Lower Maturation Rate Research Articles

Growth trajectory of Yiling sheep and its related genetic parameters.

Growth traits are one of the focuses of sheep breeding, and growth curve is an effective method to describe growth traits. The body weight of Yiling sheep at 0, 120, 180, 360 and 540days of age were fitted with five common nonlinear growth models: Logistic, Gompertz, Von Bertalanffy, Brody and Negative exponential, and the growth models were evaluated by goodness of fit standard. The results showed that the Von Bertalanffy model was suitable for characterizing the growth of Yiling sheep. The estimated values of parameters A(mature weight), B(scale parameter related to the initial body weight) and K(maturity rate) in Von Bertalanffy model were 82.88 ± 7.47, 0.64 ± 0.05 and 0.0048 ± 0.0010, respectively. The parameters of the growth curve of the Von Bertalanffy model were further utilized to estimate the inflection point body weight of Yiling sheep as 24.56kg, and the inflection point age was 135days old. The absolute growth rate(AGR) first increased and then decreased, reaching the maximum value of 0.1766kg/day at 135days of age. The relative growth rate (RGR) of Yiling sheep decreased with age. The mature weight of Yiling sheep was the closest to that of common meat breeds. The Least squares estimation showed that sex had a significant (p < 0.05) effect on the growth curve parameters of Yiling sheep, with rams having a greater mature weight than ewes, while ewes had a higher maturity rate. The heritability estimates of parameters A, B and K were 0.09, 0.04 and 0.01, respectively, and the negative correlation between A and K indicated that lower maturity rate could be selected to increase mature weight.

Effects of Chlorogenic Acid on In Vitro Maturation and Vitrification Cryopreservation of Sheep Oocytes.

Chlorogenic acid (CGA) has strong antioxidant properties. In order to improve the low maturation rate and poor vitrification freezing effect of sheep oocytes caused by oxidative stress. In this study, oocytes from 200 2-3-year-old Kazakh sheep were collected, and different concentrations of CGA were added to the maturation medium and vitrification freezing solution to study the effects of CGA on the maturation rate, cleavage rate, blastocyst rate, reactive oxygen species (ROS) and glutathione (GSH) levels, mitochondrial membrane potential, and the expression levels of oxidation and apoptosis-related genes in sheep oocytes. The results showed that adding 40 μmol/L CGA to the oocyte in vitro maturation solution significantly increased the maturation rate of oocytes, adding 50 μmol/L CGA to the vitrification cryopreservative solution significantly increased the cleavage and blastocyst rates of mature oocytes activated by parthenogenetic activation after freezing. During in vitro maturation and vitrification freezing in sheep oocytes, CGA significantly reduced the level of ROS and the expression of apoptosis-related genes (Caspase-3 and Bax/Bcl-2), and significantly increased the level of glutathione (GSH), mitochondrial membrane potential, and the expression of antioxidant and anti-apoptosis-related genes (SOD-2 and GPX-3). In addition, CGA significantly increased the expression of the anti-apoptotic gene (AKT) and anti-stress gene (FOXO) during vitrification freezing of sheep oocytes. In conclusion, 40 μmol/L CGA improves the maturation rate of sheep oocytes, and 50 μmol/L CGA improves the quality of parthenogenetic activation embryos after vitrification freezing of mature oocytes in sheep. These results provide a basis for the production of sheep in vitro embryos and the establishment of a germplasm resource bank.

Oocyte maturation defect in women undergoing IVF: contributing factors and effects on mature sibling oocyte outcomes.

This study aimed to identify demographic and clinical factors associated with low maturation rates and to investigate if the rate of immature oocytes impacts the outcomes of mature sibling oocytes. Women undergoing their first IVF-ICSI cycle between 2018 and 2022 at a fertility clinic were included. Cycles were classified into five groups according to the proportion of Metaphase II stage oocytes (MII): Null (0% MII, n = 46), Poor (1-25% MII, n = 44), Low (26-50% MII, n = 453), Acceptable (51-75% MII, n = 1641), and Optimal (76-100% MII, n = 2642). Demographic characteristics and clinical outcomes were compared between the five groups. In patients with a Null/Poor maturation rate, subsequent cycle outcomes were also evaluated. A total of 4826 cycles were included in the study; 69,909 oocytes were recovered, and 53,065 were MIIs (75.9%). The Null group was older, had lower levels of anti-Müllerian hormone (AMH), needed more gonadotropins and days of stimulation, had higher follicle stimulating hormone (FSH) levels on day 3, and had less follicles > 15mm on the day of trigger. When the outcomes of mature oocytes were compared, fertilization, usable blastocyst, aneuploidy, and life birth rates were comparable among groups. A binary logistic regression model using number of oocytes, paternal age, and trigger type with live birth rate endpoint found no differences between the categories and the base line Poor category. When patients whose maturation rate was Null/Poor, 42 (47.0%) carried out a second cycle; the maturation rate increased (56.9 ± 31.5 vs. 11.6 ± 11.2%, P < 0.0001). Our data suggest that poor responders are more likely to have low rates of oocyte maturation. The proportion of immature oocytes does not impact the outcomes of mature sibling oocytes. In patients with Null/Poor maturation in their first cycle, the subsequent cycle is often associated with improved maturation rates.

Proximal tubule cell maturation rate and function are controlled by PPARα signaling in kidney organoids

Human pluripotent stem cell-derived kidney organoids are expected to be a useful tool for new drug discoveries, however, the immaturation of kidney organoids causes difficulties in recapitulating renal pharmacokinetics using organoids. Here, we performed time-course single-cell RNA sequencing of kidney organoids and revealed cell heterogeneity in the maturation rate of the proximal tubule. An unbiased analysis to identify upstream targets of genes that are expressed differentially between cells with low and high maturation rates revealed a higher activation of PPARα signaling in rapidly maturing cells. Treatment with a combination of a PPARα agonist and an RXRα agonist induced genes related to proximal tubule maturation and increased the capacity for protein uptake as well as the sensitivity to nephrotoxicity by cisplatin. This method to promote the maturation rate of proximal tubule cells has the potential to be utilized in microphysiological systems to recapitulate proximal tubule functions and to screen nephrotoxic drugs.

P-609 Impact of low versus air oxygen tension on the oocyte maturation in PCOS women undergoing capacitation IVM: a sibling oocyte pilot study

Abstract Study question Does low or air oxygen tension improve the oocyte maturation rate in biphasic capacitation IVM (CAPA-IVM)? Summary answer Low oxygen tension resulted in a lower maturation rate and two-pronuclei fertilization rate compared with air oxygen tension in the CAPA-IVM culture. What is known already CAPA-IVM, with a pre-maturation step, improves the competence of oocytes matured in-vitro. The current CAPA-IVM culture condition is under air oxygen concentration. However, the impact of oxygen tension on in-vitro oocyte maturation needs to be better understood, and it remains uncertain whether the maturation process aligns with in-vivo conditions. Study design, size, duration A sibling oocyte pilot study of 416 cumulus-oocyte complexes (COCs) recruited from fifteen polycystic ovary syndrome (PCOS) women was performed at IVFMD, My Duc Phu Nhuan Hospital, Ho Chi Minh City, Vietnam from November 2023 to January 2024. The study was approved by the Institutional Ethics Committee (22/23/DD-BVMD, dated October 12th, 2023) and conducted according to Good Clinical Practice and Declaration of Helsinki 2002 principles. All participants provided written informed consent. Participants/materials, setting, methods Women (18–37 years) diagnosed with PCOS undergoing CAPA-IVM were recruited. At collection, COCs from each woman were divided into two groups based on the oxygen tension used in the two steps of CAPA-IVM culture, low oxygen tension (5% O2, 6% CO2 at 37 °C) and air oxygen tension (20% O2, 6% CO2 at 37 °C). Embryos were cultured to blastocyst stage. Primary outcome was the oocyte maturation rate. Secondary outcomes were embryology outcomes. Main results and the role of chance A total of 416 COCs from fifteen eligible women were assigned to the low oxygen (207 COCs) and air oxygen tension (209 COCs) groups. Mean age and body max index were 29.0±2.5 years and 22.6±2.6, respectively. The maturation rate was significantly lower in the low oxygen group compared to the air oxygen group (54.1% vs. 66.5%, p = 0.01, respectively). The two-pronuclei fertilization rate per COC was significantly decreased in the low oxygen group compared to the air oxygen group (35.7% vs. 49.3%, p = 0.005, respectively). Rates of day-3 embryos, blastocyst formation rate, and good quality blastocyst rate were also lower but not significant in the low oxygen tension group as compared to the air oxygen tension group. Limitations, reasons for caution The study’s limitations include a relatively small sample size and a single ethnicity group. There was a lack of data on clinical outcomes after transferring embryos from the study. Wider implications of the findings Oocyte maturation rate and two-pronuclei fertilization rate were significantly lower when oxygen tension was reduced in the process of CAPA-IVM culture. Therefore, air oxygen tension should be considered as a standard for oocyte culture in the pre-maturation step of CAPA-IVM. Trial registration number NCT06087354

P-795 Robust and simple in vitro maturation of ovarian tissue oocytes

Abstract Study question Using in-vitro gametogenesis results in mice, can we perform simple, robust in-vitro maturation (IVM) of oocytes from human ovarian tissue? Summary answer In vitro oogenesis in mice reveals 3 phases of oocyte maturation, IVD, IVG, and IVM. In vivo IVD and IVG simplifes a more robust IVM. What is known already For fertility preservation in cancer patients, IVM of GV’s obtained from ovary tissue with live birth has been achieved, but the results are variable, and great effort has gone into trying to make the “right” culture media, and most are given booster shots of gonadotropin with questionable results. It is easy to obtain many GV oocytes from tiny follicles using open cortical dissection. But it still has remained a puzzle how to do robust IVM in the best way. Study design, size, duration A total of 146 females underwent ovary tissue cryopreservation since 1977. The most recent 38 consecutive such patients between 2 and 38 years of age underwent in-vitro maturation of oocytes (IVM) derived from their ovarian tissue. This is a prospective non-randomized pilot study based upon our results with in vitro oogenesis. Participants/materials, setting, methods After ovarian cortex dissection, the spent media is examined for cumulous complexes (CC’s), which were placed in culture with widely varying concentrations of FSH and HCG, and in a variety of different media. Based on now established mechanisms of in vitro oogenesis, we postulated that the specific media for IVM and the concentrations of HCG would not impact results. There was no prior hormone administration. Main results and the role of chance The harvested germinal vesicle oocyte developed into mature metaphase II oocytes with simple culture media just as well as with “IVM media”, and with HCG concentration from 10 mIU/l to 1000 mIU/l. Of the 38 cases, 18 had had previous chemotherapy or were pre-pubertal. None of these had CC’s. Of the 20 who were post-pubertal and had not yet undergone chemotherapy, the number of CC’s averaged 20 per patient. Of a total of 402 GV oocytes, 137 became mature MII’s (34.1%), varying between 20% and 56%. The only case with low maturation rate (9%) occurred when the cumulus was stripped immediately instead of at 24 hours. Thus mature oocytes can robustly and easily be obtained from ovary tissue with simple media and no need for ovarian stimulation. Furthermore, ovarian stimulation and ovulation are not necessary for oocyte maturation. Oocytes that have just exited IVD that have been exposed to less than 7 days of gonadotropin (IVG) will not mature. Those oocytes exposed to more than 11 days of IVG will degenerate. Thus about 35% of oocytes at any given time will have already been properly prepared in vivo for IVM. Limitations, reasons for caution This is an early proof of concept study in humans with only 30 participants. However, it is completely consistent with what we have predicted from in vitro oogenesis in the mouse. Wider implications of the findings Ovary tissue freezing along with robust IVM will at once provide cancer patients with two possibilities for fertility preservation, without having to delay cancer treatment. Four months of in vivo IVD (non-hormonal) followed by 11 days of IVG (FSH) have already prepared oocytes for IVM. Trial registration number not applicable

P-185 Shortening the time from ovulation trigger to insemination is effective in reducing multiple pronuclei formation in anti-centromere antibody-positive patients

Abstract Study question Does shortening the time from ovulation trigger to insemination reduce multiple pronuclei formation (MPF) and improve the outcome after fertilization? Summary answer Shortening the time from ovulation trigger to insemination increased the normal fertilization rate and increased the number of cycles in which good embryos were obtained. What is known already When anti-centromere antibody (ACA)-positive patients undergo IVF, low oocyte maturation rates and MPF may be observed, which tend to occur when ACA antibody titers are high. In this case, the patient tends to present with poor embryonic development. There is currently no effective treatment for ACA-positive patients. It has been reported that MPF is caused by chromosome dispersion due to significant abnormal spindle deformation. In such cases, it has been reported that the oocyte spindle is often not visualized by polarized light microscopy. The oocyte is affected by ACA during the meiosis, but the mechanism is not well understood. Study design, size, duration A retrospective study was conducted on 764 mature oocytes from 174 cycles in 45 patients who underwent oocyte retrieval and inseminated by intra-cytoplasmic sperm injection (ICSI) with consent from 2009 to 2022. Patients were limited to those who tested positive for ACA by blood antinuclear antibody testing and whose oocytes occurred MPF. Participants/materials, setting, methods Oocyte retrieval was performed 36 hours after ovulation trigger. After ICSI, embryos were cultured until day 3 or blastocyst. The group was divided into two groups: the time reduced from ovulation trigger to ICSI (S group, n = 95) and the normal protocol (N group, n = 79). The normal fertilization rate, MPF rate, the rate of good embryo on day 3, the percentage of IVF cycles with good embryo, and clinical pregnancy rate per transfer were compared. Main results and the role of chance The mean time from maturation trigger to insemination was shorter in the S group than in the N group (38.2h vs. 41.8h, P &amp;lt; 0.01). The normal fertilization rate was significantly increased in the S group compared to the N group (49.9 vs. 40.7, P &amp;lt; 0.01) and the MPF rate was significantly decreased (39.1 vs. 28.3, P &amp;lt; 0.01). The rate of transferable embryo on day 3 tended to increase in the S group compared to the N group (70.6 vs. 61.9, P = 0.09). In addition, the percentage of IVF cycles with good embryo was significantly increased in the S group compared to the N group (57.4 vs. 28.4, P &amp;lt; 0.01). There was no difference in pregnancy rate per embryo transfer. There was no difference in the age of the wife at the time of oocyte retrieval. Limitations, reasons for caution The data were conducted from a single facility. and the limited number of cases, resulting in a small sample size. The process of visualizing the oocyte spindle was not performed in this study because it is time consuming and increases the time from ovulation trigger to insemination. Wider implications of the findings Shortening the time from maturation trigger to insemination was decreased MPF, suggesting that the oocyte spindle is affected by ACA in a time-dependent manner. It is recommended that ICSI be performed as soon as possible after oocyte retrieval. Trial registration number not applicable

O-071 Stringent statistical analysis of 108,684 oocyte donation cycles identifies 2.6% of donors with specific oocyte/embryo developmental arrest phenotypes for genome association studies

Abstract Study question Can statistical modelling accurately identify an outlier group of presumed fertile donors with specific reduced embryological outcomes suggestive of an underlying infertility-related genomic condition? Summary answer Statistical modelling identified a highly specific group of donors as embryologically under-performing outliers across multiple treatment cycles for the optimisation of downstream genomic association studies. What is known already Female infertility has a wide range of clinical presentations. While some causes of infertility are well defined, the aetiology of most infertility remains unexplained. Since IVF allows for in vitro monitoring of oocyte and embryo, it is now possible to identify subtle infertility endophenotypes, characterized by specific developmental failure that would otherwise go undetected in natural conception. Currently, the lack of large and well-categorized datasets of infertility endophenotypes has limited research in the area. Therefore, a clear and methodological definition of infertility endophenotypes in IVF is the first crucial step towards a better understanding of their mechanism and biological/genetic aetiology. Study design, size, duration A retrospective analysis was performed on clinical and embryological data from 40497 egg donors, undergoing 108.684 stimulations, with 4.235.036 oocytes retrieved. The data also included information on the 85482 recipients and their 190996 oocyte reception cycles, spanning from 1999 to 2023. Infertility endophenotypes were able to be well categorized as oocyte donors are presumed fertile based on their young age and on normal ranges displayed for the parameters assessed during typical oocyte donation work-up. Participants/materials, setting, methods Donor performance was calculated according to: (I) oocyte maturation rate (MR); (ii) survival rate of cryopreserved oocytes (SR); (iii) oocyte fertilization rate (FR;2PN/Injected MII); and (iii) blastulation rate (BR;blastocyst/2PN). Donors with significantly impaired rates, considering ≥2 stimulations and/or recipients, were identified as outliers using probabilistic modelling (Binomial test, Bonferroni corrected P &amp;lt; 0.05). Outliers were computed for each rate on data subsets after applying this stringent filtering criteria and excluding inconsistent or missing data. Main results and the role of chance 2.6% of donors were identified as underperforming outliers when summing the rate observed for each embryological outcome using the stringent statistical modelling. The average MR was 79.8% (95%CI: 79.6-80). Taking into account the correlation of follicle size during ultrasound tracking and the number of mature oocytes collected, a total of 168/29768 (0.56%) donors with statistically lower MR (mean MR = 43% 95%CI:40.1-45.7) across ≥2 egg collection cycles were identified as outliers. The average SR was 88% (95%CI:87.9-88.2). A total of 176/18544 (0.95%) donors with ≥2 stimulations and ≥2 recipient cycles were deemed outliers, showing a statistically reduced SR (mean SR = 51% 95%CI:48.9-53.1). The average FR was 74% (95%CI: 73.8-74.2). 164/31657 (0.52%) donors were found to have statistically low FR (mean FR = 37% 95%CI:34.6-38.7) and deemed outliers when assessed over ≥2 stimulation cycles with ≥2 recipient cycles. The average BR was 53.4% (95%CI: 53.1-53.7). After considering only donors with ≥2 recipients and ≥2 stimulations, a total of 142/24716 (0.57%) donors with statistically low BR (mean BR = 14% 95%CI:12.4-15.3) were considered outliers. Limitations, reasons for caution The stringent criteria applied to our statistical modelling has likely resulted in underestimating the true level of outlier donors. This is also likely compounded by the selection bias associated with donor recruitment and the retention policies after the first stimulation cycle preventing the evaluation of recurrent patterns in some cases. Wider implications of the findings The analytical tools developed on this unprecedentedly large dataset are fundamental to define a methodological workflow for accurately identifying unexpectedly infertile individuals. This is crucial for investigating the genetic and biological mechanisms underlying infertility endophenotypes, leading to improvements in treatment and developing screening tool to predict infertility and related pathologies. Trial registration number not applicable

Supplementation of SkQ1 Increases Mouse In Vitro Oocyte Maturation and Subsequent Embryonic Development by Reducing Oxidative Stress.

In vitro oocyte maturation (IVM) technology is important for assisted animal and human reproduction. However, the maturation rates and developmental potential of in vitro-matured oocytes are usually lower than those of in vivo-matured oocytes. Oxidative stress is a main factor that causes the lower maturation rates and quality of in vitro-matured oocytes. The purpose of this study was to investigate the effects of treatment with SkQ1, a mitochondria-targeted antioxidant, on mouse IVM and subsequent embryonic development. The results demonstrated that the supplementation of SkQ1 during IVM improves the maturation rates of mouse oocytes and the subsequent developmental competence of in vitro-fertilized embryos. The addition of SkQ1 to the IVM medium also decreased oxidative stress and apoptosis, and increased mitochondrial membrane potential in matured mouse oocytes. This study provides a new method through which to enhance the maturation rates and the quality of in vitro-matured mouse oocytes, thus promoting the application and development of assisted animal and human reproductive technology.

Cathepsin G promotes arteriovenous fistula maturation by positively regulating the MMP2/MMP9 pathway

Background Arteriovenous fistula (AVF) is currently the preferred vascular access for hemodialysis patients. However, the low maturation rate of AVF severely affects its use in patients. A more comprehensive understanding and study of the mechanisms of AVF maturation is urgently needed. Methods and results In this study, we downloaded the publicly available datasets (GSE119296 and GSE220796) from the Gene Expression Omnibus (GEO) and merged them for subsequent analysis. We screened 84 differentially expressed genes (DEGs) and performed the functional enrichment analysis. Next, we integrated the results obtained from the degree algorithm provided by the Cytohubba plug-in, Molecular complex detection (MCODE) plug-in, weighted gene correlation network analysis (WGCNA), and Least absolute shrinkage and selection operator (LASSO) logistic regression. This integration allowed us to identify CTSG as a hub gene associated with AVF maturation. Through the literature search and Pearson’s correlation analysis, the genes matrix metalloproteinase 2 (MMP2) and MMP9 were identified as potential downstream effectors of CTSG. We then collected three immature clinical AVF vein samples and three mature samples and validated the expression of CTSG using immunohistochemistry (IHC) and double-immunofluorescence staining. The IHC results demonstrated a significant decrease in CTSG expression levels in the immature AVF vein samples compared to the mature samples. The results of double-immunofluorescence staining revealed that CTSG was expressed in both the intima and media of AVF veins. Moreover, the expression of CTSG in vascular smooth muscle cells (VSMCs) was significantly higher in the mature samples compared to the immature samples. The results of Masson’s trichrome and collagen I IHC staining demonstrated a higher extent of collagen deposition in the media of immature AVF veins compared to the mature. By constructing an in vitro CTSG overexpression model in VSMCs, we found that CTSG upregulated the expression of MMP2 and MMP9 while downregulating the expression of collagen I and collagen III. Furthermore, CTSG was found to inhibit VSMC migration. Conclusions CTSG may promote AVF maturation by stimulating the secretion of MMP2 and MMP9 from VSMCs and reducing the extent of medial fibrosis in AVF veins by inhibiting the secretion of collagen I and collagen III.

Oblique versus Straight skin incision: The impact of incision type on postoperative outcomes in distal radiocephalic arteriovenous fistula creation

Aim: The type of skin incision utilized in arteriovenous (AV) fistula creation may significantly influence the patency, maturation, and overall outcomes of the fistulas. An oblique skin incision may offer distinct advantages in preventing early AV fistula failure by reducing skin tension and minimizing pressure on the newly formed anastomosis. This study aims to compare the postoperative early period outcomes of oblique versus straight skin incision techniques, with a focus on their impact on AV fistula success. Material and Methods: 2020 to 2023, all patients with created AV fistula via distal antebrachial vessels with oblique and straight skin incisions were enrolled. Retrospectively, the patients’ demographics, comorbidities were collated from the hospital data. All patients were operated with local anesthesia for radiocephalic AV fistula creation. The characteristics of the vessels were determined through preoperative Doppler ultrasonography mapping. Evaluations were conducted on primary patency, early maturation, and the incidence of early thrombosis. Results: In Total, 182 patients were operated for AV fistula creation. The mean age was 57±17 years and there was a male predominance with %68. We performed 74 patients AVF creation via straight skin incision and 108 via oblique skin incision. Despite there being no differences in comorbidities and demographic characteristics between the two groups, the group with a straight skin incision exhibited a higher incidence of early thrombosis (p=0.03), a lower maturation rate (p=0.03), and reduced primary patency (p=0.01). Conclusion: Oblique skin incision could be a feasible alternative to conventional straight incision with a higher early AVF patency and lower thrombosis rates. Reducing the pressure on and keeping the incision away from the new anastomosis may be crucial for achieving better outcomes.

Immune-Related Genes and Immune Cell Infiltration Characterize the Maturation Status of Arteriovenous Fistulas: An Integrative Bioinformatics Study and Experimental Validation Based on Transcriptome Sequencing.

Arteriovenous fistula (AVF) is the preferred vascular access for hemodialysis, but the low maturation rate is concerning. Immune cells' impact on AVF maturation lacks bioinformatics research. The study aims to investigate the potential predictive role of immune-related genes and immune cell infiltration characteristics in AVF maturation. We analyzed the high-throughput sequencing dataset to identify differentially expressed genes (DEGs). Then, we performed enrichment analyses (GO, KEGG, GSEA) on immune-related genes and pathways in mature AVF. We focused on differentially expressed immune-related genes (DEIRGs) and constructed a PPI network to identify hub genes. These hub genes were validated in other databases and experiments, including qPCR and immunohistochemistry (IHC). The immune cell infiltration characteristics in native veins, failed AVFs, and matured AVFs were analyzed by cibersortX. Partial experimental validation was conducted using clinical samples. Our results showed that immune-related genes and signaling pathways are significantly enriched in mature AVF. We validated this in other databases and ultimately identified three hub genes (IL1B, IL6, CXCR4) in combination with experiments. Significant differences in immune cell infiltration characteristics were observed among native veins, failed AVFs, and matured AVFs. Immune cell infiltration analysis revealed that accumulation of CD4+ T cells, dendritic cells, mast cells and M2 macrophages contribute to AVF maturation. These immune-related genes and immune cells have the potential to serve as predictive factors for AVF maturation. We partially validated this experimentally. From a bioinformatics perspective, our results have identified, for the first time, a set of immune-related genes and immune cell infiltration features that can characterize the maturation of AVF and significantly impact AVF maturation. These features hold potential as predictive indicators for AVF maturation outcomes.

Dual trigger for final oocyte maturation in expected normal responders with a high immature oocyte rate: a randomized controlled trial.

To evaluate whether dual trigger could improve reproductive outcomes in women with low oocyte maturation rates compare to human chorionic gonadotropin (hCG) trigger. This study included expected normal ovarian responders younger than 40 years old whose immature oocyte rate in the previous cycle was more than 50% at the reproductive center from July 2021 to November 2022. A total of 73 patients were enrolled at trigger, including 34 in the hCG trigger group and 39 in the dual trigger group (co-administration of gonadotrophin releasing hormone (GnRH) agonist and hCG, 40 and 34 h prior to oocyte retrieval, respectively). The primary outcome was oocyte maturation rate. There was no significant difference in the number of oocytes retrieved between the two study groups, but the oocyte maturation rate was higher in dual trigger group (84.0% [14.0%] vs. 55.5% [19.8%], p < 0.001). Moreover, there were also higher cumulative pregnancy rate (69.4% vs. 40.0%, p = 0.035) and cumulative live birth rate (66.7% vs. 36.0%, p = 0.022) in dual trigger group. For normal responders with low oocyte maturation rates, the dual trigger may be more effective than the conventional hCG trigger. ClinicalTrials.gov, identifier ChiCTR2100049292.

Melatonin Improves Turbot Oocyte Meiotic Maturation and Antioxidant Capacity, Inhibits Apoptosis-Related Genes mRNAs In Vitro.

High-quality eggs are essential for the sustainability of commercial aquaculture production. Melatonin is a potent candidate for regulating the growth and maturation of oocytes. Therefore, research on the effect of melatonin on marine fish oocytes in vitro has been conducted. The present study successfully established a culture system of turbot (Scophthalmus maximus) oocytes in vitro and investigated the effect of melatonin on oocyte meiotic maturation, antioxidant capacity, and the expression of apoptosis-related genes. The cultures showed that turbot Scophthalmus maximus late-vitellogenic denuded oocytes, with diameters of 0.5-0.7 mm, had a low spontaneous maturation rate and exhibited a sensitive response to 17α, 20β-dihydroxyprogesterone (DHP) treatment in vitro. Melatonin increased by four times the rate of oocyte germinal vesicle breakdown (GVBD) in a concentration- and time-dependent manner. The mRNA of melatonin receptor 1 (mtnr1) was significantly upregulated in the oocyte and follicle after treatment with melatonin (4.3 × 10-9 M) for 24 h in vitro, whereas melatonin receptor 2 (mtnr2) and melatonin receptor 3 (mtnr3) remained unchanged. In addition, melatonin significantly increased the activities of catalase, glutathione peroxidase, and superoxide dismutase, as well as the levels of glutathione, while decreasing the levels of malondialdehyde and reactive oxygen species (ROS) levels in turbot oocytes and follicles cultures in vitro. p53, caspase3, and bax mRNAs were significantly downregulated in oocytes and follicles, whereas bcl2 mRNAs were significantly upregulated. In conclusion, the use of turbot late-vitellogenesis oocytes (0.5-0.7 mm) is suitable for establishing a culture system in vitro. Melatonin promotes oocyte meiotic maturation and antioxidative capacity and inhibits apoptosis via the p53-bax-bcl2 and caspase-dependent pathways, which have important potential to improve the maturation and quality of oocytes.

P-156 A statistical model to pinpoint oocyte donors subject to IVF outcomes significantly poorer than expected: a tool for egg banks’ management and Research

Abstract Study question What is the prevalence of egg donors resulting in oocyte maturation/fertilization/blastulation rates significantly poorer than expected? Summary answer 6.7% of the egg donors (N = 34/504) elicited outcomes significantly poorer than expected mostly due to low oocyte maturation rates (N = 27/34, 79.4%). What is known already 5-10% of donor oocyte cycles produce no blastocyst involving challenging follow-up counseling. Recent research outlined a statistical model identifying women subject to IVF outcomes poorer than expected. These infertile women underwent whole-exome-sequencing (WES) to identify genes involved in oocyte-maturation-defects (OOMD) and/or preimplantation-embryo-lethality (PREMBL). Oocyte donors are the ideal population to apply this model, because they are young, theoretically-fertile, and undergo multiple retrievals of numerous oocytes. This model can find clinical application in improving egg banks’ management, social application in pinpointing young women potentially-infertile because of oocyte defects, and improving knowledge on the molecular pathways involved in human oocyte/embryo competence. Study design, size, duration Retrospective study involving oocyte donors recruited at three private IVF centers in 2020-2021. 504 donors (25.6 ± 4.7 years; range: 18-35) undergoing 1684 retrievals (N = 32056 cumulus-oocyte-complexes, 24288 metaphase-II) and whose metaphase-II were used (N = 6996 to date) in 487 fresh and 445 frozen ICSI cycles, respectively, were included. Recipient couples affected from severe-male-factor infertility were excluded. Blastocyst culture was systematically conducted. We leveraged clinical/embryological key-performance-indicators to pinpoint outliers subject to outcomes significantly poorer than expected. Participants/materials, setting, methods We quantitatively described donors’ performance as oocyte maturation, fertilization and blastulation rates. The outlier identification was based on the probabilistic modeling of the rates as independent binomial processes, whose baseline success probabilities were estimated from the whole dataset and accounted both for donor age and oocyte type (fresh/vitrified-warmed). The model assigns low probability to individuals with unusually large numbers of immature/unfertilized oocytes or arrested embryos. Donors with low probability (p &amp;lt; 0.001) were designated as outliers. Main results and the role of chance Oocyte donors underwent 3.3 ± 1.9 retrievals (1-8) collecting 63.6 ± 44.4 cumulus-oocyte-complexes (5-241), of which 48.2 ± 34.6 (3-162) metaphase-II (maturation-rate: 75 ± 13%,19-100). 13.9 ± 10.5 (3-75) metaphase-II were used during 1.8 ± 1.3 (1-9) cycles per donor, 1.0 ± 1.0 (0-6) cycles with 7.1 ± 7.6 (0-45) fresh metaphase-II oocytes and 0.9 ± 1.0 (0-7) cycles with 6.8 ± 8.1 (0-59) vitrified-warmed metaphase-II oocytes. Overall, 10.1 ± 8.0 (0-58) 2PN-zygotes were obtained per donor (fertilization-rate: 72 ± 16%,0-100), 5.1 ± 5.8 (0-34) in fresh (fertilization-rate: 72 ± 17%,0-100) and 5.0 ± 6.1 (0-41) in frozen cycles (fertilization-rate: 74 ± 16%,20-100). Overall, 5.5 ± 5.0 (0-31) blastocysts were obtained per donor (blastulation-rate: 55 ± 25%,0-100), 3.0 ± 3.8 (0-25) in fresh (blastulation-rate: 59 ± 25%,0-100) and 2.5 ± 3.6 (0-31) in frozen cycles (50 ± 24%,0-100). The model applied to this dataset designated 34 outliers (N = 34/504,6.7%, p &amp;lt; 0.001, FDR&amp;lt;0.1%). Most of them (N = 27) showed oocyte maturation-rates significantly lower than expected (51 ± 10%,19-66). These women were 26.9 ± 5.1 years old (19-35) and underwent 4.3 ± 1.7 retrievals, collecting 91.2 ± 45.0 cumulus-oocyte-complexes (25-210), of which 49.1 ± 31 (8-127) were metaphase-II oocytes. Three outliers showed fertilization-rates significantly poorer than expected (19 ± 17%,0-31). They were 21 ± 4.4 years old (18-26) and their metaphase-II oocytes (12.7 ± 4.9,7-16) were inseminated in 1.3 ± 0.6 donation cycles (1-2) resulting in 3 ± 2.6 (0-5) 2PN-zygotes. Four outliers showed blastulation-rates significantly poorer than expected (13 ± 7%,7-22SAME). They were 26.3 ± 6.2 years old (19-34) and their oocytes resulted in 28.5 ± 20.5 (14-58) 2PN-zygotes across 4.5 ± 3.1 (2-9) donation cycles that developed into 4.5 ± 5.7 (1-13) blastocysts. Limitations, reasons for caution Euploidy, another pivotal measure of oocyte competence, was not assessed. Larger studies are warranted, also accounting for putative additional confounders on all embryological outcomes under investigation. Nonetheless, gathering well-structured data on the cumulative performance of donor oocytes collected across multiple retrievals and through multiple IVF cycles is challenging and remarkable. Wider implications of the findings These outlier oocyte donors could benefit from follow-up investigation aimed at underpinning OOMD/PREMBL-related genetic factors. The development of genetic prediction tools to this end will improve egg donation cycles’ management and equity and might pave the way to preventive screening strategies and precision reproductive interventions in theoretically-fertile young women. Trial registration number not applicable

P-258 In-vitro oocyte maturation (IVM) in the ART clinic – What can we learn from our failed cycles?

Abstract Study question Why do some IVM cycles remain unsuccessful? Which factors contribute to failure in producing a usable embryo in IVM cycles? Summary answer Failure to obtain usable embryos after IVM was associated with oocyte dysfunction, inappropriate patient selection and unanticipated low oocyte yield. What is known already In-vitro maturation of oocytes retrieved from antral follicles has been offered as a mild-ART-approach to subfertile women with increased functional ovarian reserve, ovarian resistance to gonadotropins and in the context of fertility preservation. When using monophasic IVM culture systems, oocyte maturation, embryological development and pregnancy outcomes are generally lower compared to conventional ART (cART). Women who yield high numbers of cumulus oocyte complexes from a single egg retrieval procedure, such as those with polycystic ovary syndrome (PCOS), have been considered the best candidates for IVM, to compensate for the lower maturation rate and lower embryo yield from IVM cycles. Study design, size, duration This was a single-tertiary center, retrospective cohort study between May 2018 and February 2022 including 277 patients: 53 patients with failed IVM and 224 patients with a successful IVM cycle (inter-patient analysis). All ART cycles from patients with one failed IVM were investigated (intra-patient analysis). Failed IVM cycles were grouped: (i) ≤20% oocyte maturation; (ii) fertilization failure; (iii) embryo development failure. Patients with FSH resistance, fertility preservation or egg donation were excluded. Participants/materials, setting, methods Only patients with AMH &amp;gt;2.27 ng/ml were included. Oocytes retrieved in minimally stimulated non-hCG triggered IVM cycles were matured in a monophasic IVM system. Embryos were vitrified at cleavage stage and transferred in a hormonally substituted (HRT) cycle. Baseline patient characteristics, endocrinology and embryological data were analyzed. Intra-patient analysis was done using all ART cycles (failed IVM, IVM and cART) performed in one patient. Main results and the role of chance Inter-patient analysis revealed similar age, BMI and free testosterone, whereas AMH was lower in patients with a failed cycle (8.8 ± 3.5 vs 10.9 ± 6.4 ng/mL, p = 0.03). As expected, maturation, fertilization and useable embryos differed significantly in failed vs successful cycles: respectively 31% vs 48%; 40% vs 65%; 0% vs 50%; p &amp;lt; 0.0001. Out of 53 patients with a failed IVM cycle, 6 had oocyte maturation problems, 11 had failed fertilization and 36 had impaired embryological development. Intra-patient analysis revealed that in the failed oocyte maturation group, 2/6 patients had maturation problems that persisted in cART cycles, indicating oocyte dysfunction. Similarly, recurrent fertilization failure was noted in 3/11 patients, due to insufficient sperm quality (n = 2). In 8/36 patients, insufficient embryo quality was observed both after IVM and cART. Hence 13/53 (24.5%) failed IVM cycles were attributed to intrinsic patient characteristics. A subgroup of patients with a failed IVM cycle had normal maturation (3/6), fertilization (7/11) or good embryos (18/36) in other IVM or cART cycles. Factors associated with cycle failure included technical issues during egg retrieval resulting in unanticipated low oocyte yield (n = 14/40), and suboptimal inclusion criteria (no PCOS, n = 3/40). Limitations, reasons for caution This was a retrospective study and holds the possibility of unmeasured confounding factors. Wider implications of the findings Risk factors for IVM failure should be identified (e.g. non-PCOS, poor operator experience) and any underlying intrinsic patient conditions should be managed within a multi-disciplinary team. Optimal patient selection is key. Analysis of failed cycles helps clinicians to estimate the prognosis of future ART cycles in women with elevated AMH. Trial registration number not applicable

Brilliant Cresyl Blue Staining Enhances the Morphological Quality Evaluation of Porcine Oocytes-a Preliminary Study

Abstract Brilliant cresyl blue (BCB) staining is a method used for selection of developmentally competent porcine oocytes, in addition to the standard morphological classification. The aim of the current study was to assess the relationship between BCB staining in porcine oocytes with high and low morphological characteristics and its maturation rate. In the first part of the study, cumulus-oocytes complexes (COCs) (n=434) were aspirated from 60 ovaries. According to the morphological classification and BCB staining, they were divided in 4 groups: COC+/BCB+, COC−/BCB+, COC+/BCB−, and COC−/BCB−. In the second experiment, COCs (n=445) were categorized into 3 groups: control, BCB+, and BCB−. Significant differences in the maturation rate were observed between COC+/BCB+ and COC−/BCB− (66% vs. 23%), and between COC+/BCB+ and COC−/BCB+ (66% vs. 38%) (p&lt;0.0001). Significant differences were also registered between COC−/BCB+ and COC−/BCB-(38% vs. 23%) and between COC+/BCB− and COC−/BCB− (53% vs. 23%) (p&lt;0.01). Results from the second experiment showed that only BCB− oocytes had a significantly lower maturation rate (28%) compared to the control (63%) and BCB+ oocytes (59%) (p&lt;0.001). These findings showed that COCs with high morphological characteristics had higher developmental ability compared to COCs with low morphology. The BCB-staining in high-quality oocytes did not have a significant impact on their maturation ability compared to a control group, but it might be useful for selecting developmentally competent oocytes with low morphology characteristics.

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.

Effect of pentobarbital as a euthanasia agent on equine in vitro embryo production

Postmortem and pre-euthanasia oocyte retrieval provides the last opportunity to preserve the genetic material in mares. Pentobarbital (PB) is the most common euthanasia agent; however, its effect on the developmental competence of oocytes has not been determined. Here, we evaluated the concentration of PB in equine follicular fluid (FF) and investigated its effect on the developmental competence of oocytes using a bovine IVF model to overcome the low availability of equine oocytes. The concentration of PB was measured by gas-chromatography/mass-spectrometry in FF collected from mare ovaries immediately after euthanasia (n = 10), 24 h post-euthanasia (n = 10), and from the ovaries collected by ovariectomy (negative control; n = 10). The serum concentration of PB was also evaluated as a positive control. PB was detected in all FF samples with an average concentration of 56.5 μg/ml. Next, bovine cumulus-oocyte complexes (COC) were held in holding media with PB for 6 h at 60 μg/ml (H60, n = 196), 164 μg/ml (H164, n = 215) or without PB (control; n = 212). After holding, the oocytes were matured and fertilized in vitro, followed by in vitro culture to the blastocyst stage. The cumulus expansion grade, cleavage rate, blastocyst rate, embryo kinetic rate and the blastocyst cell numbers were compared among the experimental groups of bovine COC. Higher rates of Grade 1 cumulus expansion were found in controls (54%, 32–76%; median, min-max) in comparison to H60 and H164 (24%,11–33% and 13%, 8–44%; P < 0.001). The cleavage rate was higher in the controls than in H164 (64% vs. 44%; P < 0.01). Blastocyst rates (blastocyst/cleaved oocytes) and total cell number were not different among the groups (control 29%, H60 25%, and H164 24%). In a preliminary study, equine oocytes (n = 28) were exposed to PB in vitro for 6 h followed by intracytoplasmic sperm injection (ICSI) and in vitro embryo production. Exposed oocytes showed a numerically lower maturation rate (43% Vs 52%; P > 0.05) in comparison to the laboratory-established rate during the same timepoints. Overall, we showed that PB reaches the FF immediately after euthanasia, exposing oocytes to this drug. This exposure affected cumulus expansion and cleavage rates in a bovine model, suggesting initial damage caused by PB that may not completely impede the formation of embryos, although lower overall embryo numbers might be obtained.

Prediction of oocyte maturation rate in the GnRH antagonist flexible IVF protocol using a novel machine learning algorithm – A retrospective study

Oocyte maturation is affected by various patient and cycle parameters and has a key effect on treatment outcome. A prediction model for oocyte maturation rate formulated by using machine learning and neural network algorithms has not yet been described. A retrospective cohort study that included all women aged ≤ 38 years who underwent their first IVF treatment using a flexible GnRH antagonist protocol in a single tertiary hospital between 2010 and 2015. 462 patients met the inclusion criteria. Median maturation rate was approximately 80%. Baseline characteristics and treatment parameters of cycles with high oocyte maturation rate (≥80%, n = 236) were compared to cycles with low oocyte maturation rate (<80%, n = 226). We used an XGBoost algorithm that fits the training data using decision trees and rates factors according to their influence on the prediction. For the machine training phase, 80% of the cohort was randomly selected, while rest of the samples were used to evaluate our model’s accuracy. We demonstrated an accuracy rate of 75% in predicting high oocyte maturation rate in GnRH antagonist cycles. Our model showed an operating characteristic curve with AUC of 0.78 (95% CI 0.73–0.82). The most predictive parameters were peak estradiol level on trigger day, estradiol level on antagonist initiation day, average dose of gonadotropins per day and progesterone level on trigger day. A state-of-the-art machine learning algorithm presented promising ability to predict oocyte maturation rate in the first GnRH antagonist flexible protocol using simple parameters before final trigger for ovulation. A prospective study to evaluate this model is needed.