test=0.1). Overall, 0.5±0.7 (0-4) MI-derived blastocysts were obtained adding up to the 3.2±1.7 MII-derived ones, thereby resulting in a + 20.8±40.1% (0 to + 300%) relative increase per cycle. Overall, 268 (94%), 247 (89.7%), and 188 (66.2%) cycles had ≥1, ≥2 and ≥3 MII-derived blastocyst(s), respectively. These rates showed a + 1.1% (N = +3), +4.9% (N = +12) and +7.4% (N = +24) relative increase due to MI-derived blastocysts use. To date, 19 out of 63 MI-derived blastocysts transferred resulted in a live-birth (30.2%, 95%CI 19.6-43.2) versus 124 out of 399 MII-derived ones (31.1%, 95%CI 26.6-35.9; p = 0.99). We reported 4 miscarriages out of 27 clinical pregnancies (14.8%, 95%CI 4.8-34.6) versus 38 out 162 (23.5%, 95%CI 17.3-30.9, p = 0.45), respectively. To date, 70% (N = 199/284) of the cycles was concluded, with a cumulative-live-birth-rate of 61.3% (N = 122/199). This rate showed a + 9.5% (N = +18) relative increase due to MI-derived blastocysts use. Among 85 non-completed cycles, 19 (22.4%) have MI-derived blastocysts still available. Limitations, reasons for caution Retrospective study. A cost-effectiveness analysis is warranted, especially because MI-oocytes clinical use involves additional workload and costs. 31% (N = 44/140) of patients with ≥1 live-birth have supernumerary cryopreserved MI-derived blastocysts. Many cycles are not concluded. Gestational/perinatal outcomes were not reported. More data with own eggs and/or including chromosomal testing are needed. Wider implications of the findings Rescuing MI-oocytes might increase the number of blastocysts available per cycle, and possibly the cumulative-live-birth-rate. Nevertheless, this practice involves a higher workload and the production of more supernumerary blastocysts. Mostly poor prognosis couples might benefit from their use; therefore a couple-specific decision-making process is desirable. Trial registration number n/a

PARP12 regulates mouse oocyte meiotic maturation.

Poly(ADP-ribosyl)ation (PARylation) is an important post-translational modification of proteins that involves the transfer of ADP-ribose moieties, and plays important roles in many biological processes including DNA repair, gene expression, RNA processing, ribosome biogenesis, and protein translation. Though it is accepted that PARylation is crucial for oocyte maturation, little is known about how Mono(ADP-ribosyl)ation (MARylation) regulates this process. Here, we report that Parp12,a mon(ADP-ribosyl) transferase of poly(ADP-ribosyl) polymerase (PARP) family, was highly expressed at all stages of oocytes during meiotic maturation. At germinal vesicle (GV) stage, PARP12 was mainly distributed in cytoplasm. Interestingly, PARP12 formed granular aggregation near to spindle poles during metaphase I (MI) and metaphase II (MII). PARP12 depletion results in abnormal spindle organization and chromosome misalignment in mouse oocytes. Chromosome aneuploidy frequency in PARP12 knockdown oocytes was significantly increased. Importantly, PARP12 knockdown triggers activation of spindle assembly checkpoint as shown by active BUBR1 in PARP12-KD MI oocytes. Besides, F actin was significantly attenuated in PARP12-KD MI oocytes which may affect the asymmetric division process. Transcriptomic analysis demonstrated that PARP12 depletion disrupts transcriptome homeostasis. Collectively, our results showed that the maternally expressed mono(ADPribosyl) transferases PARP12 was essential for oocyte meiotic maturation in mouse.

Hexavalent Chromium Disrupts Oocyte Development in Rats by Elevating Oxidative Stress, DNA Double-Strand Breaks, Microtubule Disruption, and Aberrant Segregation of Chromosomes.

Environmental and occupational exposure to hexavalent chromium, Cr(VI), causes female reproductive failures and infertility. Cr(VI) is used in more than 50 industries and is a group A carcinogen, mutagenic and teratogenic, and a male and female reproductive toxicant. Our previous findings indicate that Cr(VI) causes follicular atresia, trophoblast cell apoptosis, and mitochondrial dysfunction in metaphase II (MII) oocytes. However, the integrated molecular mechanism of Cr(VI)-induced oocyte defects is not understood. The current study investigates the mechanism of Cr(VI) in causing meiotic disruption of MII oocytes, leading to oocyte incompetence in superovulated rats. Postnatal day (PND) 22 rats were treated with potassium dichromate (1 and 5 ppm) in drinking water from PND 22-29 and superovulated. MII oocytes were analyzed by immunofluorescence, and images were captured by confocal microscopy and quantified by Image-Pro Plus software, Version 10.0.5. Our data showed that Cr(VI) increased microtubule misalignment (~9 fold), led to missegregation of chromosomes and bulged and folded actin caps, increased oxidative DNA (~3 fold) and protein (~9-12 fold) damage, and increased DNA double-strand breaks (~5-10 fold) and DNA repair protein RAD51 (~3-6 fold). Cr(VI) also induced incomplete cytokinesis and delayed polar body extrusion. Our study indicates that exposure to environmentally relevant doses of Cr(VI) caused severe DNA damage, distorted oocyte cytoskeletal proteins, and caused oxidative DNA and protein damage, resulting in developmental arrest in MII oocytes.

Specific processing of meiosis-related transcript is linked to final maturation in human oocytes.

Human meiosis in oocytes entails an intricate regulation of the transcriptome to support late oocyte growth and early embryo development, both crucial to reproductive success. Currently, little is known about the co- and post-transcriptional mRNA processing mechanisms regulating the last meiotic phases, which contribute to transcriptome complexity and influence translation rates. We analyzed gene expression changes, splicing and pre-mRNA processing in an RNA sequencing set of 40 human oocytes at different meiotic maturation stages, matured both in vivo and in vitro. We found abundant untranslated region (UTR) processing, mostly at the 3' end, of meiosis-related genes between the germinal vesicle (GV) and metaphase II (MII) stages, supported by the differential expression of spliceosome and pre-mRNA processing related genes. Importantly, we found very few differences among GV oocytes across several durations of IVM, as long as they did not reach MII, suggesting an association of RNA processing and successful meiosis transit. Changes in protein isoforms are minor, although specific and consistent for genes involved in chromosome organization and spindle assembly. In conclusion, we reveal a dynamic transcript remodeling during human female meiosis, and show how pre-mRNA processing, specifically 3'UTR shortening, drives a selective translational regulation of transcripts necessary to reach final meiotic maturation.

Expression pattern and subcellular localization of p62/SQSTM 1 during mouse oocyte maturation

Objective: Autophagy is a survival mechanism, and it is initiated by several factors such as oxidative stress. Cells give autophagy response against oxidative stress through Nrf2-Keap1-p62 pathway. p62 or Sequestosome 1 (SQSTM 1) which takes place during autophagy, and it is showed that p62 plays critical role during primordial follicle formation. During oocyte maturation, Germinal Vesicle (GV) oocytes resume meiosis with the beginning of puberty and progress through Metaphase I and Metaphase II stages. Metaphase II (MII) oocytes are ready to be fertilized and until fertilization they are arrested at metaphase II stages. It is known that oxidative stress is increasing during oocyte maturation. However, it is not shown that spatial and temporal expression of p62 throughout oocyte maturation. Thus, the aim of the study was to reveal expression pattern and the subcellular localization of the p62 protein in mouse GV, MI and MII oocytes. Methods: GV oocytes were received from female Balb/C mice at 4 weeks aged and GV oocytes were cultured for 8h to develop into MI oocytes and for 14h to mature into MII oocytes. Then, expression of p62 protein was observed in oocytes at GV, MI and MII maturation stages by using immunofluorescence method. Results: Our results showed that there is a significant increasing p62 expression when GV oocytes are compared to MI and MII oocytes, but there is no difference between MI and MII oocytes. Moreover, we revealed that p62 is localized in cytoplasm of GV oocytes, while it is localized around chromosomes in MI and MII oocytes. Conclusion: In conclusion, our results indicate that further study is mandatory to understand the participation of p62 during meiosis, and the impact of p62 during oocyte maturation.

The developmental potential of mature oocytes derived from rescue in vitro maturation

To examine the developmental competence of immature oocytes in stimulated cycles, that matured after rescue invitro maturation (IVM) compared with their sibling invivo matured oocytes. Retrospective cohort study. IVF clinic. A total of 182 patients underwent 200 controlled ovarian stimulation cycles with intracytoplasmic sperm injection cycles in which immature oocytes were retrieved and at least one mature oocyte was obtained through rescue IVM. Invitro culture of immature germinal vesicle (GV) and metaphase I (MI) oocytes, retrieved in stimulated cycles. Fertilization rate, cleavage rate, blastulation rate, ploidy of embryos evaluated using preimplantation genetic testing for aneuploidy, morphokinetic parameters and pregnancy outcomes. In total, 2,288 oocytes were retrieved from 200 cycles. After denudation, 1,056 of the oocytes (46% ± 16%) were classified as metaphase II (MII). A total of 333/375 (89%) of MI oocytes and 292/540 (54%) of GV oocytes matured overnight and underwent intracytoplasmic sperm injection. The fertilization rates of matured oocytes from MI rescue IVM (R-MI) and from GV rescue IVM (R-GV) were comparable with those of their sibling MII oocytes (71% vs. 66%; 66% vs. 63%, respectively). Early cleavage rates (80% ± 35% vs. 92% ± 20%; 80% ± 42% vs. 95% ± 28%, respectively) and blastulation rates (32 ± 40% vs. 62 ± 33%; 24 ± 37% vs. 60 ± 35%, respectively) were significantly decreased in rescue IVM matured oocytes (R-oocytes)-derived zygotes, but the blastocyst (BL) euploidy rate and "good quality" BL rate were comparable with those of MII sibling-derived embryos. In addition, rescue IVM embryos showed significantly higher levels of multinucleation at the 2- and 4-cell stages, as well as higher rates of zygote direct cleavage from one to 3 to 4 cells. Overall, 21 transfers of rescue IVM embryos resulted in 3 healthy live births. For patients with a low maturation rate and/or low numbers of mature oocytes at retrieval, rescue IVM may contribute more competent oocytes and additional viable BLs for transfer from the same stimulation cycle, maximizing the chances for pregnancy and live birth.

Physiological intracytoplasmic sperm injection does not improve the quality of embryos: A cross-sectional investigation on sibling oocytes.

This study aimed to compare the efficacy of physiological intracytoplasmic sperm injection (PICSI) and intracytoplasmic sperm injection (ICSI) in terms of the fertilization rate and embryo quality using sibling oocyte cycles. This prospective, cross-sectional study collected data from 76 couples who underwent their first cycle at the Hue Center for Reproductive Endocrinology and Infertility, Vietnam, between May 2019 and November 2021. The inclusion criteria were cycles with at least eight oocytes and a sperm concentration of 5×106/mL. Sperm parameters, sperm DNA fragmentation (SDF), fertilization, and the quality of cleavage-stage embryos on day 2 and blastocysts on day 5 were examined. From 76 ICSI cycles, 1,196 metaphase II (MII) oocytes were retrieved, half of which were randomly allocated to either the PICSI (n=592) or ICSI (n=604) treatment group. The results showed no significant difference between the two groups in terms of fertilization (72.80% vs. 75.33%, p=0.32), day 2 cleavage rate (95.13% vs. 96.04%, p=0.51), blastulation rate (52.68% vs. 57.89%), and high-quality blastocyst rate (26.10% vs. 31.13%, p=0.13). However, in cases where SDF was low, 59 cycles consisting of 913 MII oocytes produced a considerably higher blastulation rate with PICSI than with ICSI (50.49% vs. 35.65%, p=0.00). There were no significant differences between the pregnancy outcomes of the PICSI and ICSI embryo groups following embryo transfer. Using variable sperm quality provided no benefit for PICSI versus ICSI in terms of embryo outcomes. When SDF is low, PICSI appears to be able to produce more blastocysts.