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Can the best culture medium be determined by mouse embryo testing? Zygotes/embryos of four different mouse strains: inbred Balb/c (n = 79) and C57Bl/6 (n = 95), F1 hybrid DBA/2*C57Bl/6 (n = 133), and outbred ICR (n = 69) were incubated in vitro in four culture media: 1-Step Culture Medium (VitaVitro), CSC medium (Irvine Scientific), Sage 1-Step medium (Origio), G-TL medium (Vitrolife). The embryos were cultured under standard conditions at atmospheric oxygen: 37℃; 6% CO2, 100% humidity. Standard MEA-test parameters (number of expanded blastocysts on E4.5), as well as additional characteristics (embryo development on E2.5-E4.5, number of hatching and hatched blastocysts on E4.5, embryo arrest and degeneration) were assessed for each of the strain-medium combinations. The results were compared across strains as well as across media. Embryo development depended on both the media and the mouse strain. Embryos from inbred and outbred mice were more sensitive to suboptimal culture conditions compared to the hybrid strain; this was reflected in reduced blastocyst and hatching rates, as well as an increased percentage of arrested and degraded embryos. The choice of optimal media depended strongly on the strain: CSC was better for Balb/c, Sage 1-step for C57Bl/6, while both were preferred for hybrids, and G-TL for outbreds. VitaVitro was quite good for hybrids and performed worse for other strains. Overall, the embryos of each strain behaved differently in different media, and it was not possible to make a real preference for any of them over human embryos based on the results of any single mouse strain. Only a pooled sample of different mouse strains can be used for comprehensive media MEA testing. MEA testing with any strain of mice is doomed to error due to their specific culture requirements. Hybrid mice are too reproductively efficient and cannot be used to distinguish media based on their subtle differences. Outbred and inbred mice are too sensitive and may be delayed or degraded in culture media that is well suited to the requirements of human embryos. Combined analysis of multiple mouse strains should be used to test media more fully and may serve as a model for the heterogeneity of human embryos in IVF clinics., Competing Interests: Declarations. All manipulations with animals were carried out in accordance with the European Convention for the Protection of Vertebrate Animals, Strasbourg, March 18.III.1986, and the Rules of Laboratory Practice in the Russian Federation. Approval was obtained from the Bioethics committee of Institute of Gene Biology of Russian Academy of Sciences (IGB RAS). Conflict of Interest: None of the authors has any conflict of interest to declare., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Can a set of metabolites present in embryo culture media correlate with embryo implantation? Case-control study in two phases: discovery phase (101 samples) and validation phase (169 samples), collected between 2018 and 2022, with a total of 218 participants. Culture media samples with known implantation outcomes were collected after blastocyst embryo transfer (including both PGT and non-PGT cycles) and were analyzed using chromatography followed by mass spectrometry. The spectra were processed and analyzed using statistical and machine learning techniques to identify biomarkers associated with embryo implantation, and to develop a predictive model. In the discovery phase, 148 embryo implantation biomarkers were identified using high resolution equipment, and 47 of them were characterized. Our results indicate a significant enrichment of tryptophan metabolism, arginine and proline metabolism, and lysine degradation biochemical pathways. After transferring the method to a lower resolution equipment, a model able to assign a Metabolite Pregnancy Index (MPI) to each embryo culture media was developed, taking the concentration of 36 biomarkers as input. Applying this model to 20% of the validation samples (N=34) used as the test set, an accuracy of 85.29% was achieved, with a PPV (Positive Predictive Value) of 88% and a NPV (Negative Predictive Value) of 77.78%. Additionally, informative results were obtained for all the analyzed samples. Metabolite concentration in the media after in vitro culture shows correlation with embryo implantation potential. Furthermore, the mathematical combination of biomarker concentrations using Artificial Intelligence techniques can be used to predict embryo implantation outcome with an accuracy of around 85%., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Purpose: To explore if a day 7 blastocyst is predictive of the reproductive potential of sibling day 5 or day 6 blastocysts?, Methods: Retrospective cohort of autologous frozen embryo transfers (FET), February 2019 to April 2022. Cycles divided into groups 1 to 5, according to the day of embryo cryopreservation and the presence of a day 7 blastocyst sibling within the cohort: group 1/group 2-day 5 blastocyst without/with a day 7 sibling, group 3/group 4-day 6 blastocyst without/with a day 7 sibling, group 5-day 7 blastocyst. Clinical, ongoing pregnancy and miscarriage rates, cycle, and patient characteristics are reported. Multivariable generalized estimating equations (GEE) logistic regression analysis accounts for confounders and assesses the effect of a sibling day 7 blastocyst on ongoing pregnancy rates of day 5 or day 6 blastocyst FETs., Results: Ongoing pregnancy rates are 38.4%, 59.5%, 30.8%, 32.7%, and 4.4% in groups 1-5, respectively. When correcting for maternal age, number of oocytes retrieved and discarded per cohort, and ploidy, embryos cryopreserved on either day 6 or day 7 have reduced odds of ongoing pregnancy after FET compared to day 5 blastocysts (OR = 0.76, IQR [0.61-0.95], p-value = 0.01). However, the presence of a day 7 sibling does not significantly affect odds of ongoing pregnancy of day 5 or day 6 blastocysts compared to the same-day blastocyst without a day 7 sibling (p-value = 0.20 and 0.46, respectively). This finding is consistent within both the Preimplantation Genetic Testing for Aneuploidy (PGT-A) unscreened and screened (euploid) embryo subgroups., Conclusions: Day of embryo cryopreservation significantly affects ongoing pregnancy rates. However, day 7 embryos within a cohort do not affect the reproductive potential of sibling day 5 and day 6 blastocysts, suggesting that slow embryo development is an embryo-specific trait., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

IL-6 plays an important role in oogenesis in humans. However, at the preimplantation stage, IL-6 production and the role in embryo development remain unclear. In this study, IL-6 concentrations in single-embryo media were analyzed. In addition, the association between IL-6 production and blastocyst formation was investigated. Single-embryo culture media from 194 embryos were collected on day 6 after fertilization and divided into four groups according to the developmental stage of the corresponding embryo, as follows: cleavage stage group, morula-early blastocyst group, unavailable full blastocyst group, and available full blastocyst group. IL-6 concentrations were significantly lower in the cleavage stage group than in the morula-early blastocyst group (p = 0.009), in the unavailable full blastocyst group (p = 0.003), and in the available full blastocyst group (p < 0.001). Logistic regression analysis showed that IL-6 concentration in single-embryo medium was significantly associated with blastocyst formation (odds ratios β1 = 1.876, 95% CI 1.433 to 2.644, p < 0.0001). Therefore, IL-6 was produced by human preimplantation embryos throughout the preimplantation stage and may play a role in embryo development., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Purpose: Does vitrification/warming affect the mitochondrial DNA (mtDNA) content and the gene expression profile of blastocysts?, Methods: Prospective cohort study in which 89 blastocysts were obtained from 50 patients between July 2017 and August 2018. mtDNA was measured in a total of 71 aneuploid blastocysts by means of real-time polymerase chain reaction (RT-PCR). Transcriptomic analysis was performed by RNA sequencing (RNA-seq) in an additional 8 aneuploid blastocysts cultured for 0 h after warming, and 10 aneuploid blastocysts cultured for 4-5 h after warming., Results: A significant decrease in mtDNA content just during the first hour after the warming process in blastocysts was found (P < 0.05). However, mtDNA content experimented a significantly increased along the later culture hours achieving the original mtDNA levels before vitrification after 4-5 h of culture (P < 0.05). Gene expression analysis and functional enrichment analysis revealed that such recovery was accompanied by upregulation of pathways associated with embryo developmental capacity and uterine embryo development. Interestingly, the significant increase in mtDNA content observed in blastocysts just after warming also coincided with the differential expression of several cellular stress response-related pathways, such as apoptosis, DNA damage, humoral immune responses, and cancer., Conclusion: To our knowledge, this is the first study demonstrating in humans, a modulation in blastocysts mtDNA content in response to vitrification and warming. These results will be useful in understanding which pathways and mechanisms may be activated in human blastocysts following vitrification and warming before a transfer., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Our objective was to design an automated deep learning model that extracts the morphokinetic events of embryos that were recorded by time-lapse incubators. Using automated annotation, we set out to characterize the temporal heterogeneity of preimplantation development across a large number of embryos., Methods: To perform a retrospective study, we used a dataset of video files of 67,707 embryos from four IVF clinics. A convolutional neural network (CNN) model was trained to assess the developmental states that appear in single frames from 20,253 manually-annotated embryos. Probability-weighted superposition of multiple predicted states was permitted, thus accounting for visual uncertainties. Superimposed embryo states were collapsed onto discrete series of morphokinetic events via monotonic regression of whole-embryo profiles. Unsupervised K-means clustering was applied to define subpopulations of embryos of distinctive morphokinetic profiles., Results: We perform automated assessment of single-frame embryo states with 97% accuracy and demonstrate whole-embryo morphokinetic annotation with R-square 0.994. High quality embryos that had been valid candidates for transfer were clustered into nine subpopulations, as characterized by distinctive developmental dynamics. Retrospective comparative analysis of transfer versus implantation rates reveals differences between embryo clusters as marked by poor synchronization of the third mitotic cell-cleavage cycle., Conclusions: By demonstrating fully automated, accurate, and standardized morphokinetic annotation of time-lapse embryo recordings from IVF clinics, we provide practical means to overcome current limitations that hinder the implementation of morphokinetic decision-support tools within clinical IVF settings due to inter-observer and intra-observer manual annotation variations and workload constrains. Furthermore, our work provides a platform to address embryo heterogeneity using dimensionality-reduced morphokinetic descriptions of preimplantation development., (© 2023. The Author(s).)

The presence of cell-free DNA in spent embryo culture media (SECM) has unveiled its possible utilization for embryonic ploidy determination, opening new frontiers for the development of a non-invasive pre-implantation genetic screening technique. While a growing number of studies have shown a high concordance between genetic screening using cell-free DNA (cfDNA) and trophectoderm (TE), the mechanism pertaining to the release of cfDNA in SECM is largely unknown. This review aims to evaluate research evidence on the origin and possible mechanisms for the liberations of embryonic DNA in SECM, including findings on the self-correction abilities of embryos which might contribute to the presence of cfDNA. Several databases including EMBASE, PUBMED, and SCOPUS were used to retrieve original articles, reviews, and opinion papers. The keywords used for the search were related to the origins and release mechanism of cfDNA. cfDNA in SECM originates from embryonic cells and, at some levels, non-embryonic cells such as maternal DNA and exogenous foreign DNA. The apoptotic pathway has been demonstrated to eliminate aneuploid cells in developing mosaic embryos which might culminate to the release of cfDNA in SECM. Nonetheless, there is a recognized need for exploring other pathways such as cross-talk molecules called extracellular vesicles (EVs) made of small, round bi-layer membranes. During in vitro development, embryos physiologically and actively expel EVs containing not only protein and microRNA but also embryonic DNA, hence, potentially releasing cfDNA of embryonic origin into SECM through EVs., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

To study the impact of culture media on preimplantation morphokinetics used for predicting clinical outcomes. All IVF and ICSI cycles performed between 2012 and 2017 with time-lapse information available were included. In November 2014, culture medium was changed from Vitrolife G-1 PLUS to SAGE 1-Step. Each embryo was retrospectively assigned a morphokinetic-based KIDScore for prediction of implantation. Clinical outcomes were retrieved from medical records. Linear mixed models were used to study differences in morphokinetic parameters, a proportional odds model for KIDScore ranking and logistic regression for differences in clinical outcomes. All analyses were adjusted for patient and treatment characteristics. In 253 (63.1%) cycles, embryos (n = 671) were cultured in Vitrolife, and in 148 (36.9%) cycles, embryos (n = 517) were cultured in SAGE. All cleavage divisions occurred earlier for SAGE embryos than for Vitrolife embryos (2-cell: -2.28 (95%CI: -3.66, -0.89), 3-cell: -2.34 (95%CI: -4.00, -0.64), 4-cell: -2.41 (95%CI: -4.11, -0.71), 5-cell: -2.54 (95%CI: -4.90, -0.18), 6-cell: -3.58 (95%CI: -6.08, -1.08), 7-cell: -5.62 (95%CI: -8.80, -2.45) and 8-cell: -5.32 (95%CI: -9.21, -1.42) hours, respectively). Significantly more embryos cultured in SAGE classified for the highest KIDScore compared to embryos cultured in Vitrolife (p < 0.001). No differences were observed in clinical outcomes. Our results demonstrate an impact of culture medium on preimplantation embryo developmental kinetics, which affects classification within the KIDScore algorithm, while pregnancy outcomes were comparable between the groups. This study underscores the need to include the type of culture medium in the development of morphokinetic-based embryo selection tools., (© 2022. The Author(s).)

Purpose: To evaluate the effect of two-step denudation on maternal contamination, ploidy concordance between spent embryo culture medium (SCM) and trophectoderm, blastocyst formation, and clinical outcome., Methods: Sibling embryos of the same couple were re-denuded (treatment) and not re-denuded (control) on day 3, and trophectoderm biopsy and SCM collection were performed on day 5/6. Sex chromosomes of 20 pairs of SCM and biopsy samples were analyzed to determine the reduction in maternal contamination. Blastocyst formation, implantation, and ongoing pregnancy rates were analyzed by recruiting 565 cleavage embryos on day 3. A total of 113 SCM samples and their corresponding trophectoderm results were collected for ploidy concordance analysis., Results: The detection rate of XX between the treatment and control groups was significant (12/20 (60.0%) versus 19/20 (95.0%), p = 0.02). Concordance of sex chromosomes between the two groups was significant (17/20 (85.0%) versus 8/19 (42.1%), p = 0.003). There were no significant differences in blastocyst formation rate, implantation rate, and ongoing pregnancy rate between the two groups. Among the 113 pairs of SCM and its corresponding trophectoderm, 37 cases (33.33%) were completely concordant, 39 cases (35.14%) were partially concordant, and 35 cases (31.53%) were discordant., Conclusion: Our results suggest that re-denudation on day 3 reduces the influence of maternal contamination and improves the accuracy of cfDNA detection. Moreover, the procedure had no significant effect on blastocyst formation, implantation, and ongoing pregnancy rates. In addition, the ploidy concordance approached 70% compared with biopsy, which is acceptable but still not ideal., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: The study aims to summarize current knowledge on the use of oil in embryo culture systems, with a focus on proper management of different types of oil and possible impact on culture systems., Methods: PubMed was used to search the MEDLINE database for peer-reviewed English-language original articles and reviews concerning the use of oil in embryo culture systems. Searches were performed by adopting "embryo," "culture media," "oil," and "contaminants" as main terms. The most relevant publications were assessed and discussed critically., Results: Oils used in IVF are complex mixtures of straight-chain hydrocarbons, cyclic and aromatic hydrocarbons, and unsaturated hydrocarbons, whose precise composition influences their chemical and physical properties. Possible presence of contaminants suggests their storage at 4 °C in the dark to prevent peroxidation. Washing, generally performed by manufacturers prior to commercialization, may remove trace chemical contaminants. Oils reduce evaporation from culture media at rates depending on their chemical physical properties, culture system parameters, and incubator atmosphere. Contaminants - mainly metal ion and plastic components derived from refinement processes and storage - can pass to the aqueous phase of culture systems and affect embryo development., Conclusions: Oils are essential components of culture systems. Their original quality and composition, storage, handling, and use can affect embryo development with significant efficiency and safety implications., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To compare morphokinetic parameters in embryos obtained from women with and without endometriosis., Methods: We evaluated a total of 3471 embryos resulting from 434 oocyte retrievals performed at a single academic center. One thousand seventy-eight embryos were obtained from women affected by endometriosis and 2393 came from unaffected controls. All embryos were cultured in a time-lapse incubator chamber for up to 6 days. IVF cycle outcomes and morphokinetic parameters collected prospectively were retrospectively reviewed., Results: Morphokinetic data suggest that embryo development is impaired in embryos obtained from women with endometriosis (EE). EE were slower to achieve the 2-8 cell stages compared to control embryos (CE) (p < 0.001); additionally, time to compaction was delayed compared to CE (p = 0.015). The timing of late developmental events, including morulation and blastulation was also delayed in the endometriosis cohort (p < 0.001). In addition to demonstrating delayed cell cycle milestones, EE were less likely than controls to progress to morula, blastocyst, and expanded blastocyst stages (p < 0.001). Furthermore, a smaller proportion of embryos in the endometriosis group fell into optimal kinetic ranges for cc2 (p = 0.003), t5 (p = 0.019), tSB (p < 0.001), and tEB (p = 0.007). There were no significant differences in clinical pregnancy or live birth rates between groups., Conclusion: Embryos from endometriosis patients demonstrate impairments in both early and late developmental events, and progress to the morula, blastocyst, and expanded blastocyst stages at lower rates than control embryos. Despite these differences, IVF outcomes are similar for patients with and without endometriosis., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To assess the effect of high ovarian response on oocyte quality and ovarian stimulation cycle outcomes., Methods: A retrospective cohort study conducted at three IVF units. The high ovarian response (HOR) and polycystic ovary syndrome (PCOS) with HOR (PCOS HOR) groups included 151 and 13 women who underwent controlled ovarian stimulation (COS) resulting in more than 15 retrieved oocytes, for a total of 1863 and 116 cultured embryos, respectively. The normal ovarian response (NOR) group comprised 741 women with 6-15 retrieved oocytes, resulting in 4907 cultured embryos. Data collected included fresh cycle data and pregnancy rates, in addition to annotation of morphokinetic events from time of pronuclei fading to time of initiation of blastocyst formation of embryos cultured in a time lapse incubator, including occurrence of direct unequal cleavage at first cleavage (DUC-1) (less than 5 h from two to three blastomeres). Comparison was made between morphokinetic parameters between the 3 groups. Cycle outcomes were compared in the high vs. normal ovarian response groups., Results: Oocyte maturation rate was significantly lower in the HOR vs. NOR groups (56.5% vs. 90.0%, p < 0.001), while the fertilization rates were similar (60.2% vs. 58.1%, p = 0.397). The prevalence of DUC-1 embryos was higher in the PCOS HOR and the HOR groups as compared to the NOR group (22.7% vs. 16.2% and 12.0%, respectively, p < 0.001). After exclusion of DUC-1 embryos, remaining embryos from the NOR and HOR groups reached the morphokinetic milestones at similar rates, with comparable implantation and clinical pregnancy rates, while the PCOS HOR showed shorter time to 5 blastomeres compared to the NOR and HOR groups., Conclusions: High ovarian response might be associated with decreased oocyte quality, manifested as a higher proportion of immature oocytes and higher rate of direct uneven cleavage embryos, while embryos exhibiting normal first cleavage have similar temporal milestones and implantation potential., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: A retrospective study examining the effects of embryo re-expansion before transfer on pregnancy outcomes for frozen embryo transfers (FET)., Methods: A total of 486 FET cycles from November 2017 through December 2019 were studied. These cycles included patients using autologous, donor oocytes, and donor embryo with patients ranging from ages 23 to 48 years with infertility diagnoses. Programmed FET priming was performed with exogenous estrogen and progesterone. All blastocysts were cultured in trigas incubators for 20 min to 4 h and 42 min. Pictures of each blastocyst after thaw and before transfer were taken utilizing the Hamilton Thorne Zilos laser software (Beverly, MA). The longest portion of the embryo was measured in µm. Pregnancy was defined by a positive hCG, and ongoing clinical pregnancy was defined by the presence of fetal cardiac activity. Wilcoxon rank sum tests were used to access differences in change parameters., Results: There is no significant difference in the amount of embryo expansion or contraction to achieve an ongoing pregnancy. The difference remained non-significant when stratified by embryo expansion or contraction. The amount of change over time and percent change from the first measurement were also not associated with achieving an ongoing pregnancy. This remained true after adjustment for patient age and whether or not a biopsy was performed., Conclusions: Embryos that do not re-expand after warming appear to have a similar chance of achieving a successful pregnancy as those that do re-expand., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To understand the clinical factors associated with embryo survival after vitrification in a cohort of human blastocysts screened by preimplantation genetic testing for aneuploidy (PGT-A)., Methods: Patient demographic, embryo, and cycle characteristics associated with failed euploid blastocyst survival were compared in a cohort of women (n = 6167) who underwent IVF-PGT-A., Results: Compared to those that survived warming, vitrified euploid embryos that failed to survive after warming came from IVF cycles with significantly higher estradiol levels at time of surge (2754.8 ± 1390.2 vs. 2523.1 ± 1190.6 pg/mL, p = 0.03), number of oocytes retrieved (19.6 ± 10.7 vs. 17.5 ± 9.8, p = 0.005), and basal antral follicle count (BAFC) (15.3 ± 8.5 vs. 13.9 ± 7.2, p = 0.05). Euploid embryos were less likely to survive warming if they came from cycles before 2015 (24.6% vs. 13.2%, p < 0.001), were cryopreserved on day 7 versus day 5 or 6 (9.1% vs. 3.0%, p < 0.001), underwent two trophectoderm biopsies (6.9% vs. 2.3%, p < 0.001), had a grade C inner cell mass (15.4% vs. 7.7%, p < 0.001), or were fully hatched (41.1% vs. 12.2%, p < 0.001). In the multivariate model, which controlled for relevant confounders, the association between decreased survival and increased BAFC, year of IVF cycle, double trophectoderm biopsy, and fully hatched blastocysts remained statistically significant., Conclusion: Euploid embryos that are fully hatched at time of vitrification, come from patients with high ovarian reserve, or require repeat trophectoderm biopsy are less likely to survive vitrification-warming. Our results provide a framework for reproductive counseling and offer realistic expectations to patients about the number of embryos needed to achieve family building goals., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Previous studies have reported that the mitochondrial DNA (mtDNA) contents of cumulus cells (CCs) in ovarian follicular fluid are correlated with embryo quality. Quantification of mtDNA CCs has been suggested as a biomarker of embryo viability. The aim of this study was to determine the relationship between mitochondrial DNA (mtDNA)/genomic DNA (gDNA) ratio in CCs and IVF outcomes such as fertilization rates and embryo quality in infertile women. This is an observational study on 144 cumulus-oocyte complexes obtained from 144 patients undergoing IVF-intracytoplasmic sperm injection (ICSI) at a single fertility center. The CCs in ovarian follicular fluid from patients undergoing IVF-ICSI were collected by ovum pick-up. A relative copy number quantification was used to determine mtDNA/gDNA ratio. Quantitative real-time PCR for various markers (β2M and mtMinArc gene) was used to determine average mtDNA/gDNA ratio of CCs. Investigation of the correlation between mtDNA/gDNA ratio in CCs and IVF outcomes showed no statistically significant correlation between the mtDNA/gDNA ratio in CCs and fertilization rates. However, mtDNA/gDNA ratio and embryo quality showed a statistically significant positive correlation. A significantly higher mtDNA/gDNA ratio was observed in the good quality embryo group compared with the poor quality embryo group (P < 0.05). In addition, the mtDNA/gDNA ratio showed negative correlation with the patient's age (correlation coefficient= -0.228, P < 0.05). Results of this study demonstrate a negative correlation of mtDNA/gDNA ratio in CCs with patient's age, and a low copy number of mtDNA in CCs may have adverse effects on embryo quality in IVF cycles. These results suggest that the ratio of mtDNA/gDNA in CCs may serve as a biomarker in predicting IVF outcomes., (© 2021. Society for Reproductive Investigation.)

It remained unknown whether HDAC6 affected the histone deacetylation of in vitro maturation oocytes and the reprogramming of nuclear transplantation in pig. Our results indicated that HDAC6 specific inhibition did not affect overall HDAC activity and meiosis process, which increased histone H3K9/K14 and H4K8 acetylation of porcine in vitro maturation oocytes and pseudo-pronucleus embryos. HDAC6 inhibition also significantly enhanced the cleavage and blastocyst of nuclear transfer embryos (0.81 ± 0.12 vs. 0.68 ± 0.12 and 0.46 ± 0.19; 0.73 ± 0.13 vs. 0.63 ± 0.18 and 0.40 ± 0.16, P<0.05). The inhibition of HDAC6 significantly enhanced histone H3K9/K14 and H4K8 acetylation, and upregulated the OCT4 and CDX2 expressions (1.83 ± 0.16 vs. 1.00 ± 0.00 %; 2.07 ± 0.09 vs. 1.00 ± 0.00; P<0.05) in porcine SCNT blastocysts. Interestingly, HDAC6 inhibition significantly increased the pseudo-pronucleus volume during somatic cell reprogramming. Thus, HDAC6 was required for porcine histone deacetylation during the in vitro maturation and pseudo-pronucleus stages. HDAC6 inhibition improved the in vitro development of nuclear transfer embryos. HDAC6 may restrict the reprogramming of somatic nuclear transfer by regulating pseudo-pronucleus expansion. We need further research to confirm this in the future., (© 2021. Society for Reproductive Investigation.)

This study aimed to examine the effects of adding growth hormone (GH) into the in vitro maturation (IVM) culture medium of mouse oocytes on pregnancy outcomes. Cumulus-oocyte complexes (COCs) were cultured in a medium with (GH group, 100 ng/mL) or without (Con group) GH. Thereafter, chromosome morphology, spindle morphology, and mitochondrial function were examined. Embryo development and blastocyst quality after in vitro fertilization were evaluated. After the embryo transfer, the implantation sites and pregnancy outcomes were evaluated. The oocyte maturation rate of the GH group (81.8 ± 9.6%) was compared to that of the Con group (81.3 ± 6.9%, P = 0.928). The proportion of morphologically abnormal spindles in GH-treated oocytes (7.1 ± 0.9%) was significantly lower than control oocytes (13.7 ± 1.3%, P = 0.032), whereas the proportion of morphologically abnormal chromosomes and mitochondrial distribution was similar between the groups. The mitochondrial membrane potential (P < 0.001) and ATP concentration (P < 0.001) in GH-exposed oocytes were higher than those in control oocytes. After fertilization, the blastocyst rate in the GH group (33.8 ± 13.2%) was significantly higher than the Con group (16.2 ± 2.0%, P = 0.003). In addition, inner cell mass (ICM) number (13.91 ± 3.48 vs. 7.00 ± 1.91, P < 0.001), total cell number (47.45 ± 8.39 vs. 37.71 ± 4.15, P = 0.007), and the ratio of ICM/total cell number (29.9 ± 8.2% vs. 18.6 ± 5.0%, P = 0.002) of blastocyst were all higher in GH group. The implantation rate (71.2 ± 1.9% vs. 39.4 ± 16.4%, P < 0.001) and litter size (8.50 ± 3.99 vs. 3.00 ± 1.22, P = 0.018) were significantly higher in the GH group. Although addition of GH into IVM culture medium does not improve oocyte maturation rate, it improves oocyte and embryo quality, which leads to better embryo development and pregnancy outcomes., (© 2021. Society for Reproductive Investigation.)

Data regarding association between early embryo development and maternal age is limited and inconclusive. This study has two aims: to evaluate differences in the cleavage stage of embryos in young versus advanced maternal age (AMA) women. To compare the early embryonic development of embryos that result in pregnancy versus no pregnancy. A retrospective study of early embryonic development which was recorded and analyzed using time-lapse imaging was conducted. The kinetic markers of time to pronuclei fading (tPNf) and appearance of two to eight cells (t2-t8) were assessed. For embryos cultured to blastocyst, times to morula (tM), start of blastulation (tSB) cavitated, and expanded blastocyst (tB, tEB) were also recorded. A total of 2021 oocytes from 364 intracytoplasmic sperm injection (ICSI) cycles were evaluated, of which 1223 (60.5%) were derived from young patients and 798 (39.5%) from those of AMA. The mean time points to t3, t4, t5, t6, tSB, tB, and tEB were significantly shorter for embryos derived from younger women, as compared to older women (p < 0.05). Overall, women who conceived presented a faster embryonic development, for both age groups. The mean time points of t2 and t8 were significantly shorter in patients who conceived versus not conceived (p < 0.05). We concluded that older women's age is associated with delayed embryonic development. Embryos that yielded pregnancy cleaved faster compared to those which did not, in both age groups. Thus, when considering which embryo to transfer to women of AMA, selecting the faster-developing embryos may improve the chances of conception., (© 2021. Society for Reproductive Investigation.)

Purpose: Is the presence of cytoplasmic strings (CS) in human blastocysts associated with the probability of clinical pregnancy with fetal heart (CPFH) after transfer., Methods: This case-control study involved 300 single blastocyst transfers. 150 of these resulted in a CPFH (cases) while 150 did not (controls). All embryos were cultured in Embryoscope+ and AI software (IVY) was used to select the blastocyst with the highest score from the cohort for transfer. An embryologist, blind to the transfer outcome, recorded the CS number, location, and duration of their activity., Results: There was a significant difference in the number of blastocysts that contained CS, with 97.3% of women's blastocysts resulting in +CPFH containing the CS compared to 88.7% of blastocysts in women who did not have a pregnancy (p = 0.007, OR; 4.67, CI 95% 1.5-14.2). CS appeared 2.4 h earlier in embryo development in the +CPFH group compared to their negative counterparts (p = 0.007). There was a significant difference in the average number of CS/blastocyst with a higher number being present in those that achieved a clinical pregnancy (mean: 6.2, SD 2.9) compared to those that did not (mean: 4.6, SD 3.0) (p ≤ 0.0001). There was a significant increase in the number of vesicles seen traveling along the CS with more seen in the blastocysts resulting in a +CPFH (mean: 4.3 SD 2.1) compared to those in the -CPFH group (mean: 3.1, SD 2.1)., Conclusion: This study has shown that the presence of cytoplasmic strings in human blastocysts is associated with the probability of clinical pregnancy with fetal heart., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nlrp2 encodes a protein of the oocyte subcortical maternal complex (SCMC), required for embryo development. We previously showed that loss of maternal Nlrp2 in mice causes subfertility, smaller litters with birth defects, and growth abnormalities in offspring, indicating that Nlrp2 is a maternal effect gene and that all embryos from Nlrp2-deficient females that were cultured in vitro arrested before the blastocysts stage. Here, we used time-lapse microscopy to examine the development of cultured embryos from superovulated Nlrp2-deficient and wild-type mice after in vivo and in vitro fertilization. Embryos from Nlrp2-deficient females had similar abnormal cleavage and fragmentation and arrested by blastocyst stage, irrespective of fertilization mode. This indicates that in vitro fertilization does not further perturb or improve the development of cultured embryos. We also transferred embryos from superovulated Nlrp2-deficient and wild-type females to wild-type recipients to investigate if the abnormal reproductive outcomes of Nlrp2-deficient females are primarily driven by oocyte dysfunction or if a suboptimal intra-uterine milieu is a necessary factor. Pregnancies with transferred embryos from Nlrp2-deficient females produced smaller litters, stillbirths, and offspring with birth defects and growth abnormalities. This indicates that the reproductive phenotype is oocyte-specific and is not rescued by development in a wild-type uterus. We further found abnormal DNA methylation at two maternally imprinted loci in the kidney of surviving young adult offspring, confirming persistent DNA methylation disturbances in surviving offspring. These findings have implications for fertility treatments for women with mutations in NLRP2 and other genes encoding SCMC proteins.

Endometrial angiogenesis plays crucial roles in determining the endometrial receptivity. Defects in endometrial receptivity often cause repeated implantation failure, which is one of the major unmet needs for infertility and contributes a major barrier to the assisted reproductive technology. Despite the numerous extensive research work, there are currently no effective evidence-based treatments to prevent or cure this condition. As a non-invasive treatment strategy, botulinum toxin A (BoTA) was administered into one side of mouse uterine horns, and saline was infused into the other side of horns for the control. Impact of BoTA was assessed in the endometrium at 3 or 8 days after infusion. We demonstrated that BoTA administration enhances the capacity of endothelial cell tube formation and sprouting. The intrauterine BoTA administration significantly induced endometrial angiogenesis displaying increased numbers of vessel formation and expression levels of related marker genes. Moreover, BoTA intrauterine application promoted the endometrial receptivity, and the rates of embryo implantation were improved with BoTA treatment with no morphologically retarded embryos. Intrauterine BoTA treatment has a beneficial effect on vascular reconstruction of functional endometrium prior to embryo implantation by increasing endometrial blood flow near the uterine cavity suggesting BoTA treatment as a potential therapeutic strategy for patients who are suffering from repeated implantation failure with the problems with endometrial receptivity.

The oviducts (fallopian tubes in mammals) function as the site of fertilization and provide necessary support for early embryonic development, mainly via embryonic exposure to the tubal microenvironment. The main objective of this study was to create an oviduct-specific extracellular matrix (oviECM) hydrogel rich in bioactive components that mimics the native environment, thus optimizing the developmental trajectories of cultured embryos. Rabbit oviducts were decellularized through SDS treatment and enzymatic digestion, and the acellular tissue was converted into oviductal pre-gel extracellular matrix (ECM) solutions. Incubation of these solutions at 37 °C resulted in stable hydrogels with a fibrous structure based on scanning electron microscopy. Histological staining, DNA quantification and colorimetric assays confirmed that the decellularized tissue and hydrogels contained no cellular or nuclear components but retained important components of the ECM, e.g. hyaluronic acid, glycoproteins and collagens. To evaluate the ability of oviECM hydrogels to maintain early embryonic development, two-cell rabbit embryos were cultured on oviECM-coated surfaces and compared to those cultured with standard techniques. Embryo development was similar in both conditions, with 95.9% and 98% of the embryos reaching the late morula/early blastocyst stage by 48 h under standard culture and oviECM conditions, respectively. Metabolomic analysis of culture media in the presence or absence of embryos, however, revealed that the oviECM coating may include signalling molecules and release compounds beneficial to embryo metabolism.

Objective: The primary objective of this study was to test the hypotheses that compared to IVF cycles undergoing preimplantation genetic testing for aneuploidy (PGT-A) with or without testing for monogenic disorders (PGT-M), IVF cycles undergoing PGT for structural rearrangements (PGT-SR) will have (1) a poorer blastocyst conversion rate and (2) fewer usable blastocysts available for transfer. Secondarily, the study aimed to compare pregnancy outcomes among PGT groups., Patients: Retrospective cohort study including cycles started from January 1, 2012, to March 30, 2020, with the intent of pursuing PGT-A, PGT-A with PGT-M, and PGT-SR, with trophectoderm biopsy on days 5 or 6., Results: A total of 658 women underwent 902 cycles, including 607 PGT-A, 216 PGT-A&M, and 79 PGT-SR cycles. When compared with the blastocyst conversion rate for the PGT-A group (59.4%), and after adjustment for patient age, total number of mature oocytes, BMI, and ICSI, there were no significant differences for either the PGT-A&M (69.7%, aRR 1.03, 95% CI 0.96-1.10) or PGT-SR (63.2%, aRR1.04, 95% CI 0.96-1.13) groups. Compared to the PGT-A group, the proportion of usable blastocysts was statistically significantly lower in the PGT-SR group: 35.1% versus 24.4% (aRR 0.57, 95% CI 0.46-0.71) and the PGT-A&M group: 35.1% versus 31.5% (aRR 0.68, 95% CI 0.58-0.81). Implantation, pregnancy, and miscarriage rates were equivalent for all groups., Conclusion: Patients with structural rearrangements have similar blastocyst development but significantly fewer usable blastocysts available for transfer compared to PGT-A testers. Nevertheless, with the transfer of a usable embryo, PGT-SR testers perform as well as those testing for PGT-A.

To determine whether embryo developmental stage or morphological grading can predict live birth rate (LBR) from a single blastocyst in nonbiopsied and biopsied frozen embryo transfer (FET) cycles. This retrospective study included 1336 nonbiopsied and 360 euploid FET cycles. Blastocysts were divided according to developmental stage (day 5 [D5] and day 6 [D6]) and morphology (good quality and low quality). Nonbiopsied cycles in which D5 blastocysts were transferred were associated with a significantly higher LBR than those in the D6 group (48.5 vs. 24.3%; p < 0.001), as well as in good-quality embryo transfer cycles than that in low-quality embryo cycles (52.6 vs. 25.3%; p < 0.001). Embryos reaching good-quality blastocysts on D5 yielded significantly higher LBR than those similar quality blastocysts on D6. The same trend was seen in low-quality embryos. Concerning only D5 or D6 blastocyst transfer, the LBRs of good-quality embryos were still superior to those of low-quality embryos. In the case of euploid embryo transfers, the LBR (48.9 vs. 44.9%, p = 0.444) of D5 blastocysts did not significantly differ from that of D6 blastocysts. Good-quality embryos showed a higher LBR than low-quality embryos (51.6 vs. 40.0%, p = 0.030); the adjusted odds ratio remained insignificant after controlling for confounders (aOR 1.56; 95% CI 0.99-2.45; p = 0.056). The LBRs in the same developmental stage or morphology subgroups were not statistically significant. Embryo developmental stage and morphological grade are useful predictors of LBR in nonbiopsied FET cycles. However, no association was found in euploid transfer cycles.

In order to maintain pregnancy rates following single embryo transfer, optimisation of embryo culture and selection is vital. Time-lapse monitoring (TLM) has the potential to play a crucial role by providing sequential images of embryo development and minimal disturbance. Therefore, in this study morphometric assessment of blastocyst area and maximum width was performed in order to evaluate if these parameters are associated with pregnancy outcomes in IVF/ICSI cycles. This is a retrospective study of 664 patients who had elective single blastocyst transfer (eSBT). The EmbryoScope drawing tools were used to measure specific variables such as the maximum blastocyst width and blastocyst area. Our results show that women who were pregnant had significantly (P < 0.01) larger blastocyst width [median (range) μm] 184 (125-239) versus non-pregnant, 160 (120-230)] and area [median (range) μm 2 ] 26099 (12101-45,280) versus non-pregnant women, 22,251 (10992-37,931)]. A univariate logistic regression performed showed that blastocyst width [(OR = 1.026, 95% CI = (1.019, 1.033)] was significant (P < 0.01) and for every μm increase of blastocyst width, the odds of clinical pregnancy increase by 2.6%. A univariate logistic regression performed showed that blastocyst area [(OR = 1.00008, 95% CI = (1.00006, 1.00011)] was significant with P < 0.01. For every μm 2 increase of blastocyst area, our data showed the odds of clinical pregnancy increase by 0.008%. Hosmer-Lemeshow tests of calibrations were performed to verify calibration. Although our findings show a clear correlation between blastocyst dimensions and the clinical pregnancy rate, further studies are necessary to confirm these observations.

With the recent increased utilization of oocyte vitrification for the purpose of fertility preservation, information regarding the future fertility potential of the frozen oocytes is mandatory. Nowadays, there is a relative lack of data about prediction of assisted reproductive technique (ART) success relying on the retrieved oocytes. In the present study, we therefore aimed to investigate whether oocyte diameter might predict the quality of the developing embryo. A retrospective, single-center cohort study. Oocytes retrieved following controlled ovarian hyperstimulation cycles during 2016 and incubated in a time-lapse incubator system were analyzed. Oocytes were grouped by mean oocyte diameter (MOD) and incubated for 5 days before the final morphological evaluation done by an expert embryologist. A total of 471 cycles which yielded 3355 metaphase II oocytes were included in the analysis. Embryos developed from oocytes with MOD close to the average (Average 1SD < MOD < Average + 1SD) had increased good-quality blastulation rates compared with embryos that developed from very small or very large oocytes. Oocytes with MOD between 105.96 and 118.69 μm have better probability of becoming top-quality D5 blastocysts (17.1-17.4% grade 1 embryos). There is a correlation between oocyte's MOD and the embryo quality at day 5. The oocytes with near average MOD have a better chance to develop to a good-quality embryo. Therefore, the study suggests that MOD might serve as a predictor for embryo grading at day 5.

Purpose: Laevo (l)-carnitine plays important roles in reducing the cytotoxic effects of free fatty acids by forming acyl-carnitine and promoting beta-oxidation, leading to alleviation of cell damage. Recently, the mitochondrial functions in morula has been shown to decrease with the maternal age. Here, we assessed the effect of l-carnitine on mitochondrial function in human embryos and embryo development., Methods: To examine the effect of L-carnitine on mitochondrial function in morulae, 38 vitrified-thawed embryos at the 6-11-cell stage on day 3 after ICSI were donated from 19 couples. Each couple donated two embryos. Two siblings from each couple were divided randomly into two groups and were cultured in medium with or without 1 mM L-carnitine. The oxygen consumption rates (OCRs) were measured at morula stage. The development of 1029 zygotes cultured in medium with or without L-carnitine was prospectively analyzed., Results: Addition of L-carnitine to the culture medium significantly increased the OCRs of morulae and improved the morphologically-good blastocyst formation rate per zygote compared with sibling embryos. Twenty healthy babies were born from embryos cultured in L-carnitine-supplemented medium after single embryo transfers., Conclusion(s): L-carnitine is a promising culture medium supplement that might be able to counteract the decreased mitochondrial function in human morula stage embryos.

Cathecolestrogens are estradiol metabolites produced during folliculogenesis in the mammalian ovary. 2-Hydroxyestradiol (2-OHE 2 ) is one of the most abundant although its role remains unknown. The aim of this study is to investigate whether the presence of 2-OHE 2 during the germinal vesicle-to-metaphase II transition affects oocyte meiotic and preimplantation developmental competence. Mouse cumulus-oocyte complexes (COCs), isolated from fully grown antral follicles, were in vitro-matured (IVM) in the presence of 2-OHE 2 (0.1, 1, 10 or 100 nM) for 6 or 15 h; then, their meiotic and developmental competence was evaluated using a number of cytological quality markers. With the exception of the highest dose (100 nM), the addition of 2-OHE 2 to the IVM medium, did not alter, compared with untreated control, the frequency of oocytes that reached the MII stage. Instead, IVM in the presence of 1 nM 2-OHE 2 highly increased the rate of preimplantation development and blastocyst quality. To understand whether this positive effect could be attributed to the events occurring during meiosis resumption, we analysed a number of specific cytological quality markers of the asymmetric division, such as PB-I volume and position, presence and extension of the cortical F-actin cap, meiotic spindle shape and area, and microtubule organisation centre localisation. The results highlighted how the presence of 1 nM 2-OHE 2 significantly improved the overall cytological organisation required for a correct asymmetric division. Our results contribute a first step to acknowledge a potential role of this estradiol metabolite during the GV-to-MII transition, contributing to the acquisition of oocytes developmental competence.

Purpose: To determine whether maternal age has an impact on monozygotic twinning (MZT) rates in women undergoing single embryo transfer (SET)., Methods: This is a retrospective cohort study analyzed for the incidence of MZT of all clinical pregnancies after a single embryo transfer was carried out between 2014 and 2018. The effect of different assisted reproductive technology (ART) parameters on the incidence of MZT was evaluated., Results: There were a total of 8459 cycles resulting in pregnancy during the study period. Of these pregnancies, 8236 were singletons and 223 were MZT. The preterm birth rate, miscarriage rate, and cesarean section rate were higher in MZT. Birth weight and gestational age at delivery were lower and smaller. In the univariate analysis, the risk of MZT was decreased with frozen embryo transfer (ET). A nonlinear relationship was observed between maternal age and MZT. A negative relationship between maternal age and MZT was observed in the patients' age ≥ 36 years., Conclusion: Advanced maternal age was associated with a lower rate of MZT. A threshold female age of 36 years existed for lower MZT.

Purpose: This study aimed to determine the role of Wnt pathway in mouse embryonic stem cell (mESC) derivation from single blastomeres isolated from eight-cell embryos and in the pluripotency features of the mESC established., Methods: Wnt activator CHIR99021, Wnt inhibitor IWR-1-endo, and MEK inhibitor PD0325901 were used alone or in combination during ESC derivation and maintenance from single blastomeres biopsied from eight-cell embryos. Alkaline phosphatase activity, FGF5 levels, expression of key pluripotency genes, and chimera formation were assessed to determine the pluripotency state of the mESC lines., Results: Derivation efficiencies were highest when combining pairs of inhibitors (15-24.7%) than when using single inhibitors or none (1.4-10.1%). Full naïve pluripotency was only achieved in CHIR- and 2i-treated mESC lines, whereas IWR and PD treatments or the absence of treatment resulted in co-existence of naïve-like and primed-like pluripotency features. IWR + CHIR- and IWR + PD-treated mESC displayed features of primed pluripotency, but IWR + CHIR-treated lines were able to generate germline-competent chimeric mice, resembling the predicted properties of formative pluripotency., Conclusion: Wnt and MAPK pathways have a key role in the successful derivation and pluripotency features of mESC from single precompaction blastomeres. Modulation of these pathways results in mESC lines with various degrees of naïve-like and primed-like pluripotency features.

Purpose: This study aimed to determine the effects of drilling and thinning treatment of laser-assisted hatching on the expression and methylation of imprinted gene IGF2/H19 in embryos and offspring., Methods: The prehatching blastocysts with treatment of drilling or thinning, or control prehatching blastocysts, were transplanted in surrogate uteri. The DNA methylation of IGF2/H19 imprinting control region (ICR) and the expression of IGF2 and H19 were respectively evaluated using bisulfite conversion-mediated sequencing and real-time PCR., Results: The drilling group showed a significant increase in the development rate of hatched blastocysts in comparison with the control and thinning group. DNA methylation level of IGF2/H19 ICR of hatched blastocysts in the thinning group was 27.33% in comparison with the 38.67% and 36% observed in the control and drilling group. The thinning treatment increased the DNA methylation level of IGF2/H19 ICR in the placenta in comparison with the control and drilling group. The drilling and thinning treatment decreased the expression level of H19 mRNA in prehatching and hatched blastocysts as well as placenta, while a significant increase in the expression level of H19 mRNA of offspring was observed in the thinning group. The thinning treatment increased the expression level of IGF2 mRNA of prehatching blastocysts and offspring and a significant decrease in placenta, while the drilling treatment resulted in a significant increase in the expression level of IGF2 mRNA of hatched blastocysts and placenta., Conclusion: These observations suggested that drilling used for hatching of in vitro cultured mouse blastocysts may improve the production of offspring.

Purpose: To establish blastocyst freezing criteria for day 7 blastocyst (day 7 BL) for single vitrified-warmed blastocyst transfer (SVBT) by examining the diameter of blastocysts., Methods: Patients who underwent day 7 BL transfer cycles (1143 cycles, mean age: 38.5 ± 3.5) and randomly selected patients after 1:1 matching who underwent day 6 BL transfer cycles and day 2-single-embryo transfer (SET) cycles were used for analysis. Comparison of the miscarriage (per clinical pregnancy) and live birth rates were made among day 2-SET, day 7 BL, and day 6 BL. These blastocyst groups were stratified into six groups based on blastocyst diameter, namely, 180 μm, 190 μm, 200 μm, 210 μm, over 220 μm, and hatched, for making the freezing criteria., Results: For each diameter, 180 μm, 190 μm, 200 μm, 210 μm, over 220 μm, and hatched, the live birth rates of day 7 BL after SVBT were 9.0%, 11.9%, 11.5%, 15.6%, 20.0%, and 19.9%, respectively. Compared with the 14.6% live birth rate of the day 2-SET group, the live birth rate of 220 μm day 7 BL was significantly higher (P < 0.05) and was around the same in other diameter groups., Conclusion: Our study demonstrates that sufficient live birth rates can be obtained after SVBT even from blastocysts on day 7 when blastocysts were vitrified at expanded blastocyst stage of over 180 μm of diameter or at hatched blastocyst stage and were transferred at the optimal time. This is the first study to establish a day 7 blastocyst freezing criteria using blastocyst diameter, which is an objective assessment way.

Purpose: miRNAs have been suggested as biomarkers of embryo viability; however, findings from preliminary studies are divergent. Furthermore, the presence of other types of small RNA molecules remains to be investigated. The purpose of this study was to perform a comprehensive analysis of small non-coding RNA levels in spent and unconditioned embryo culture media, along with miRNA levels in blastocoelic fluid samples from human embryos., Methods: miRNAs in unconditioned culture medium from 3 different manufacturers, along with miRNA from day 5 conditioned culture medium, control medium, and corresponding blastocoel fluid from 10 human blastocysts were analyzed with array-based q-PCR analysis. Subsequently, deep sequencing of total and small RNA in day 5 spent culture medium from 5 human blastocysts and corresponding controls was performed., Results: In spite of using state-of-the-art sensitive detection methods, no miRNAs were found to be reliably present in the spent culture medium or the blastocoel fluid. C t values were above the recommended limit for detection in the array-based analysis, a finding that was confirmed by deep sequencing. The majority of miRNAs identified by deep sequencing were expressed in all samples including control media and seem to originate from sources other than conditioned IVF media., Conclusions: Our findings question the use of miRNAs as a reliable biomarker and highlight the need for a critical methodological approach in miRNA studies. Interestingly, tiRNA fragments appear to be overexpressed in conditioned IVF media samples and could potentially be a novel biomarker worthy of investigation.

Purpose: Embryo genotyping in IVF clinics aims to identify aneuploid embryos, and current methodologies rely on costly, invasive and time-consuming approaches such as PGT-A screening. MALDI-ToF-based mass spectral analysis of embryo culture has been demonstrated to be a non-invasive, affordable and accurate technique that is able to capture secretome profiles from embryo culture media extremely quick. Thus, aneuploid embryo genotypes can be distinguished from euploids from these profiles towards the development of novel embryo selection tools., Methods: A retrospective cohort study, including 292 spent media samples from embryo cultures collected from a single IVF clinic in USA. There were 149 euploid and 165 aneuploid embryos previously analysed by PGT-A next-generation sequencing techniques. Secretome mass spectra of embryos were generated using MALDI-ToF mass spectrometry in the UK. Data was systematically analysed using a fully automated and ultra-fast bioinformatic pipeline developed for the identification of mass spectral signatures., Results: Distinct spectral patterns were found for euploid and aneuploid genotypes in embryo culture media. We identified 12 characteristic peak signatures for euploid and 17 for aneuploid embryos. Data analysis also revealed a high degree of complementarity among regions showing that 22 regions are required to differentiate between genotypes with a sensitivity of 84% and a false positive rate of 18%., Conclusion: Ultra-fast and fully automated screening of an embryo genotype is possible based on multiple combinations of specific mass spectral peak signatures. This constitutes a breakthrough towards the implementation of non-invasive and ultra-fast tools for embryo selection immediately prior to transfer.

The objective of this study was to reduce the negative effects of oxidative stress by decreasing the levels of reactive oxygen species (ROS) through supplementation of the major antioxidants present in elderberries: kuromanin and cyanidin. Oocytes (n = 1150) were supplemented with 100 or 200 μM of kuromanin or cyanidin during maturation, and then evaluated for ROS levels or fertilized and evaluated for penetration, polyspermic penetration, male pronucleus formation, and embryonic development. The ROS levels and incidence of polyspermic penetration were lower (P < 0.05) in oocytes supplemented with 100 μM cyanidin when compared with other treatments. Supplementation of 100 μM cyanidin increased (P < 0.05) MPN and blastocyst formation compared with other treatments. However, supplementation of 100 μM kuromanin did not have significant effects on the criteria evaluated, and supplementation of 200 μM kuromanin had significant (P < 0.05) detrimental effects for each criterion. Additional oocytes (n = 1438) were supplemented with 100 μM cyanidin during maturation and evaluated for glutathione, glutathione peroxidase, catalase, and superoxide dismutase activity. Supplementation of 100 μM cyanidin increased (P < 0.05) catalase activity and intracellular GSH levels compared with no supplementation of cyanidin. These results indicate that supplementing cyanidin during maturation reduces oxidative stress by reducing ROS levels and increasing GSH concentrations within the oocyte.

Research Question: To investigate whether patient factors influence the decision to freeze a blastocyst with low implantation potential., Design: This experimental study assessed 170 practicing embryologists from a variety of countries who were recruited via an online survey. Participants were currently practicing embryologists, who grade blastocysts as part of this role. The survey presented decision-making 'vignettes' to participants. These included specific patient information, as well as an image of an expanded blastocyst that was of borderline quality for inner cell mass and trophectoderm, for which the embryologist selected whether or not to freeze. High/low maternal age, the presence/absence of other top quality blastocysts, and the presence/absence of previously unsuccessful IVF cycles were systematically varied within the patient information in a 2 × 2 × 2 design. Participants reported how likely they would be to freeze a particular blastocyst on a scale of 1 (Extremely Unlikely) to 7 (Extremely Likely), and whether or not they would ultimately freeze each blastocyst (Yes or No)., Results: Lower maternal age, no other high-quality blastocysts within the cohort, and multiple unsuccessful IVF cycles were associated with greater likelihood of recommending to freeze (P < .001). Furthermore, significant interactions among all three patient factors were noted., Conclusion: This study provides evidence suggesting that when faced with an uncertain blastocyst, factors pertaining to the patient (maternal age, the presence/absence of other top quality blastocysts, and the presence/absence of previously unsuccessful IVF cycles) influence the decision to freeze.

Purpose: The purpose was to investigate the association between embryonic development or implantation and the content of interleukin-6 and 10 (IL-6, IL-10) and tumor necrosis factor-α (TNF-α) in single-blastocyst conditioned medium (SBCM)., Methods: Thirty-eight SBCM samples (SBCMs) were collected from blastocysts with different morphological scores. IL-6, IL-10, and TNF-α concentration in 38 SBCMs was detected by Single Molecule Array and compared according to the blastocyst quality: top-quality (TQ) and non-top quality (NTQ), or blastulation time: day 5 (D5) and day 6 (D6). In another experiment, 61 SBCMs were collected from TQ blastocyst transplanted on D5, and IL-6 concentration in SBCM was compared based on whether embryos are implanted or not (implanted and non-implanted)., Results: In the first experiment, IL-6, IL-10, and TNF-α concentration was not significantly different between the TQ-SBCM and NTQ-SBCM. The D6-SBCM had a higher IL-6 concentration compared with the D5-SBCM, while IL-10 and TNF-α concentration was not significantly different between the D5-SBCM and D6-SBCM. The IL-6 concentration in D5-NTQ or D6-TQ SBCM was higher than that in D5-TQ or D6-NTQ SBCM (P < 0.05), respectively. Furthermore, the spearman analysis demonstrated that IL-6 concentration in SBCM was negatively correlated with the blastocyst quality on D5 and positively correlated with the blastocyst quality on D6. In the second experiment, no significant difference in IL-6 concentration was found between SBCM from implanted and non-implanted blastocyst., Conclusion: IL-6 concentration in SBCM was associated with embryo quality depending on the blastulation time, although it might not be associated with the blastocyst implantation.

Purpose: The aim of this study was to compare the levels of hyperglycosylated human chorionic gonadotropin (hCG-H) secreted from balanced and unbalanced human embryos., Methods: Single-step culture media samples from 155 good quality embryos, derived from 90 good prognosis patients undergoing intracytoplasmic sperm injection (ICSI), were collected on the fifth day of embryo cultivation. All embryos were tested by next-generation sequencing (NGS) technique. The hCG-H levels in the culture media were evaluated by ELISA kit (Cusabio Biotech, CBS-E15803h) according to the manufacturer's instructions. Statistical analysis was performed using SPSS v.21 (IBM Corp., Armonk, NY, USA)., Results: The NGS analysis revealed that 36% of the embryos (n = 56) were balanced, and 64% of the embryos were unbalanced (n = 99). The presence of hCG-H was confirmed in all embryo culture media samples but was absent in the negative control. In addition, hCG-H concentration was significantly higher in the culture media from unbalanced embryos compared with the balanced ones (0.72 ± 0.30 mIU/ml vs. 0.62 ± 0.12 mIU/ml, p = 0.02, respectively). Furthermore, the mean levels of hCG-H were significantly increased in the samples from embryos with multiple abnormalities. Finally, the highest levels of hCG-H were expressed from embryos with monosomy of chromosome 11 (1.28 ± 0.04 mIU/ml) and those with trisomies of chromosomes 21 (2.23 mIU/ml) and 4 (1.02 ± 0.35 mIU/ml)., Conclusion: Our results suggest that chromosomal aberrations in human embryos are associated with an increased secretion of hCG-H. However, hCG-H concentration in embryo culture media as a single biomarker is not sufficient for an accurate selection of balanced embryos.

Purpose: To investigate the between-laboratory reproducibility of embryo selection/deselection effectiveness using qualitative and quantitative time-lapse parameters., Methods: A systematic search was performed on MEDLINE, EMBASE, and the Cochrane Library (up to February 2020) without restriction on date, language, document type, and publication status. Measuring outcomes included implantation, blastulation, good-quality blastocyst formation, and euploid blastocyst., Results: We detected 6 retrospective cohort studies externally validating the first clinical time-lapse model (Meseguer) emphasizing quantitative parameters, of which 3 (including one involving 2 independent centers) were included for the pooled analysis. Receiver operating characteristics analysis showed reduced predictive power of the model when either including or not including sister clinic validation. Fifteen cohort studies evaluating qualitative parameters were included for meta-analysis, and the mean Newcastle-Ottawa Scale was 5.3. Overall, meta-analysis showed significantly adverse association between the presence of ≥ 1 cleavage abnormalities and embryo implantation rates (11 studies, n = 7266; RR = 0.39[0.28, 0.55] 95% CI ; I 2  = 57%). Further analysis showed adverse impacts of direct cleavage (7 studies, n = 7065; RR = 0.28 [0.15, 0.54] 95% CI ; I 2  = 46%), reverse cleavage (2 studies, n = 3622; RR = 0.16 [0.03, 0.75] 95% CI ; I 2  = 0%), chaotic cleavage (2 studies, n = 3643; RR = 0.11 [0.02, 0.69] 95% CI ; I 2  = 24%), and multinucleation (5 studies, n = 2576; RR = 0.59 [0.50, 0.69] 95% CI ; I 2  = 0%), but not the < 6 intercellular contact points at the 4-cell stage (1 study, n = 185; RR = 0.17 [0.02, 1.15] 95% CI )., Conclusions: Qualitative time-lapse parameters are reliably associated with embryo developmental potential among laboratories, whereas the reproducibility of time-lapse embryo selection model that emphasizes quantitative parameters may be compromised when externally applied.

To assess whether morphokinetic features at the cleavage stage together with specific gene expression in cumulus cells (CCs) may be used to predict whether human embryos are able to achieve the expanded blastocyst stage on day 5. Eighty-one embryos were cultured using the Geri plus® time-lapse system. Twenty-seven embryos progressing to the expanded blastocyst stage (BL group) were compared with thirty-five embryos showing developmental arrest (AR group) and nineteen reaching the stage of early or not fully expanded blastocyst (nBL group). The analyzed morphokinetic variables were pronuclear appearance (tPNa), pronuclear fading (tPNf), and completion of cleavage to two, three, four, and eight cells (t2, t3, t4, and t8). CCs were analyzed by RT-qPCR for bone morphogenetic protein 15 (BMP15), cytochrome c oxidase subunit II (COXII), ATP synthase subunit 6 (MT-ATP6), connexin 43 (Cx43), and heme oxygenase-1 (HO-1). Embryos of BL group showed a significantly faster kinetic. BMP15, COXII, and MT-ATP6 mRNA expression was significantly higher in CCs of BL group embryos, whereas Cx43 and HO-1 mRNA levels were higher in AR group. Kinetic parameters and gene expression were not significantly different between either the BL and nBL groups or the AR and nBL groups. ROC curves showed that the most predictive cut-offs were t2 < 26.25 for morphokinetics and COXII > 0.3 for gene expression. Multivariable logistic regression analysis showed that morphokinetic variables and gene expression were both valuable, independent predictors of embryo development to expanded blastocyst. Our results suggest the possibility of developing integrated prediction models for early embryo selection at the cleavage stage.

Purpose: To evaluate whether mtDNA content at the blastocyst stage differs between embryos derived from fresh or vitrified sibling oocytes., Material and Methods: A retrospective analysis was performed between March 2017 and September 2018, including 504 blastocysts from 94 couples undergoing preimplantation genetic testing for aneuploidies (PGT-A), using fresh oocytes together with previously vitrified oocytes. Trophectoderm biopsies were performed and subjected to next generation sequencing., Results: On average, 1.8 ± 1.0 oocyte vitrification cycles were performed per patient. Between fresh and vitrified cycles, no difference was observed between the number of fertilized oocytes (5.3 ± 4.2 versus 5.5 ± 3.0). Blastulation rate on day 5 per fertilized oocyte was significantly higher in the fresh group (62% ± 29% versus 44% ± 31%; p < 0.001). For the 504 biopsied blastocysts, 294 fresh versus 210 vitrified, no significant differences were found in the euploid rate, 40.5% versus 38.6% (p = 0.667), and mtDNA content, 30.1 (± 10.6) versus 30.0 (± 12.5) (p = 0.871), respectively. Regardless of the origin of the oocytes, aneuploid blastocysts contained significantly higher mtDNA values compared with the euploid ones (31.4 versus 28.0; p = 0.001). Furthermore, top-quality blastocysts had a significantly lower mtDNA content compared with moderate and poor-quality blastocysts (p < 0.001) and blastocysts biopsied on day 5 showed significantly lower mtDNA content compared with day 6 or day 7 blastocysts (p < 0.001). However, when analyzing the blastocyst mtDNA content according to the ploidy state, no differences were found for blastocyst quality or day of biopsy between blastocysts originating from fresh or vitrified oocytes., Conclusion: Oocyte vitrification does not affect the mtDNA content of trophectoderm biopsies.

The mouse embryo assay (MEA) has been used in the field of human in vitro fertilization (IVF) for multiple purposes such as developing embryo culture media, quality control within the laboratory, and procedural training and proficiency testing for embryology staff. In addition, manufacturing companies use the MEA as a means of quality control for the development of embryo culture media and medical devices and to meet the standards of testing for FDA approval of new products. It has long been considered by embryologists and laboratory scientists whether the MEA is an accurate or sensitive test in the quality assessment of culture media and medical devices or if use of this testing is more an obligation. There is no uniformly accepted gold standard method for IVF lab quality control or FDA approval. This review aims to revisit the role of the use of mouse embryos in the formulation of IVF media for clinical use and the different methods of employing the mouse embryo assay for quality control. In addition, we will review the use of the MEA as an important adjunct in the training for embryology staff and fellows in training in reproductive endocrinology and infertility (REI), as well as alternatives to the use of the MEA for these purposes.

Purpose: To evaluate the relationship between elective blastocyst transfer, freezing strategy, and the abandonment of frozen embryos with a storage time limit of 10 years as specified in the National Assisted Reproduction Act of Taiwan., Methods: This two-phase retrospective cohort study was conducted at a single tertiary center, Kaohsiung Chang Gung Memorial Hospital (KCGMH), in 2019. Participants were selected from a data registry containing 4167 fresh IVF cycles, including phase 1 cycles from 1999 to 2009 and phase 2 cycles from 2010 to 2014, at KCGMH., Results: In phase 1, embryo abandonment was associated with the production of more mature oocytes and embryos, the freezing of more embryos, young female age, blastocyst transfer, and positive pregnancy results. After adjustment for confounding factors, only positive pregnancy results (adjusted odds ratio [aOR] 4.38, 95% confidence interval [CI] 3.17, 6.04), the freezing of ≥ 2 embryos (aOR 3.68, 95% CI 3.10, 4.38), the production of ≥ 6 embryos (aOR 1.68, 95% CI 1.03, 2.73), and the use blastocyst transfer (aOR 2.46, 95% CI 1.64, 3.69) remained significantly associated with embryo abandonment. The factors associated with embryo abandonment or possible abandonment were similar in phase 2., Conclusion: For elective blastocyst stage transfer and a freezing strategy performed according to the Taiwan National Assisted Reproduction Act, a young female age ≤ 35 with positive pregnancy status due to the original IVF treatment, the production of ≥ 6 embryos, and the cryopreservation of ≥ 2 blastocysts may increase the likelihood of abandoning embryos in the future.

The frequency of congenital malformations is 3-5 times higher in mothers with pregestational diabetes mellitus than in general population. Apparently, this problem is due to change in the expression of apoptotic and antiapoptotic genes induced by the oxidative stress derived from the diabetes/hyperglycemia. One of these genes is Bcl-2, which is associated with the control and inhibition of apoptosis. The purpose of the present work was to study the effect of polyamine addition over expression of Bcl-2 gene in a model of diabetic embryopathy. For this, gestational day 10.5 (GD10.5) rat embryos were incubated at 37°C for 24 h in control medium, medium with high glucose, or medium with high glucose and supplemented with spermidine or spermine. Post-cultured embryos were harvested and observed to obtain morphological scores; some of them were subjected to molecular biology studies: DNA isolation plus conventional PCR or RNA isolation plus RT-PCR; other embryos were fixed with paraformaldehyde and used for immunohistochemical detection of Bcl-2 protein. Although Bcl-2 mRNA was similarly expressed in all rat embryo treatments, Bcl-2 protein was found only in control-incubated embryos. In conclusion, it seems that the inhibition of Bcl-2 gene expression induced by glucose was not reversed by polyamines.

Leptin, a metabolic hormone, regulates the reproductive functions responding to both nutritional and body conditions. Embryonic stem cells play important roles in reproductive technology, but their derivation can be challenging. In this study, we evaluated the derivation rates of mouse embryonic stem cell (mESC) line from blastocysts developing in embryo culture media supplemented with different leptin concentrations. The results showed that addition of leptin into the embryo culture medium supported the in vitro development of mouse embryo. The mESC line derivation rates for media treated with 0, 10, 50, and 100 ng/ml of leptin were 61.24 % (54/88), 84.96 % (42/50), 81.79 % (61/76), and 85.78 % (56/67), respectively. In addition, leptin treatment of blastocysts upregulated the expression levels of the trophectoderm marker Cdx2, whereas inner cell mass markers Oct-4 and Nanog were not affected. mESC lines derived after leptin treatment demonstrated hallmarks of pluripotency, such as alkaline phosphatase activity, expression of, OCT4, NANOG, and SSEA1, as well as the ability to form embryoid bodies and well-differentiated teratomas. In conclusion, leptin has a positive effect on the derivation rate of mouse embryonic stem cell lines which may be, in part, due to its effects on the development of the trophectoderm cell lineage in the embryo.

Purpose: This study was to evaluate if spent culture media (SCM) of embryos could be used as a non-invasive tool to achieve aneuploidy screening. Ploidy calls, as well as concordance rates between PGT-A results from trophectoderm (TE) and SCM, were compared. Clinical outcomes of single euploid transfers were also evaluated., Methods: The study was conducted from March 2017 to June 2018 in a university-based ART center. SCM of day 3 to the day(s) of TE biopsy of all biopsied blastocysts were collected for testing. PGT-A results of SCM were compared with the standard results of TE, with clinical relevance and outcomes examined., Results: NiPGT-A using SCM gave a sensitivity of 81.6%, specificity of 48.3%, positive predictive value of 82.6%, and negative predictive value of 46.7% in ploidy calling. The concordance rates for autosomes and sex determination were 62.1% and 82.4%, respectively. There were 14 single embryo transfer cycles of euploids as determined by TE biopsy. Clinical outcomes not only confirmed 3 false positive results from SCM but also reflected the true ploidy status of the transferred embryo in one case. If ploidy calls were dichotomized without mosaic embryos, the sensitivity and NPV would increase to 91.0% and 66.7% (p = 0.60 and p = 0.25), respectively., Conclusions: Cell-free DNA found in SCM could provide ploidy information of an embryo as in PGT-A from its TE. Given its potential to reflect the comprehensive chromosomal profile of the whole embryo, more research based on clinical outcomes is required to determine if SCM could be a reliable selection tool in PGT-A.

Purpose: We first assessed regulation of FGF2 expression in cumulus cells by FSH and oocyte-secreted factors during in vitro maturation (IVM). Then, we tested the hypothesis that FGF2 regulates meiotic progression, cumulus expansion, and apoptosis in cumulus-oocyte complexes (COC) undergoing IVM., Methods: In vitro maturation of bovine COC was utilized as a model to assess regulation of FGF2 expression by FSH and oocyte-secreted factors (via microsurgical removal of the oocyte), as well as effects of graded doses of FGF2 on meiotic progression, degree of cumulus expansion, dissociation of cumulus cells, and cumulus cells apoptosis. Expression of genes regulating functional endpoints altered by FGF2 treatment was assessed in cumulus cells by real-time PCR. Cultures were replicated 4-5 times and effects of treatments were tested by ANOVA., Results: FGF2 mRNA expression was increased by FSH and oocyte-secreted factors during IVM. Addition of FGF2 to the IVM medium advanced meiosis resumption, decreased the ease with which cumulus cells were dissociated, and inhibited cumulus cells apoptosis. Decreased cumulus dissociation was accompanied by decreased expression of TNFAIP6., Conclusions: This is the first study showing that FGF2 expression is regulated by the oocyte in cumulus cells. Moreover, we report novel effects of FGF2 on cumulus cell survival and extracellular matrix (ECM) quality during IVM that may favor acquisition of developmental competence and suggest physiological roles during the final steps of COC differentiation.