Aged Oocytes Research Articles

O-158 Prediction of implantation of vitrified-warmed blastocysts using a deep learning algorithm on a single post-warming image of each embryo

Abstract Study question Can a deep learning algorithm determine the implantation likelihood of vitrified blastocysts from single time-point post-warming time-lapse images obtained from EmbryoScope? Summary answer A significant positive correlation was found between the score given by the deep learning algorithm and the implantation rate of the frozen blastocysts analyzed. What is known already Despite the safety and success of the vitrification technique, many of the best morphologically classified embryos prior to vitrification do not maintain their characteristics when returned to room temperature. Since artificial intelligence (AI) has been shown to be useful in assessing the quality of fresh embryos at different stages, it could also be used to predict the outcome of frozen embryo transfers. This is the first attempt to distinguish vitrified blastocysts that will implant from those that will not by applying deep learning on a single image taken in the period between warming and transfer. Study design, size, duration This retrospective single-centre study included 689 blastocysts with known implantation data. All of them were vitrified and warmed using the Cryotop method (Kitazato, Biopharma, Japan). Warmed embryos were assessed by experienced embryologists according to the ASEBIR morphological scoring system and the degree of expansion. Immediately after warming, blastocysts were placed in EmbryoScope (Vitrolife, Denmark) time-lapse incubators for 2-5 hours to let them re-expand until embryo transfer. Participants/materials, setting, methods A pre-transfer embryo image was provided to the algorithm (Life Whisperer Viability), which returned scores from 0 to 10. Data were analyzed by chi-square test or one-way ANOVA. Multivariate logistic regression analysis was performed including embryo score, oocyte origin (donated vs. autologous), oocyte age, patient age, oocyte handling (fresh vs. vitrified eggs), and day of vitrification (5 vs. 6). The area under the receiver operating characteristic (ROC) curve (AUC) was used to calculate evaluation performance. Main results and the role of chance There was a significant difference between the scores given to vitrified implanted and non-implanted embryos. The mean value was 4.98 [95% CI 4.70-5.26]* for non-implanted embryos and 6.16 [95% CI 5.84-6.48]* for implanted embryos. When dividing the embryo score by quartiles, the implantation rate for each quartile was 27.7% for Q1, 33.7% for Q2 (Odds ratio (OR)=1.31 [0.82–2.09]), 44.8% for Q3 (OR = 2.05 [1.30–3.22]*) and 48.3% for Q4 (OR = 2.46 [1.56–3.87]*). The contribution of each increased unit of the algorithm score to the implantation (OR = 1.16 [95% CI: 1.09–1.23]*) was statistically significant. The deep learning algorithm score successfully predicted implantation with an AUC of 0.65 [95% CI: 0.60–0.69]*. Moreover, the algorithm performed better than the ASEBIR morphological classification, which achieved an AUC of 0,62 [95% CI: 0.58–0.66] in predicting implantation. *P < 0.001. Limitations, reasons for caution Our clinic was not involved in the development of the algorithm. Furthermore, it was designed to assess fresh embryos prior to vitrification but not thawed ones, so this study should be considered an external trial. A post-warming full video analysis by AI could possibly provide additional information. Wider implications of the findings The use of predictive models on vitrified cycles may help to select the best frozen embryo for transfer and predict which embryos will result in implantation failure early enough to thaw another one with a better chance of success. It also indicates that AI can generalize to thawed embryo assessment. Trial registration number -

P-198 Evolution of artificial intelligence-based embryo selection models: a massive external validation on 70,456 embryos

Abstract Study question How do three artificial intelligence-based embryo selection models that have evolved over the years work? Summary answer The three automatic scores were positively associated with clinical outcomes, and the iDAScore v2 showed the best performance in conventional treatments with patient oocytes. What is known already Artificial intelligence (AI) models have been introduced in in vitro fertilization laboratories in recent years as an adjunct to clinical decision-making. The most initial function of the AI involved guiding embryologists in the annotations of embryonic events. This was followed by the development of embryo selection models based on machine learning, requiring annotations (i.e., KIDScore). Finally, the latest in this field are models based on deep learning, which analyze raw time-lapse video (i.e., iDAScore). In this study, the evolution of three AI-based models have been analyzed on the same large set of embryos. Study design, size, duration This single-center study includes 6,737 patients who underwent in vitro fertilization treatments for 6 consecutive years. A total of 7,722 cycles were analyzed, resulting in 70,456 embryos cultured in EmbryoScope® time-lapse systems. Embryos were routinely evaluated and selected according to conventional morphology (ASEBIR criteria) by senior embryologists. Retrospectively, the embryos were scored by three AI models from 1 to 9,9: KIDScore D5 v3 (n = 32,784), iDAScore v1 (n = 68,440) and iDAScore v2 (n = 68,471). Participants/materials, setting, methods Automatic embryo scores were compared with conventional morphology, ploidy, and clinical outcomes for single blastocyst transfers. Then, we performed multivariate logistic regression analysis (confounding factors: oocyte origin, donated-autologous; type of embryo transfer, fresh-frozen; oocyte age; patient body mass index; culture strategy, individual-group; day of embryo transfer, fifth-sixth day of embryo development) in different patient populations (PGT-A, oocyte donation program and conventional treatments with patient oocytes). Finally, the performance (AUC) was calculated for comparison. Main results and the role of chance The mean of the three scores increased as embryos had better morphological grade*. Regarding ploidy (euploid vs. aneuploid): 5,62 ± 1,78 vs. 5,00 ± 1,72 for KIDScore* (n = 6,580); 7.59 ± 1.61 vs. 6.92 ± 1.75 for iDAScorev1* (n = 7,089); and 6.31 ± 2.53 vs. 5.07 ± 2.55 for iDAScorev2* (n = 7,082). Regarding implantation (implanted vs. non-implanted): 6.24 ± 2.01 vs. 5.42 ± 2 for KIDScore* (n = 9,681); 8.36 ± 1.37 vs. 7.76 ± 1.70 for iDAScorev1* (n = 10,079); and 6.83 ± 2.18 vs. 5.75 ± 2.54 for iDAScorev2* (n = 10,068). Regarding live birth (positive vs. negative): 6.29 ± 2.01 vs. 5.51 ± 2.01 for KIDScore* (n = 9,668); 8,39 ± 1,34 vs.7,83 ± 1,68 for iDAScorev1* (n = 10,065); and 6.88 ± 2.15 vs. 5.87 ± 2.52 for iDAScorev2* (n = 10,054). In general, the multivariate analysis showed statistically significant odds ratio for the three models in predicting implantation and live birth (all patient subpopulations)*. Regarding oocyte donation program: the AUCs for predicting implantation were 0.636 [0.621−0.650] for KIDScore, 0.638 [0.624−0.652] for iDAScorev1, and 0.636 [0.622−0.650] for iDAScorev2; and the AUCs for live birth prediction were 0.630 [0.615−0.644] for KIDScore, 0.629 [0.615−0.643] for iDAScorev1, and 0.635 [0.621−0.649] for iDAScorev2. Regarding treatments with patient oocytes: the AUCs for predicting implantation were 0.667 [0.644−0.689] for KIDScore, 0.674 [0.652−0.696] for iDAScorev1, and 0.686 [0.664−0.708] for iDAScorev2; and the AUCs for live birth prediction were 0.664 [0.642−0.687] for KIDScore, 0.669 [0.647−0.692] for iDAScorev1, and 0.686 [0.664−0.708] for iDAScorev2. *p<0.001 Limitations, reasons for caution A major limitation of our study is its retrospective nature. Although ours is the largest external validation ever performed with an unselected ICSI population, its single-center design should be considered for the universal application of the models. Also, specific culture conditions should be addressed when considering the generalized application. Wider implications of the findings Our results showed a positive association between automatic scores and the success of IVF. Despite observing similar AUCs between the three embryo selection models, the use of the most advanced automatic algorithms should improve workflow, standardize the process between laboratories and allow embryologists to spend their time on other tasks. Trial registration number PI21/00283

P-195 Can we predict blastulation, implantation and live birth in early stages of embryo development? Clinical validation of a classification algorithm based on automatic morphokinetic annotations

Abstract Study question Can a classification algorithm based on automatic annotations of early morphokinetic timings improve embryo selection in ICSI cycles? Summary answer The automatic scoring resulted significantly predictive of blastulation, implantation and live birth, especially when combined with morphological evaluation, but not of euploidy status. What is known already The Eeva Test® is an imaging system that automatically annotates morphokinetic parameters of the early embryo development. The latest version features the Xtend® algorithm, which classifies embryos on day 3 of development from highest (1) to lowest (5) potential to reach blastocyst stage, based on four parameters: the automatically recorded parameters P2 (t3-t2) and P3 (t4-t3), plus oocyte age and number of cells. Previous bibliography suggests that the Eeva scoring, in combination with morphologic evaluation, might also be predictive of implantation and live birth potential. However, the efficacy and reproducibility of this algorithm need to be validated with external data. Study design, size, duration Retrospective, observational cohort study, performed over 3736 embryos from oocyte donation cycles and 1291 embryos from autologous cycles with preimplantation genetic testing for aneuploidies (PGT-A) performed in a single IVF clinic over 3 years. All embryos were scored by the Eeva Test on day 3. The performance of the Eeva-Xtend scoring system as predictor of blastocyst development, euploidy, implantation and live birth was assessed. Participants/materials, setting, methods Embryo development was monitored by a time-lapse system. Embryo selection was performed by morphological (ASEBIR) criteria, resulting in the transfer of 959 embryos. The correlation between the Eeva-Xtend scoring, individually and combined with morphological classification, and the study outcomes was quantified by generalized estimating equations (GEE) including main possible confounders, and expressed in terms of odds ratio (OR). The performance of the GEEs was quantified and compared through the Area Under the ROC Curves (AUCs). Main results and the role of chance A positive association (P<0.001) was confirmed between lower Eeva scores and higher odds of reaching blastocyst stage (OR (1 vs 5) = 15.849, 95%CI 12.510-20.078; OR (2 vs 5) = 11.592, 95%CI 9.229-14.560; AUC = 0.768 (95%CI 0.754−0.783)), and consistent in both oocyte donation and PGT-A cycles. Lower morphokinetic embryo scores also resulted significantly associated with higher chances of implantation (OR (1 vs 5) = 3.385, 95%CI 1.507-7.605; P = 0.003) and live birth (OR (1 vs 5) = 5.132, 95%CI 2.089-12.605; P<0.001) in oocyte donation cycles, but not in embryos subjected to PGT-A: OR (1 vs 5) = 2.089, 95%CI 0.321-13.614, P = 0.441 for implantation and OR (1 vs 5) = 0.916, 95%CI 0.161-5.205, P = 0.921 for live birth. In embryos subjected to PGT-A, the automatic scoring system did not result significantly associated with the euploidy status: OR (1 vs 5) = 0.755 (95%CI 0.255−0.981; P = 0.489). In global, the Eeva Test had similar performance than morphological evaluation for implantation (AUC = 0.610 (95%CI 0.572−0.648) vs 0.606 (95%CI 0.568−0.644), respectively) and live birth (AUC = 0.622 (95%CI0.584−0.660) vs 0.609 (95%CI 0.571−0.647)) prediction. However, the highest performance was achieved when combining both scoring systems: AUC for implantation = 0.629 (95%CI 0.592−0.666), AUC for live birth = 0.636 (95%CI 0.598−0.673). Limitations, reasons for caution The retrospective nature of the study introduces some variability in the characteristics of the sample. However, the large sample size partially overcomes said limitation. The automatic annotations of the morphokinetic parameters were not validated in this study. Wider implications of the findings Our results support the applicability and reproducibility of this automatic early embryo evaluation system. Furthermore, our findings support that the use of this automatic embryo evaluation system in combination with morphological evaluation can potentially reduce subjectivity, improve embryo evaluation and increase success rates in IVF treatments. Trial registration number not applicable

P-268 Artificial intelligence (AI) image analysis outperforms patient age as a surrogate marker for oocyte quality, demonstrating an increased accuracy in predicting blastocyst development

Abstract Study question Can an AI image analysis tool (VIOLET) provide better predictions of oocyte potential than the current standard, patient age, as it relates to blastocyst development? Summary answer While increasing patient age slightly correlates with decreases in blastocyst development, VIOLET provides personalized assessments of individual oocytes correlated to blastocyst development with improved accuracy. What is known already Increasing patient age correlates to decreases in oocyte competency, leading to greater challenges in successful fertility treatment. Clinically, patient age is used to estimate probabilities of success based on national databases. Such as the case in oocyte cryopreservation cycles, considering only age and number of mature oocytes vitrified. However, oocyte quality may vary widely between patients of the same age, and even within each cohort of oocytes. VIOLET is an AI tool that assesses images of mature denuded oocytes to provide an analysis shown to significantly correlate with subsequent blastocyst development and quality, consistently outperforming embryologists in this task. Study design, size, duration This large-scale retrospective study assessed 9,120 mature denuded oocytes retrieved during 2014-2022, representing 1,384 patients between ages 19-49, attending 7 fertility clinics across 5 countries. The VIOLET prediction model is based on image analysis of mature denuded oocytes, without incorporating clinical variables, such as age. Patient age was used to build a separate predictive model of blastocyst development (10,947 training and 3,750 validation samples) to assess the predictive value of age in comparison to VIOLET. Participants/materials, setting, methods Blastocyst development was determined by embryos achieving a Gardner grade by Day5/6 post-ICSI. Blastocyst rates per oocyte cohort were calculated by number of blastocysts divided by total number of mature oocytes retrieved. Various machine learning techniques were trialed to build the predictive model strictly using age; with Random Forest model providing the best-balanced performance. VIOLET and the age model assessed the images and age of 9,120 mature denuded oocytes, respectively, providing predictions of blastocyst development. Main results and the role of chance Blastocyst development was significantly different between patients <35 years old compared to those ≥35 years old [47% vs 42%; p < 0.05 by Two Proportion Z-test]; however, not when the older age group was stratified further. On a patient level, the cohort of oocytes was evaluated by the blastocyst development ratio. Among four age groups (<35, 35-37, 37-40, >40), the blastocyst development ratios per cohort were very similar, with overlapping distributions of 84-94% using Kernel Density estimates. Thus, patient age group does not provide enough information to explain blastocyst development success for an individual oocyte or within an oocyte cohort. In comparison, VIOLET probability is significantly correlated to blastocyst development (p < 0.05; Welch’s Two sample t-test). And blastocyst development rates display a stepwise positive correlation that is significantly different between VIOLET probability quartiles [24% vs 39% vs 47% vs 53%; p < 0.05 by Two Proportion Z-test], providing meaningful information on individual oocytes. Additionally, the predictive model built with age as the only feature had poor ability to predict blastocyst success of an individual oocyte with a limited area-under-the-curve (AUC) of 0.5—unable to separate positive and negative classes. This was outperformed by VIOLET, which displayed an AUC of 0.62 on the same unseen dataset. Limitations, reasons for caution Increasing maternal age causes increased chromosomal abnormalities in oocytes, which translates to lower efficacy with treatment outcomes; therefore, further research to assess VIOLET correlation with PGT-A and implantation outcomes is needed and underway. Wider implications of the findings Patient age correlates with blastocyst development on a general level; however, it does not provide meaningful insights to distinguish rates of blastocyst success on an individual oocyte or oocyte cohort level. VIOLET augments oocyte understanding over the current standard of care, which can be utilized to support personalized clinical decision-making. Trial registration number Not applicable.

P-016 WHO 2021-based comprehensive appraisal of sperm factor parameters’ association with embryological and clinical outcomes. A single center study of 4013 PGT-A cycles

Abstract Study question What is the impact of sperm parameters and paternal age on all embryological and clinical outcomes during ICSI-cycles with aneuploidy testing? Summary answer Reduced basal/post swim-up motility and patients with concentration+morphology+motility <5th-percentile (based on WHO-2021) are associated with poorer embryological outcomes and cumulative-live-birth-rate per concluded PGT-A cycle. What is known already Previous studies reported an association between oligoasthenoteratozoospermia (OAT), obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) and poorer fertilization/blastulation rates, but no impact on euploidy rates at the blastocyst stage. Similarly, the clinical outcomes were mostly independent from sperm characteristics when euploid blastocysts are transferred. The data about paternal age effect are controversial, varying between absent and severe embryological/clinical impact. Moreover, little is known about the association between each semen analysis parameters and embryological/clinical outcomes. Lastly, most of the evidence to date was based on WHO 2010 criteria, recently updated by WHO 2021, and did not mention cumulative-live-birth-rates (CLBR) per cycle. Study design, size, duration Retrospective study at a single private center involving 4013 ICSI+PGT-A cycles with own-oocytes conducted by 3101 couples (2013-2021). The primary embryological outcome was euploid-blastocyst-rate (EBR) per cohort of inseminated oocytes. The primary clinical outcome was CLBR per concluded cycle (i.e., LB achieved or no transferable/all transferred blastocysts). Intermediate embryological/clinical outcomes were also tested. All parameters were analyzed as continuous variables. For some analyses we categorized paternal age (<38/38-44/>44 years) and sperm factor (WHO-2021 parameters<5th percentile). Participants/materials, setting, methods GnRH-antagonist ovarian-stimulation, ICSI, trophectoderm biopsy without zona-pellucida drilling in day3, qPCR/NGS-based analysis to report non-mosaic aneuploidies (segmental aneuploidies reported from 2017 onwards; N = 1991 cycles) and vitrified-warmed euploid single-blastocyst-transfers were conducted. Semen analysis (volume, concentration, total sperm per ejaculate, basal/post swim-up motility, morphology), sperm characteristics (ejaculated/FNA/TESE; fresh/frozen), DNA-fragmentation-index (DFI, outlined via Tunel-test; N = 188 cycles), paternal BMI and age were investigated for their association with embryological/clinical outcomes adjusted for confounders via linear/logistic regressions. Main results and the role of chance Maternal and paternal age were 38.9±3.2(range:23-45) and 41.9±5.7(25-72) years, 10±6.3(1-44) cumulus-oocyte-complexes were retrieved and 7.3±4.6(1-32) metaphase-II oocytes inseminated. Cycles showing all parameters >5th-percentile were 47.1%(N = 1890), concentration<5th-percentile were 3.7%(N = 149), motility<5th-percentile were 3.9%(N = 155), morphology<5th-percentile were 10.1%(N = 405), concentration+motility<5th-percentile were 1.7%(N = 70), concentration+morphology<5th-percentile were 6.2%(N = 248), motility+morphology<5th-percentile were 5.0%(N = 70), concentration+motility+morphology<5th-percentile were 19.9%(N = 797), OA and NOA were 1.4%(N = 56) and 1.0%(N = 41), respectively. The only confounder upon EBR per cohort of metaphase-II oocytes was maternal age. Only basal/post swim-up motility was associated with this outcome. When categorized, only concentration+morphology<5th-percentile (-2.7%,95%CI -5.1 to -0.3%, adjusted-p=0.03), concentration+morphology+motility<5th-percentile (-4.0%,95%CI -5.5 to -2.6%, adjusted-p<0.01), OA (-5.5%,95%CI -10.3 to -0.7%, adjusted-p=0.03) and NOA (-5.8%,95%CI -11.4 to -0.1%, adjusted-p=0.05) showed significantly poorer results. Maternal age and number of metaphase-II oocytes were the confounders upon the chance to obtain ≥1 euploid blastocyst and ≥1 LB among concluded cycles. Only basal/post swim-up motility was associated with this outcome. When categorized, only concentration+morphology+motility<5th-percentile showed significantly lower chances (multivariate-OR:0.74,95%CI 0.61-0.90, adjusted-p<0.01 and multivariate-OR:0.74,95%CI 0.59-0.92, adjusted-p<0.01). No association was reported between semen parameters/sperm factor categories and 1PN/³3PN rates, day5-7 blastocyst-rate, morphological quality, outcomes per euploid transfer. Paternal age categories were associated with day5-blastocyst (N = 1131/3308,34.2%, N = 1179/3644,32.4%, and N = 919/3127,29.4%) and AA-blastocyst rates (N = 1732/3308,52.4%, N = 1815/3644,49.8%, and N = 1387/3127,44.4%) in men <38/38-44/>44 years, respectively (p < 0.01). Limitations, reasons for caution Some confounders were unbalanced among the study groups but accounted for in linear/logistic regressions. 9% of the cycles were not concluded by the time of abstract drafting. The sample size in the groups of surgically-retrieved sperm is limited, in part because NOA patients due to genetic causes were not included. Wider implications of the findings By comprehensively accounting for sperm factor characteristics, this report provides reproductive professionals with useful figures to counsel infertile couples about their chance of success during IVF. These estimates are useful for the decision-making process regarding the most effective clinical strategies to apply. Further studies including more NOA/OA patients are warranted. Trial registration number not applicable

P-233 Novel, non-invasive imaging techniques in quality assessment of mammalian eggs

Abstract Study question Can Optical Coherence Microscopy (OCM) imaging and Cytoplasmic Movement Velocity (CMV) analysis be useful in quality assessment of mature mammalian oocytes? Summary answer OCM allows for assessment of oocytes’ metaphase spindles, and CMV – the actomyosin cytoskeleton functionality, therefore they are potentially useful in oocyte selection for IVF. What is known already The success rate of IVF has significantly improved over the recent years, yet its efficiency plummets with the increasing age of female patients. Therefore, novel approaches to oocyte/embryo quality assessment are in high demand. Among the most promising new methods are OCM, a label-free, 3D tool for subcellular structure visualization, such as the meiotic spindle in mature oocytes or nuclear structure in immature oocytes, and the analysis of CMV, a time-lapse imaging-based method for studying properties of the actomyosin cytoskeleton, crucial for a plethora of cellular process. Both these methods provide unique insights into oocyte competence. Study design, size, duration MII spindle dimensions were measured in OCM-imaged freshly ovulated oocytes collected from young mice (FO-MII, n = 108) and correlated with the outcome of preimplantation development achieved after the oocytes’ activation. Spindles of FO-MII (n = 45) were also compared to spindles of oocytes collected from mice in advanced maternal age (AMA-MII, n = 44). CMV was compared in time-lapse imaged FO-MII (n = 24), postovulatory aged oocytes collected from young mice (PA-MII, n = 16), and AMA-MII (n = 19). Participants/materials, setting, methods Oocytes were collected from hormonally primed mice and imaged using a customized OCM protocol. They were then activated parthenogenetically and cultured for 5 days, followed by immunofluorescence staining to assess the total number of cells and number of cells in the first embryonic cell lineages. FO-MII, PA-MII, and AMA-MII were time-lapse imaged (6 frames per minute; 15 min). The images were subjected to particle image velocimetry analysis and the mean CMV was calculated. Main results and the role of chance Oocytes that achieved the blastocyst stage had smaller spindle volume (p < 0.001) and width (p < 0.05) as compared to those failing to do so. Univariate logistic regression analysis indicated that a 1 μm rise in the spindle width decreases the odds of blastocyst formation by 28% and the number of cells on average by 8, whereas a 100 μm3 rise in the volume – by almost 10% and 2.7 cells respectively. Moreover, the spindle volume is also related to the number of trophectodermal cells, and the spindle length - to the number of primitive endoderm cells in the blastocysts. Noteworthily, young oocytes’ spindles had significantly smaller volume (p < 0.0001), as well as were narrower (p < 0.0001) compared to their counterparts from aged females. A multivariate linear regression model combining spindle length with three morphokinetic parameters (t3, cc2b, tSB) explained 60% of the variability in the total number of cells in 5-day-old embryos (R2 = 0.604) while models based solely on a single morphokinetic parameters explained only up to approx. 54% (R2 = 0.537, for tSB). On the other hand, CMV analysis revealed that freshly ovulated eggs display faster cytoplasmic movements than those postovulatory-aged (p < 0.0001). Reduced cytoplasmic speed was also observed in oocytes collected from aged mice (p < 0.0001). Limitations, reasons for caution All studies presented here were performed on a mouse model, so the obtained data needs to be verified in the target species for validation of these methods as novel tools in oocyte selection protocols. Wider implications of the findings Our results suggest that OCM-based assessment of the MII spindle and short, 15-minute CMV imaging allow for the selection of higher quality oocytes and could potentially find application in assisted reproductive technologies, as they are non-invasive and may enhance the universally acclaimed oocyte/embryo quality assessment protocols. Trial registration number not applicable

P-277 Can a validated, livebirth predictive, in-house morphokinetic algorithm, score embryos according to maternal age; providing more information, for patients, regarding chance of success?

Abstract Study question How does a livebirth (LB) prediction algorithm, built in-house on data from 6228 known outcomes from all ages, perform when categorised by female age? Summary answer The algorithm scored and ranked embryos accurately for clinical outcome (FH) regardless of female age; with proportions of poor prognosis embryos highest in older patients. What is known already Time-lapse incubation enables development of predictive algorithms using key morphokinetic variables to score embryos according to potential for clinical outcomes. Maternal age is associated with embryo quality and implantation. Understanding whether kinetic events are influenced by increasing age may benefit embryo selection and patient information provision. There is little evidence of a correlation between advancing maternal age and morphokinetics, although incidence of abnormal cleavage is linked to aneuploidy and reduced blastulation. Predictive algorithms are generally applied to embryos irrespective of oocyte age but are they effective in selecting the optimum embryo for transfer comparatively across differing maternal age groups? Study design, size, duration A retrospective, comparative analysis of 10034 transferred embryos from 2011 to 2022 in 11 sister fertility clinics. Embryos were cultured for 4 - 6 days in time-lapse incubation before transfer (Embryoscope, Vitrolife). A score was derived using a prospectively validated in-house statistical algorithm, assessing 6 manually annotated morphokinetic variables (t3,t4,t5,t8,tSB,and tB) and trophectoderm grade. Embryos were scored 1 to10 for increasing live birth (LB) potential. Participants/materials, setting, methods All transferred embryos were assessed for age of the patient or donor at treatment, traceable to an algorithm score (1-10) with known implantation, defined by presence of fetal heart (KID-FH). Embryos were assigned to two age groups, < 38 years (n6948) or > = 38 years (n3086) and compared according to the distribution of algorithm scores. Comparative significance of the two groups, against each other and the whole cohort, was assessed for significance using chi-squared. Main results and the role of chance Comparison of the KID-FH per score, for age groups revealed increasing implantation rates and effective ranking per score in each age group, <38 KID-FH scores 1 to10; 9.6%,17.8%, 25.7%, 24.3%, 28.3%, 38.2%, 41.4%, 48.4%, 54.6% and 57.8% and for > = 38 score 1-10; 2.0%, 3.8%, 5.9%, 9.9%, 10.8%, 17.9%, 22.2%, 22.0% 31.6% and 36.8%. KID-FH increased incrementally with increasing score in both maternal age groups, demonstrating that the algorithm is predictive irrespective of maternal age. The percentage incidence of scores were compared, <38 years, score 1 to 10; 3.8%, 4.6%, 4.5%, 5.0%, 6.2%, 7.5%, 9.3%, 12.4%, 16.8%, 29.8% versus > = 38 years 1 to 10; 8.0%, 9.4%, 7.1%, 9.2%, 8.7%, 9.4%,10.5%,11.0%, 11.2%,15.4%. There were significantly higher numbers of embryos with lower scores (p = 0.01) in the > = 38 age group (scores 1-6) than the <38 age group. The younger ages conversely, had significantly greater high scoring embryos 9-10, (p = 0.01), score 7 to 8 were comparable across the age groups. The algorithm score is effective in determining implantation potential irrespective of maternal age. Embryos with poorer implantation potential are present in significantly greater numbers in the older age group and this age group attains significantly fewer high potential embryos when compare to younger patient embryos. Limitations, reasons for caution The data for this analysis was determined from multiple clinics within a fertility group operating under uniform practices. The derived algorithm variables and outcomes may not be transferable to another clinic setting. Follow up is required for known live birth outcomes to confirm comparative prediction to KID-FH per age group. Wider implications of the findings The algorithm score infers embryo competence irrespective of age with differences existing only in the prevalence of low and high scores. This algorithm differentiation promises to inform clinicians and patients regarding their chance of success, when utilised to assess embryos. Morphology alone does not have this breadth or predictive capability Trial registration number N/A

P-710 Single-cell proteomic analysis of human oocytes reveals a decreased abundance of meiosis and proteostasis regulators with advanced maternal age

Abstract Study question How does maternal age affect the proteomic content of her oocytes during their final meiotic progression? Summary answer Compared to young oocytes, oocytes obtained from women of advanced maternal age (AMA) show a decreased abundance of meiosis and proteostasis regulators. What is known already It is well established that AMA leads to a decline in oocyte number and quality, key limiting factors of female reproductive success. Poor oocyte quality is tied to aneuploidies and alterations in cytoplasmic fitness, however the precise mechanisms that underlie oocyte aging remain unknown. Currently knowledge has been largely restricted to transcriptomic studies with no information on the proteome of oocytes obtained from women of AMA. Single-cell proteomic studies may shed light on these mechanisms, as proteins and (post)-translational modifications regulate events unfolding at the oocyte-to-embryo transition in transcriptionally silent oocytes. Study design, size, duration A total of 67 oocytes, obtained from 52 women undergoing controlled ovarian stimulation cycles between May 2021 and May 2022, were included in the study. We obtained 18 fresh germinal vesicle oocytes (GVs) and 17 vitrified in vivo matured metaphase II oocytes (MIIs) from young women (oocyte donors), and 18 GVs and 14 MIIs from AMA. Participants/materials, setting, methods The oocytes were obtained from oocyte donors (n = 27) and AMA patients (n = 25) with a mean age of 24 ± 3.9 years and 39 ± 1.8 years respectively. Single denuded oocytes were snap frozen and their proteomic profiles were generated using the mass spectometry based method plexDIA . Mann-Whitney U test (fold change >|1.5|, adjusted p-value <0.05), Spearman correlation (rs ≥|0.4|, p-value <0.05) and ggplot were used in R to compare the different groups. Main results and the role of chance A total of 2105 proteins were quantified across oocytes stages and age groups. Most of proteins were present in oocytes independently of their maturation state and related to vital processes, such as cellular respiration and energy metabolism. Fifty-four proteins varied in abundance between immature and mature oocytes, including RS14, YBOX2 and RL10A (translational regulation), CLUS and AHSA1 (post-translational modifications) and proteins involved in meiosis progression (WEE2, AURKA, BUB1B, SPDLY). Notably, we observed a negative correlation between protein levels and maternal age for meiosis regulators (e.g.1433E, CDK1; rs: -0.7 and -0.5), meiotic spindle proteins (e.g. DYL2, CAPZB, ARP3, GCP3; rs: -0.6, -0.6, -0.5, -0.5 respectively) and for 5 proteasome subunits (PRS8, PRS6A, PRS10, PSA6, PSMF1; rs: -0.4 to -0.7). The proteasome is a degradation machinery with multiple roles, including meiosis regulation and proteostasis maintenance. Additional proteins of the proteostasis network were found to decline with age (e.g., TCPH, TCPA; rs: -0.5, -0.5). Loss of proteostasis is a common feature of aging and diseases. We also observed a positive correlation between mitochondrial and stress response proteins and maternal age (e.g. ATP5L, TMX1; rs: 0.6, 0.5). Limitations, reasons for caution Unlike GVs that were collected fresh, MII oocytes underwent vitrification and warming before being included in the study due to clinical protocols. These procedures may have unknown effects on the proteome. Wider implications of the findings We show that a woman’s age correlates with changes in her oocyte proteome, which may contribute to the deterioration of oocyte quality through dysregulation of meiosis and disturbance of proteostasis. Proteins showing differential expression in oocytes from women of AMA, such as proteasome, may become targets for improving oocyte quality. Trial registration number Not applicable

P-481 Additional sub-cutaneous progesterone: a putative strategy to counteract poor clinical outcomes after vitrified-warmed euploid blastocyst transfer?

Abstract Study question Is there an association between additional subcutaneous progesterone (a-sP) during luteal phase support for vitrified-warmed euploid blastocyst transfers and the clinical outcomes? Summary answer a-sP was mainly administered to worse-prognosis patients:positive-pregnancy-test (PPT), but also biochemical-pregnancy-loss (BPL) rates were lower than control, thereby involvingsimilar live-birth-rate (LBR) per transfer. What is known already Frozen-embryo-transfer (FET) implementation is increasing worldwide, and luteal-phase-support is a hot-topic. Serum/uterine progesterone levels are key in eliciting immunological tolerance and uterine quiescence, while supporting the processes underlying embryo implantation. Yet, a consensus is missing on progesterone administration (formulation, route, dosage, and duration). Endometrial biopsy studies showed increased progesteroneconcentration after vaginal administration, while sP has excellent pharmacokinetics with faster peak serum concentration.Combining both administration routes might compensate cases ofpoor vaginal absorption and contribute to reaching adequate systemic/uterine levels. This synergy might be beneficial especially in RIF-patients, subject to lower LBRs also in the context of euploid ET. Study design, size, duration Retrospective analysis of 775 vitrified-warmed euploid blastocysttransfers conducted January2020-March2021. Three of 12 gynecologists administrated a-sP as luteal phase support mostly to patients with a worse reproductive history (i.e., longer duration of infertility and/or higher number of previous failed IVF). We assessed the clinical outcomes (PPT, BPL, miscarriage and LBR) in the group of patients who used a-SP (N = 128) versus the control(N = 647), adjusting for confounders in logistic regression analyses. Participants/materials, setting, methods Only euploid non-mosaic blastocysts were transferred. All patients had normal uterine cavities and thyroid function. Endometrial preparation was performed with either hormone-replacement-therapy (estradiol valerate 6mg/die plus vaginal micronized progesterone 800mg/die) or modified-natural-cycle (hCG administration plus vaginal micronized progesterone 400mg/die). In the a-sP group, the supplementation was started 3 days before FET. In case of pregnancy, the therapy was continued until the 8thweek. Main results and the role of chance The patients in the control and a-sP groups were similar for oocyte age (37.6±2.7yr versus 37.5±2.7yr), age at ET (37.7±3.2yr versus 37.7±3.4yr), and body-mass-index (22.2±2.4 versus 22.0±2.8yr). Conversely, the patients in the a-SP group experienced longer duration of infertility (3.5±1.9yr in the control versus 4.1±2.6yr, p = 0.05), and had already undergone ≥1 previous IVF cycle (37%) and ≥2 failed ETs (22%) more frequently than the control (27% and 12%, respectively;p=0.05). The endometrial preparation protocol was similar in the two groups (76% hormone-replacement-therapy and 24% modified-natural-cycle). Also, blastocyst quality and day of transfer were similar. In the study period, the prevalence of a-sP administration increased from first to second-third ETs (13% to 29%). The confounders identified on the clinical outcomes were blastocyst quality, day of transfer and consecutive number of ET. Therefore, these features were included in multivariate logistic regressions. PPT rates were 61.1% (N = 395/647) and 49.2% (N = 63/128, p = 0.01) in the control and a-SP groups, respectively (multivariate-OR:0.65, 95%CI 0.44-0.97;adjusted-p=0.04). BPL rates were 9.1% (N = 36/395) and 1.6% (N = 1/63, p = 0.04), respectively (multivariate-OR:0.14, 95%CI 0.02-0.99;adjusted-p=0.05). Miscarriage rates were 14.2% (N = 51/359) and 6.5% (N = 4/62;p=0.1), respectively (multivariate-OR:0.4, 95%CI 0.14-1.16;adjusted-p=0.1). Lastly, LBRs were 47.6% (N = 308/647) and 45.3% (N = 58/128), respectively (multivariate-OR:1.04, 95% 0.7-1.55;adjusted-p=0.8). Limitations, reasons for caution Retrospective analysis. Progesterone levels are not routinely assessed in our clinical practice. A larger prospective study only in poor prognosis patients is required to assess the putative benefit of a-sP. Wider implications of the findings About 30% of patients undergoing FET suffer from inadequate progesterone levels, possibly impacting the clinical outcomes, yetscarcely detectable through its serum levels. In these women, vaginal progesterone could be insufficient, and a-sP can act as a rescue strategy against reduced endometrial receptivity via lower BPL. Trial registration number Not applicable

Near-infrared fluorophore IR-61 delays postovulatory aging of mouse oocytes through suppressing oxidative stress mediated by mitochondrial protection.

Postovulatory aging can trigger deterioration of oocyte quality and subsequent embryonic development, and thus reduce the success rates of assisted reproductive technology (ART). The molecular mechanisms underlying postovulatory aging, and preventative strategies, remain to be explored. The near-infrared fluorophore IR-61, a novel heptamethine cyanine dye, has the potential for mitochondrial targeting and cell protection. In this study, we found that IR-61 accumulated in oocyte mitochondria and reduced the postovulatory aging-induced decline in mitochondrial function, including mitochondrial distribution, membrane potential, mtDNA number, ATP levels, and mitochondrial ultrastructure. In addition, IR-61 rescued postovulatory aging-caused oocyte fragmentation, defects in spindle structure, and embryonic developmental potential. RNA sequencing analysis indicated that the postovulatory aging-induced oxidative stress pathway might be inhibited by IR-61. We then confirmed that IR-61 decreased the levels of reactive oxygen species and MitoSOX, and increased GSH content in aged oocytes. Collectively, the results indicate that IR-61 may prevent postovulatory aging by rescuing oocyte quality, promoting successful rate in ART procedure.

Protective effect of onion peel extract on ageing mouse oocytes.

Mammalian oocytes not fertilized immediately after ovulation can undergo ageing and a rapid decline in quality. The addition of antioxidants can be an efficient approach to delaying the oocyte ageing process. Onion peel extract (OPE) contains quercetin and other flavonoids with natural antioxidant activities. In this study, we investigated the effect of OPE on mouse oocyte ageing and its mechanism of action. The oocytes were aged in vitro in M16 medium for 16 h after adding OPE at different concentrations (0, 50, 100, 200, and 500 μg/ml). The addition of 100 μg/ml OPE reduced the oocyte fragmentation rate, decreased the reactive oxygen species (ROS) level, increased the glutathione (GSH) level, and improved the mitochondrial membrane potential compared with the control group. The addition of OPE also increased the expression of SOD1, CAT, and GPX3 genes, and the caspase-3 activity in OPE-treated aged oocytes was significantly lower than that in untreated aged oocytes and similar to that in fresh oocytes. These results indicated that OPE delayed mouse oocyte ageing by reducing oxidative stress and apoptosis and enhancing mitochondrial function.

Decreased LONP1 expression contributes to DNA damage and meiotic defects in oocytes.

Meiotic defects in oocytes are the primary reason for decreased female fertility with advanced maternal age. In this study, we revealed that decreased expression of ATP-dependent Lon peptidase 1 (LONP1) in aged oocytes and oocyte-specific depletion of LONP1 disrupt oocyte meiotic progression accompanying with mitochondrial dysfunction. In addition, LONP1 downregulation increased oocyte DNA damage. Moreover, we demonstrated that splicing factor proline and glutamine rich directly interacts with LONP1 and mediate the effect of LONP1 depletion on meiotic progression in oocytes. In summary, our data suggest that decreased expression of LONP1 is involved in advanced maternal age-related meiosis defects and that LONP1 represents a new therapeutic target to improve aged oocyte quality.

Effects of calcium-free ageing on ethanol-induced activation and developmental potential of mouse oocytes.

Although ethanol treatment is widely used to activate oocytes, the underlying mechanisms are largely unclear. Roles of intracellular calcium stores and extracellular calcium in ethanol-induced activation (EIA) of oocytes remain to be verified, and whether calcium-sensing receptor (CaSR) is involved in EIA is unknown. This study showed that calcium-free ageing (CFA) in vitro significantly decreased intracellular stored calcium (sCa) and CaSR expression, and impaired EIA, spindle/chromosome morphology and developmental potential of mouse oocytes. Although EIA in oocytes with full sCa after ageing with calcium does not require calcium influx, calcium influx is essential for EIA of oocytes with reduced sCa after CFA. Furthermore, the extremely low EIA rate in oocytes with CFA-downregulated CaSR expression and the fact that inhibiting CaSR significantly decreased the EIA of oocytes with a full complement of CaSR suggest that CaSR played a significant role in the EIA of ageing oocytes. In conclusion, CFA impaired EIA and the developmental potential of mouse oocytes by decreasing sCa and downregulating CaSR expression. Because mouse oocytes routinely treated for activation (18 h post hCG) are equipped with a full sCa complement and CaSR, the present results suggest that, while calcium influx is not essential, CaSR is required for the EIA of oocytes.

Boosting mitochondrial function and metabolism in aging female germ cells with dual ROCK/ROS inhibition

The decline in oocyte quality with age is an irreversible process that results in low fertility. Reproductive aging causes an increase in oocyte aneuploidy leading to a decrease in embryo quality and an increase in the incidence of miscarriage and congenital defects. Here, we show that the dysfunction associated with aging is not limited to the oocyte, as oocyte granulosa cells also show a range of defects related to mitochondrial activity. The addition of Y-27632 and Vitamin C combination drugs to aging germ cells was effective in enhancing the quality of aging cells. We observed that supplement treatment significantly decreased the production of reactive oxygen species (ROS) and restored the balance of mitochondrial membrane potential. Supplementation treatment reduces excessive mitochondrial fragmentation in aging cells by upregulating mitochondrial fusion. Moreover, it regulated the energy metabolism within cells, favoring oxygen respiration and reducing anaerobic respiration, thereby increasing cellular ATP production. In an experiment with aged mice, supplement treatment improved the maturation of oocytes in vitro and prevented the buildup of ROS in aging oocytes in culture. Additionally, this treatment resulted in an increased concentration of anti-mullerian hormone (AMH) in the culture medium. By improving mitochondrial metabolism in aging females, supplement treatment has the potential to increase quality of oocytes during in vitro fertilization.

Circadian regulation of mTORC1 signaling via Per2 dependent mechanism disrupts folliculogenesis and oocyte maturation in female mice.

mTOR (mammalian target of Rapamycin) is an important signaling pathway involved in several crucial ovarian functions including folliculogenesis and oocyte maturation. The circadian rhythm regulates multiple physiological processes and PER2 is one of the core circadian rhythm components. mTOR is regulated by the circadian clock and in turn, the rhythmic mTOR activities strengthen the clock function. Our current study aims to investigate a possible interconnection between the circadian clock and the mTORC1 signaling pathway in folliculogenesis and oocyte maturation. Here we demonstrate that the circadian system regulates mTORC1 signaling via Per2 dependent mechanism in the mouse ovary. To investigate the effect of constant light on ovarian and oocyte morphology, animals were housed 12:12h L:D group in standard lightening conditions and the 12:12h L:L group in constant light for one week. Food intake and body weight changes were measured. Ovarian morphology, follicle counting, and oocyte aging were evaluated. Afterward, western blot for mTOR, p-mTOR, p70S6K, p-p70S6K, PER2, and Caspase-3 protein levels was performed. The study demonstrated that circadian rhythm disruption caused an alteration in their food intake and decrease in primordial follicle numbers and an increase in the number of atretic follicles. It caused an increase in oxidative stress and a decrease in ZP3 expression in oocytes. Decreased protein levels of mTOR, p-mTOR, p70S6K, and PER2 were shown. The results showed that the circadian clock regulates mTORC1 through PER2 dependent mechanism and that decreased mTORC1 activity can contribute to premature aging of mouse ovary. In conclusion, these results suggest that the circadian clock may control ovarian aging by regulating mTOR signaling pathway through Per2 expression.

Elevated serum progesterone levels before frozen embryo transfer do not negatively impact reproductive outcomes: a large retrospective cohort study

To evaluate whether patients with high-serum progesterone levels before frozen embryo transfer (FET) under hormonal replacement therapy present with worse reproductive outcomes. A cohort retrospective study. A university-affiliated fertility center. A total of 3,183 FET cycles in patients receiving hormonal replacement therapy between March 2009 and December 2020 were included. The luteal phase was covered with 200 mg per 8 hours of vaginal micronized progesterone either alone or in combination with a daily subcutaneous injection of 25 mg of progesterone. A total of 1,360 cycles corresponded to frozen homologous embryo transfer (ET) (hom-FET), 1,024 were euploid ET (eu-FET) after preimplantation genetic testing for aneuploidies, and 799 cycles were frozen heterologous ET (het-FET). All patients had adequate serum progesterone levels (≥10.6 ng/mL) before the procedure. Frozen embryo transfer cycles. Clinical pregnancy, miscarriage, and live birth rates (LBRs). Median (P25; P75) serum progesterone level before FET was 14.39 (12.43-17.49) ng/mL. Progesterone levels were significantly higher in the group under vaginal plus subcutaneous progesterone (15.96 [13.74-21.60] vs. 14.09 [12.19-16.95]). No differences in clinical pregnancy, miscarriage, and LBR were observed based on the use of vaginal or vaginal plus subcutaneous progesterone for each of the groups (hom-FET, eu-FET, and het-FET). Live birth rates were comparable among patients in the highest centile of serum progesterone levels (≥p90) (22.33 ng/mL) and the rest of the patients (p<90) (43.9% vs. 41.3%). Patients with progesterone levels ≥p90 presented lower body mass index than those in the lower centiles (<p90) (22.62 ± 3.82 vs. 23.32 ± 4.06). After dividing patients into deciles based on serum progesterone levels, no differences in LBRs were observed among the groups. No association was observed using a generalized additive model between progesterone levels and LBR. A multivariable logistic regression adjusted by oocyte age, type of treatment, body mass index, type of luteal phase support, and the number of embryos transferred was applied for centile 90 and centile 95 of progesterone and showed that serum progesterone in their highest levels did not negatively impact LBR. Elevated serum progesterone levels before FET do not impair reproductive outcomes in patients receiving artificially prepared cycles with vaginal or vaginal plus subcutaneous progesterone.

Serotonergic signaling plays a deeply conserved role in improving oocyte quality

Declining germline quality is a major cause of reproductive senescence. Potential remedies could be found by studying regulatory pathways that promote germline quality. Several lines of evidence, including a C. elegans male pheromone ascr#10 that counteracts the effects of germline aging in hermaphrodites, suggest that the nervous system plays an important role in regulating germline quality. Inspired by the fact that serotonin mediates ascr#10 signaling, here we show that serotonin reuptake inhibitors recapitulate the effects of ascr#10 on the germline and promote healthy oocyte aging in C. elegans. Surprisingly, we found that pharmacological increase of serotonin signaling stimulates several developmental processes in D. melanogaster, including improved oocyte quality, although underlying mechanisms appear to be different between worms and flies. Our results reveal a plausibly conserved role for serotonin in maintaining germline quality and identify a class of therapeutic interventions using available compounds that could efficiently forestall reproductive aging.

Alpha-ketoglutarate supplementation ameliorates ovarian reserve and oocyte quality decline with aging in mice

Assisted reproductive technology is widely accepted as an effective treatment to improve female fertility, but the decline of aging oocyte quality remains an important factor in the decrease of female fecundity. However, the effective strategies for improving oocyte aging are still not well understood. In the study, we demonstrated that ROS content and abnormal spindle proportion were increased and mitochondrial membrane potential was decreased in aging oocytes. However, supplementation of α-ketoglutarate (α-KG), an immediate metabolite in the tricarboxylic acid cycle (TCA), for 4 months to aging mice, significantly increased the ovarian reserve showed by more follicle numbers observed. In addition, the oocyte quality was significantly improved, as demonstrated by reduced fragmentation rate and decreased reactive oxygen species (ROS) levels, in addition to a lower rate of abnormal spindle assembly, thereby improving the mitochondrial membrane potential. Consistent with the in vivo data, α-KG administration also improved the post-ovulated aging oocyte quality and early embryonic development by improving mitochondrial functions and reducing ROS accumulation and abnormal spindle assembly. Our data revealed that α-KG supplementation might be an effective strategy to improve the quality of aging oocytes in vivo or in vitro.

Kinetochore deterioration concommitant with centromere weakening during aging in mouse oocyte meiosis-I.

Age-related oocyte aneuploidy occurs as a result of chromosome segregation errors in female meiosis-I and meiosis-II, and is caused by a progressive age-related deterioration of the chromosome segregation machinery. Here, we assess the impact of age upon the kinetochore, the multi-protein structure that forms the link between the chromosome and spindle microtubules. We find that in meiosis-I the outer kinetochore assembles at germinal vesicle breakdown, but that a substantially smaller outer kinetochore is assembled in oocytes from aged mice. We show this correlates with a weaker centromere in aged oocytes and, using nuclear transfer approaches to generate young-aged hybrid oocytes, we show that outer kinetochore assembly always mirrors the status of the centromere, regardless of cytoplasmic age. Finally, we show that weaker kinetochores in aged oocytes are associated with thinner microtubule bundles, that are more likely to be mis-attached. We conclude that progressive loss of the centromere with advancing maternal age underpins a loss of the outer kinetochore in meiosis-I, which likely contributes to chromosome segregation fallibility in oocytes from older females.

Maternal epigenetic factors in embryonic and postnatal development.

Maternal factors present in oocytes and surrounding granulosa cells influence early development of embryos. In this study, we searched for epigenetic regulators that are expressed in oocytes and/or granulosa cells. Some of the 120 epigenetic regulators examined were expressed specifically in oocytes and/or granulosa cells. When their expression was examined in young versus aged oocytes or granulosa cells, many were significantly up- or downregulated in aged cells. The maternal role of six genes in development was investigated by generating oocyte-specific knock-out (MKO) mice. Two genes (Mllt10, Kdm2b) did not show maternal effects on later development, whereas maternal effects were evident for Kdm6a, Kdm4a, Prdm3, and Prdm16 for MKO female mice. Offspring from Kdm6a MKO mice underwent perinatal lethality at a higher rate. Pups derived from Prdm3;Prdm16 double MKO showed a higher incidence of postnatal death. Finally, embryos derived from Kdm4a MKO mice showed early developmental defects as early as the peri-implantation stage. These results suggest that many of maternal epigenetic regulators undergo differential expression upon aging. Some, such as Kdm4a, Kdm6a, Prdm3, and Prdm16, have maternal role in later embryonic or postnatal development.