This study aimed to evaluate the effect of chemical gasification and HEPES as alternative systems to pH control during in vitro maturation on bovine oocytes competence. Groups of 20 bovine cumulus oocytes complexes (COCs) were randomly distributed and cultured for 24 h in one of the following experimental groups: (i) chemical reaction (ChRG) system: CO2 generated from sodium bicarbonate and citric acid reaction (ii) culture media TCM-HEPES (HEPES-G); and (iii) control group (CNTG) in conventional incubator. After in vitro maturation (IVM), the COCs were in vitro fertilized (IVF), and in vitro cultivated (IVC) in a conventional incubator. We evaluated oocyte nuclear maturation, cleavage and blastocyst rates, in addition to the relative mRNA expression of BAX, BMP-15, AREG and EREG genes in oocytes and cumulus cells. The proportion of oocytes in metaphase II was higher in CNTG and ChRG (77.57% and 77.06%) than in the HEPES-G (65.32%; p = .0408 and .0492, respectively). The blastocyst production was similar between CNTG and ChRG (26.20% and 28.47%; p = .4232) and lower (p = .001) in the HEPES-G (18.71%). The relative mRNA expression of BAX gene in cumulus cells was significantly higher (p = .0190) in the HEPES-G compared to the CNTG. Additionally, the relative mRNA expression of BMP-15 gene was lower (p = .03) in oocytes from HEPES-G compared to the CNTG. In conclusion, inadequate atmosphere control has a detrimental effect on oocyte maturation. Yet, the use of chemical gasification can be an efficient alternative to bovine COCs cultivation.

This study used the brilliant cresyl blue (BCB) staining method to group buffalo oocytes (BCB+ and BCB-) and perform in vitro maturation, in vitro fertilization and embryo culture. At the same time, molecular biology techniques were used to detect gap junction protein expression and oxidative stress-related indicators to explore the molecular mechanism of BCB staining to predict oocyte developmental potential. The techniques of buffalo oocytes to analyse their developmental potential and used immunofluorescence staining to detect the expression level of CX43 protein, DCFH-DA probe staining to detect ROS levels and qPCR to detect the expression levels of the antioxidant-related genes SOD2 and GPX1. Our results showed that the in vitro maturation rate, embryo cleavage rate and blastocyst rate of buffalo oocytes in the BCB+ group were significantly higher than those in the BCB- group and the control group (p < .05). The expression level of CX43 protein in the BCB+ group was higher than that in the BCB- group both before and after maturation (p < .05). The intensity of ROS in the BCB+ group was significantly lower than that in the BCB- group (p < .05), and the expression levels of the antioxidant-related genes SOD2 and GPX1 in the BCB+ group were significantly higher than those in the BCB- group (p < .05). Brilliant cresyl blue staining could effectively predict the developmental potential of buffalo oocytes. The results of BCB staining were positively correlated with the expression of gap junction protein and antioxidant-related genes and negatively correlated with the reactive oxygen species level, suggesting that the mechanism of BCB staining in predicting the developmental potential of buffalo oocytes might be closely related to antioxidant activity.

BACKGROUND: For in vitro fertilization (IVF), mitochondrial DNA (mtDNA) levels in the trophectodermal (TE) cells of biopsied blastocysts have been suggested to be associated with the cells\' developmental potential. However, scholars have reached differing opinions regarding the use of mtDNA levels as a reliable biomarker for predicting IVF outcomes. Therefore, this study aims to assess the association of mitochondrial copy number measured by mitoscore associated with embryonic developmental characteristics and ploidy.
METHODS: This retrospective study analyzed the developmental characteristics of embryos and mtDNA levels in biopsied trophectodermal cells. The analysis was carried out using time-lapse monitoring and next-generation sequencing from September 2021 to September 2022. Five hundred and fifteen blastocysts were biopsied from 88 patients undergoing IVF who met the inclusion criteria. Embryonic morphokinetics and morphology were evaluated at 118 h after insemination using all recorded images. Blastocysts with appropriate morphology on day 5 or 6 underwent TE biopsy and preimplantation genetic testing for aneuploidy (PGT-A). Statistical analysis involved generalized estimating equations, Pearson\'s chi-squared test, Fisher\'s exact test, and Kruskal-Wallis test, with a significance level set at P < 0.05.
RESULTS: To examine differences in embryonic characteristics between blastocysts with low versus high mitoscores, the blastocysts were divided into quartiles based on their mitoscore. Regarding morphokinetic characteristics, no significant differences in most developmental kinetics and observed cleavage dysmorphisms were discovered. However, blastocysts in mitoscore group 1 had a longer time for reaching 3-cell stage after tPNf (t3; median: 14.4 h) than did those in mitoscore group 2 (median: 13.8 h) and a longer second cell cycle (CC2; median: 11.7 h) than did blastocysts in mitoscore groups 2 (median: 11.3 h) and 4 (median: 11.4 h; P < 0.05). Moreover, blastocysts in mitoscore group 4 had a lower euploid rate (22.6%) and a higher aneuploid rate (59.1%) than did those in the other mitoscore groups (39.6-49.3% and 30.3-43.2%; P < 0.05). The rate of whole-chromosomal alterations in mitoscore group 4 (63.4%) was higher than that in mitoscore groups 1 (47.3%) and 2 (40.1%; P < 0.05). A multivariate logistic regression model was used to analyze associations between the mitoscore and euploidy of elective blastocysts. After accounting for factors that could potentially affect the outcome, the mitoscore still exhibited a negative association with the likelihood of euploidy (adjusted OR = 0.581, 95% CI: 0.396-0.854; P = 0.006).
CONCLUSIONS: Blastocysts with varying levels of mitochondrial DNA, identified through biopsies, displayed similar characteristics in their early preimplantation development as observed through time-lapse imaging. However, the mitochondrial DNA level determined by the mitoscore can be used as a standalone predictor of euploidy.

In vivo studies offer a detailed understanding of organism functioning, surpassing the insights provided by in vitro studies. These experiments are crucial for comprehending disease emergence, progression, and associated mechanisms in humans, as well as for developing treatments. When choosing experimental models, factors such as genomic similarity, physiological relevance, ethical appropriateness, and economic feasibility must be considered. Standardized protocols enhance the reliability, and reproducibility of scientific methods, promoting the assessment of research in the scientific literature. Researchers conducting embryo studies should establish and document standardized protocols for increased data comparability. Standardization is vital for scientific validity, reproducibility, and comparability in both in vivo and in vitro studies, ensuring the accuracy and reliability of experimental results and advancing scientific knowledge.

Objective: To explore the correlation between blastomere count variations \"skip value\" which extracted from by time-lapse technology (TLT) combined with artificial intelligence (AI) and morphological features of in vitro fertilization (IVF) embryo, and to test its feasibility in clinical applications. Methods: This study was a diagnostic experiment (AI reassessment of embryo transferred patients), a total of 6 545 embryos from 1 226 patients who underwent IVF at the Women and Children\'s Hospital of Chongqing Medical University from December 2020 to December 2021 were retrospectively analyzed, of which 2 869 embryos were attempted to cultured to blastocyst stage by TLT. The embryo dynamic map (EDM) was drawn by Embryo Viewer, a TLT recording software, based on embryo developmental kinetics. The self-developed AI embryo evaluation software identified and recorded the number of cleavages in real time during embryonic development, and compared with the EDM, the correlation between the skip value formed by the change of cleavage sphere counts and the outcomes of the embryos was analyzed. The correlation among skip value, morphological score of embryo, implantation rate and live birth rate were performed by Spearman and step-up logistic regression. The receiver operating characteristic (ROC) curve was selected for reporting there relationship of skip value and morphology. Finally, predicting power of skip value for implantation and live birth rate were performed by ROC analysis. Results: The total skip values extracted from the blastomere count of embryos (72 hours post-fertilization) were negatively correlated with abnormal cleavage, blastocyst formation rate, day 3 (D3)-cell score, uneven size and fragmentation (the β values were -0.268, -0.116, -0.213, -0.159 and -0.222, respectively; all P<0.001); positively correlated with D3-cell number (β=0.034; P<0.001); negatively correlated with blastocyst formation rate and implantation rate (OR=0.97, 95%CI: 0.93-0.99, P=0.034; OR=0.96, 95%CI: 0.93-0.98, P=0.044). The power of predicting implantation were similar between the order selection of skip values and traditional morphology criteria [area under curve (AUC): 0.679 vs 0.620]. Live birth rate were negatively correlated with female age (OR=0.91, 95%CI: 0.88-0.93; P<0.001), D3 general score (OR=0.77, 95%CI: 0.59-0.99; P=0.045) and order selection of skip values (OR=0.98, 95%CI: 0.96-0.99; P=0.038), while positively correlated with retrieved oocyte number and endometrial thickness in embryo transferred (OR=1.08, 95%CI:1.05-1.11, P<0.001; OR=1.09, 95%CI:1.06-0.12, P<0.001, respectively) from multivariate regression analysis, and the power of predicting live birth was 0.666 for AUC. Conclusions: The skip value and its order form is a systematic quantification of embryo development, correlated with embryo developmental quality and clinical outcome. It could be an addition parameter for embryo culture and selection.
目的： 通过胚胎时差成像技术（TLT）结合人工智能（AI）探索体外胚胎发育曲线（EDM）中的卵裂球计数曲线“跳变值”与胚胎形态学特征的相关性，并探讨其在体外受精（IVF）胚胎评估中应用的可行性。 方法： 本研究为自主研发的AI胚胎评估软件的诊断性试验，即临床数据AI再评估，为回顾性研究。收集2020年12月至2021年12月于重庆医科大学附属妇女儿童医院生殖医学中心行IVF的1 226例患者共6 545个胚胎，其中2 869个胚胎进行囊胚培养。所有胚胎通过TLT培养并进行观察，由TLT记录软件Embryo Viewer依据胚胎发育动力学绘制EDM。本课题组自主研发的AI胚胎评估软件对胚胎发育过程中的卵裂球数目进行实时识别、记录，并与EDM进行比较，分析由卵裂球计数变化而形成的跳变值与胚胎结局的相关性。采用Spearman相关性及logistic回归分析跳变值与胚胎形态学评估、胚胎着床及活产的相关性，使用受试者工作特征（ROC）曲线评估各指标对胚胎着床、活产的预测价值。 结果： Spearman相关性分析显示，受精卵受精后72 h卵裂球计数曲线提取的总跳变值与卵裂模式、囊胚形成、培养至第3天胚胎（D3胚胎）的形态学评级、D3胚胎的细胞均一度、D3胚胎的细胞碎片率均呈显著负相关（β值分别为-0.268、-0.116、-0.213、-0.159、-0.222；P均<0.001），与D3胚胎的细胞数呈显著正相关（β=0.034；P<0.001）。多因素logistic回归分析显示，胚胎总跳变值排序与囊胚形成（OR=0.97，95%CI为0.93~0.99；P=0.034）、胚胎着床（OR=0.96，95%CI为0.93~0.98；P=0.044）呈显著负相关；ROC曲线分析显示，胚胎总跳变值排序预测胚胎着床能力与传统胚胎形态学评估方法相近［其曲线下面积（AUC）分别为0.679、0.620］。多因素logistic回归分析显示，活产与患者年龄（OR=0.91，95%CI为0.88~0.93；P<0.001）、D3胚胎形态学评级（OR=0.77，95%CI为0.59~0.99；P=0.045）及胚胎总跳变值排序（OR=0.98，95%CI为0.96~0.99；P=0.038）呈显著负相关，而与获卵数（OR=1.08，95%CI为1.05~1.11；P<0.001）及移植日子宫内膜厚度（OR=1.09，95%CI为1.06~1.12；P<0.001）呈显著正相关；ROC曲线分析显示，纳入建模的以上多个因素预测活产的AUC值为0.666。 结论： 跳变值及其排序是对体外胚胎卵裂期发育的系统量化，其与胚胎发育质量及临床结局相关，可能是胚胎体外评估及选择的有益补充。.

Somatic cell nuclear transfer (SCNT) is one of the primary methods for production of genetically engineered sheep, which allows for gene editing or transgene introduction in somatic cells. The use of SCNT eliminates the risk of genetic mosaicism in embryos and animals that is commonly observed after zygote micromanipulations. This retrospective analysis of SCNT in sheep performed at Utah State University, spanning from 2016 to 2021, examined parameters that may impact pregnancy and full-term development, including donor oocytes (donor age), donor cell lines, SCNT parameters (time of oocyte activation following SCNT, number of transferred embryos, in vitro maturation and culture conditions), and recipients (surgical number and ovulatory status), as well as factors that may correlate with large offspring syndrome or abnormal offspring syndrome (LOS/AOS) in the fetuses and lambs. Our findings indicated that compared to prepubertal oocytes, the SCNT embryos produced from adult sheep oocytes had comparable in vitro maturation rates, pregnancy and full-term development rates, as well as SCNT efficiency. In addition, earlier activation time of SCNT embryos (e.g. 24-26 h post maturation) was correlated to the early pregnancy loss rate, full-term rate, and SCNT efficiency. Compared to our standard serum-containing medium, commercial serum-free culture medium showed a positive correlation with the full-term development of sheep SCNT embryos. Transferring 15-30 embryos per recipient resulted in consistently good pregnancy rates. Surgical numbers and ovulatory status (having at least one follicle between 6 and 12 mm in size or a corpus hemorrhagicum (CH)) of recipients did not affect pregnancy and full-term development rates. In summary, this retrospective analysis identified parameters for improving pregnancy and full-term development of SCNT embryos in sheep.

BACKGROUND: Time-lapse imaging systems for embryo incubation and selection might improve outcomes of in-vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) treatment due to undisturbed embryo culture conditions, improved embryo selection, or both. However, the benefit remains uncertain. We aimed to evaluate the effectiveness of time-lapse imaging systems providing undisturbed culture and embryo selection, and time-lapse imaging systems providing only undisturbed culture, and compared each with standard care without time-lapse imaging.
METHODS: We conducted a multicentre, three-parallel-group, double-blind, randomised controlled trial in participants undergoing IVF or ICSI at seven IVF centres in the UK and Hong Kong. Embryologists randomly assigned participants using a web-based system, stratified by clinic in a 1:1:1 ratio to the time-lapse imaging system for undisturbed culture and embryo selection (time-lapse imaging group), time-lapse imaging system for undisturbed culture alone (undisturbed culture group), and standard care without time-lapse imaging (control group). Women were required to be aged 18-42 years and men (ie, their partners) 18 years or older. Couples had to be receiving their first, second, or third IVF or ICSI treatment and could not participate if using donor gametes. Participants and trial staff were masked to group assignment, embryologists were not. The primary outcome was live birth. We performed analyses using the intention-to-treat principle and reported the main analysis in participants with primary outcome data available (full analysis set). The trial is registered on the International Trials Registry (ISRCTN17792989) and is now closed.
RESULTS: 1575 participants were randomly assigned to treatment groups (525 participants per group) between June 21, 2018, and Sept 30, 2022. The live birth rates were 33·7% (175/520) in the time-lapse imaging group, 36·6% (189/516) in the undisturbed culture group, and 33·0% (172/522) in the standard care group. The adjusted odds ratio was 1·04 (97·5% CI 0·73 to 1·47) for time-lapse imaging arm versus control and 1·20 (0·85 to 1·70) for undisturbed culture versus control. The risk reduction for the absolute difference was 0·7 percentage points (97·5% CI -5·85 to 7·25) between the time-lapse imaging and standard care groups and 3·6 percentage points (-3·02 to 10·22) between the undisturbed culture and standard care groups. 79 serious adverse events unrelated to the trial were reported (n=28 in time-lapse imaging, n=27 in undisturbed culture, and n=24 in standard care).
CONCLUSIONS: In women undergoing IVF or ICSI treatment, the use of time-lapse imaging systems for embryo culture and selection does not significantly increase the odds of live birth compared with standard care without time-lapse imaging.
BACKGROUND: Barts Charity, Pharmasure Pharmaceuticals, Hong Kong OG Trust Fund, Hong Kong Health and Medical Research Fund, Hong Kong Matching Fund.

Two methods for preimplantation genetic testing (PGT) have been described for equine embryos: trophoblast cell biopsy (TCB) or blastocoele fluid aspiration (BFA). While TCB is widely applied for both in vivo- and in vitro-produced embryos, BFA has been mostly utilized for in vivo-produced embryos. Alternative methods for PGT, including analysis of cell-free DNA (CFD) in the medium where in vitro-produced embryos are cultured, have been reported in humans but not for equine embryos. In Experiment 1, in vivo- (n = 10) and in vitro-produced (n = 13) equine embryos were subjected to BFA, cultured for 24 h, then subjected to TCB, and cultured for additional 24 h. No detrimental effect on embryonic diameter or re-expansion rates was observed for either embryo group (P > 0.05). In Experiment 2, the concordance (i.e., agreement on detecting the same embryonic sex using two techniques) among BFA, TCB, and the whole embryo (Whole) was studied by detecting the sex-determining region Y (SRY) or testis-specific y-encoded protein 1 (TSPY) (Y-chromosome), and androgen receptor (AR; X-chromosome) genes using PCR. Overall, a higher concordance for detecting embryonic sex was observed among techniques for in vivo-produced embryos (67-100 %; n = 14 embryos) than for in vitro-produced embryos (31-92 %; n = 13 embryos). The concordance between sample types increased when utilizing TSPY (77-100 %) instead of SRY (31-100 %) as target gene. In Experiment 3, CFD analysis was performed on in vitro-produced embryos to determine embryonic sex via PCR (SRY [Y-chromosome] and amelogenin - AMEL [X- and Y-chromosomes]). Overall, CFD was detected in all medium samples, and the concordance between CFD sample and the whole embryo was 60 % when utilizing SRY and AMEL genes. In conclusion, equine embryos can be subjected to two biopsy procedures (24 h apart) without apparent detrimental effects on embryonic size. For in vivo-, but not for in vitro-produced equine embryos, BFA can be considered a potential alternative to TCB for PGT. Finally, CFD can be further explored as a non-invasive method for PGT in in vitro produced equine embryos.

BACKGROUND: The occurrence of blastocyst collapse may become an indicator of preimplantation embryo quality assessment. It has been reported that collapsing blastocysts can lead to higher rates of aneuploidy and poorer clinical outcomes, but more large-scale studies are needed to explore this relationship. This study explored the characteristics of blastocyst collapse identified and quantified by artificial intelligence and explored the associations between blastocyst collapse and embryo ploidy, morphological quality, and clinical outcomes.
METHODS: This observational study included data from 3288 biopsied blastocysts in 1071 time-lapse preimplantation genetic testing cycles performed between January 2019 and February 2023 at a single academic fertility center. All transferred blastocysts are euploid blastocysts. The artificial intelligence recognized blastocyst collapse in time-lapse microscopy videos and then registered the collapsing times, and the start time, the recovery duration, the shrinkage percentage of each collapse. The effects of blastocyst collapse and embryo ploidy, pregnancy, live birth, miscarriage, and embryo quality were studied using available data from 1196 euploid embryos and 1300 aneuploid embryos.
RESULTS: 5.6% of blastocysts collapsed at least once only before the full blastocyst formation (tB), 19.4% collapsed at least once only after tB, and 3.1% collapsed both before and after tB. Multiple collapses of blastocysts after tB (times ≥ 2) are associated with higher aneuploid rates (54.6%, P > 0.05; 70.5%, P < 0.001; 72.5%, P = 0.004; and 71.4%, P = 0.049 in blastocysts collapsed 1, 2, 3 or ≥ 4 times), which remained significant after adjustment for confounders (OR = 2.597, 95% CI 1.464-4.607, P = 0.001). Analysis of the aneuploid embryos showed a higher ratio of collapses and multiple collapses after tB in monosomies and embryos with subchromosomal deletion of segmental nature (P < 0.001). Blastocyst collapse was associated with delayed embryonic development and declined blastocyst quality. There is no significant difference in pregnancy and live birth rates between collapsing and non-collapsing blastocysts.
CONCLUSIONS: Blastocyst collapse is common during blastocyst development. This study underlined that multiple blastocyst collapses after tB may be an independent risk factor for aneuploidy which should be taken into account by clinicians and embryologists when selecting blastocysts for transfer.

An effective tool to assess embryo quality in the assisted reproduction clinical practice will enhance successful implantation rates and mitigate high risks of multiple pregnancies. Potential biomarkers secreted into culture medium (CM) during embryo development enable rapid and noninvasive methods of assessing embryo quality. However, small volumes, low biomolecule concentrations, and impurity interference collectively preclude the identification of quality-related biomarkers in single blastocyst CM. Here, we developed a noninvasive trace multiomics approach to screen for potential markers in individual human blastocyst CM. We collected 84 CM samples and divided them into high-quality (HQ) and low-quality (LQ) groups. We evaluated the differentially expressed proteins (DEPs) and metabolites (DEMs) in HQ and LQ CM. A total of 504 proteins and 189 metabolites were detected in individual blastocyst CM. Moreover, 9 DEPs and 32 DEMs were identified in different quality embryo CM. We also categorized HQ embryos into positive implantation (PI) and negative implantation (NI) groups based on ultrasound findings on day 28. We identified 41 DEPs and 4 DEMs associated with clinical implantation outcomes in morphologically HQ embryos using a multiomics analysis approach. This study provides a noninvasive multiomics analysis technique and identifies potential biomarkers for clinical embryo developmental quality assessment.