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In the last four decades, the assisted reproductive technology (ART) field has witnessed advances, resulting in improving pregnancy rates and diminishing complications, in particular reduced incidence of multiple births. These improvements are secondary to advanced knowledge on embryonic physiology and metabolism, resulting in the ability to design new and improved culture conditions. Indeed, the incubator represents only a surrogate of the oviduct and uterus, and the culture conditions are only imitating the physiological environment of the female reproductive tract. In vivo, the embryo travels through a dynamic and changing environment from the oviduct to the uterus, while in vitro, the embryo is cultured in a static fashion. Importantly, while culture media play a critical role in optimising embryo development, a large host of additional factors are equally important. Additional potential variables, including but not limited to pH, temperature, osmolality, gas concentrations and light exposure need to be carefully controlled to prevent stress and permit optimal implantation potential. This manuscript will provide an overview of how different current culture conditions may affect oocyte and embryo viability with particular focus on human literature.

The preimplantation stage of development is exquisitely sensitive to environmental stresses, and changes occurring during this developmental phase may have long-term health effects. Animal studies indicate that IVF offspring display metabolic alterations, including hypertension, glucose intolerance and cardiac hypertrophy, often in a sexual dimorphic fashion. The detailed nature of epigenetic changes following in-vitro culture is, however, unknown. This study was performed to evaluate the epigenetic (using whole-genome bisulfite sequencing (WGBS) and assay for transposase-accessible chromatin using sequencing (ATAC-seq)) and transcriptomic changes (using RNA-seq) occurring in the inner cell mass (ICM) of male or female mouse embryos generated in vivo or by IVF. We found that the ICM of IVF embryos, compared to the in-vivo ICM, differed in 3% of differentially methylated regions (DMRs), of which 0.1% were located on CpG islands. ATAC-seq revealed that 293 regions were more accessible and 101 were less accessible in IVF embryos, while RNA-seq revealed that 21 genes were differentially regulated in IVF embryos. Functional enrichment analysis revealed that stress signalling (STAT and NF-kB signalling), developmental processes and cardiac hypertrophy signalling showed consistent changes in WGBS and ATAC-seq platforms. In contrast, male and female embryos showed minimal changes. Male ICM had an increased number of significantly hyper-methylated DMRs, while only 27 regions showed different chromatin accessibility and only one gene was differentially expressed. In summary, this study provides the first comprehensive analysis of DNA methylation, chromatin accessibility and RNA expression changes induced by IVF in male and female ICMs. This dataset can be of value to all researchers interested in the developmental origin of health and disease (DOHaD) hypothesis and might lead to a better understanding of how early embryonic manipulation may affect adult health.

Full-grown oocytes are transcriptionally quiescent. Following maturation and fertilization, the early stages of embryonic development occur in the absence (or low levels) of transcription that results in a period of development relying on maternally derived products (e.g., mRNAs and proteins). Two critical steps occur during the transition from maternal to embryo control of development: maternal mRNA clearance and embryonic genome activation with an associated dramatic reprogramming of gene expression required for further development. By combining an RNA polymerase II inhibitor with RNA sequencing, we were able not only to distinguish maternally derived from embryonic transcripts in bovine preimplantation embryos but also to establish that embryonic gene activation is required for clearance of maternal mRNAs as well as to identify putative transcription factors that are likely critical for early bovine development.

Study questionDoes the oxygen concentration in the culture medium [either physiologic (5%) or atmospheric (20%)] affect mitochondrial ultrastructure and function in preimplantation mouse embryos generated by IVF?Summary answerEmbryos cultured in 20% oxygen show increased mitochondrial abnormalities compared to embryos cultured in 5% oxygen.What is known alreadyART are widely used and have resulted in the birth of more than 8 million children. A variety of media and oxygen concentrations are used to culture embryos. Embryos cultured under physiological O2 tension (5%) reach the blastocyst stage faster and have fewer alterations in gene expression when compared with embryos cultured under atmospheric oxygen conditions (20%). The mechanisms by which oxygen tension affects preimplantation development remain unclear, but mitochondria are believed to play an important role. The aim of this study was to evaluate how mitochondrial ultrastructure and function in IVF embryos were affected by culture under physiologic (5%) or atmospheric (20%) oxygen concentrations.Study design, size, durationZygotes, 2-cell, 4-cell, morula and blastocyst were flushed out of the uterus after natural fertilization and used as controls. IVF was performed in CF1 x B6D2F1 mice and embryos were cultured in Potassium simplex optimized medium (KSOM) with amino acids (KAA) under 5% and 20% O2 until the blastocyst stage. Embryo development with the addition of antioxidants was also tested.Participants/materials, setting, methodsMitochondrial function was assessed by measuring mitochondrial membrane potential, reactive oxygen species (ROS) production, ATP levels, and the expression of selected genes involved in mitochondrial function. Mitochondria ultrastructure was evaluated by transmission electron microscopy (TEM).Main results and the role of chanceEmbryos cultured under 20% O2 had fewer mitochondria and more vacuoles and hooded (abnormal) mitochondria compared to the other groups (P < 0.05). At the blastocyst stage the mitochondria of IVF embryos cultured in 20% O2 had lower mtDNA copy number, a denser matrix and more lamellar cristae than controls. Overall IVF-generated blastocysts had lower mitochondrial membrane potential, higher ROS levels, together with changes in the expression of selected mitochondrial genes (P < 0.05). ATP levels were significantly lower than controls only under 5% O2, with the 20% O2 IVF group having intermediate levels. Unexpectedly, adding antioxidant to the culture medium did not improve development.Large scale dataN/A.Limitations, reasons for cautionFindings in mice embryos might be different from human embryos.Wider implications of the findingsThis study suggests that changes in the mitochondria may be part of the mechanism by which lower oxygen concentration leads to better embryo development and further emphasize the importance of mitochondria as a locus of reprogramming.Study funding/competing interest(s)This study was funded by R01 HD 082039 to PFR, the Department of Life, Health and Environmental Sciences, University of L'Aquila, Italy (RIA 2016-2018) and the Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University of Rome, Italy (University grants 2016-2017). The authors declare no competing interests.

Development of quantitative, safe and rapid techniques for assessing embryo quality provides significant advances in Assisted Reproductive Technologies (ART). Instead of assessing the embryo quality by the standard morphologic evaluation, we apply the phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) method to capture endogenous fluorescent biomarkers of pre-implantation embryos as a non-morphological caliber for embryo quality. Here, we identify, under hypoxic and non-hypoxic conditions, the unique spectroscopic trajectories at different stages of mouse pre-implantation development, which is referred to as the developmental, or "D-trajectory", that consists of fluorescence lifetime from different stages of mouse pre-implantation embryos. The D-trajectory correlates with intrinsic fluorescent species from a distinctive energy metabolism and oxidized lipids, as seen with Third Harmonic Generation (THG) that changes over time. In addition, we have defined a non-morphological Embryo Viability Index (EVI) to distinguish pre-implantation embryo quality using the Distance Analysis (DA), a machine learning algorithm to process the fluorescence lifetime distribution patterns. We show, under our experimental conditions, that the phasor-FLIM approach provides a much-needed non-invasive quantitative technology for identifying healthy embryos at the early compaction stage with 86% accuracy. The DA and phasor-FLIM method may provide the opportunity to improve implantation success rates for in vitro fertilization clinics.

Abstract Objective To generate an effective embryo prediction model and identify a non-invasive evaluation method by analyzing microRNAs (miRNAs) in embryo culture medium. Design Analysis of microRNA profiles from spent culture medium of blastocysts with good morphology that did or did not result in pregnancy. Setting Clinical and experimental research. Patients Sixty patients who underwent thawed embryo transfer of blastocysts after intracytoplasmic sperm injection. Intervention(s) None. Main outcome measure(s) The association of miRNA abundance levels secreted by blastocysts in culture medium and implantation success. Results Our RNA sequencing analysis found a total of 53 differentially expressed miRNAs in the culture media of pregnancy and non-pregnancy groups. Twenty-one miRNAs were analyzed for their potential to predict implantation success. Eight miRNAs (hsa-miR-191-5p, hsa-miR-320a, hsa-miR-92a-3p, hsa-miR-509-3p, hsa-miR-378a-3p, hsa-miR-28-3p, hsa-miR-512-5p, and hsa-miR-181a-5p) were further extracted from the results of a logistic regression analysis of qPCR Ct values. A prediction model for high-quality blastocysts was generated using the eight miRNAs, with an average accuracy of 0.82 by 5-fold cross validation. Conclusion We isolated blastocyst miRNAs that may predict implantation success and created a model to predict viable embryos. Increasing the number of investigated cases and further studying the effect of each miRNA on embryonic development is needed to refine the miRNA-based predictive model.

Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts. From agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genome engineering, and modeling human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors. To date, however, it remains a challenge to produce stable pluripotent bovine ESC lines. Employing a culture system containing fibroblast growth factor 2 and an inhibitor of the canonical Wnt-signaling pathway, we derived pluripotent bovine ESCs (bESCs) with stable morphology, transcriptome, karyotype, population-doubling time, pluripotency marker gene expression, and epigenetic features. Under this condition bESC lines were efficiently derived (100% in optimal conditions), were established quickly (3-4 wk), and were simple to propagate (by trypsin treatment). When used as donors for nuclear transfer, bESCs produced normal blastocyst rates, thereby opening the possibility for genomic selection, genome editing, and production of cattle with high genetic value.

Oocyte developmental competence declines in women aged 35 and older resulting in many women resorting to IVF. The present study determined whether adding Granulocyte-macrophage colony-stimulating factor (GM-CSF) during in vitro oocyte maturation (IVM) could improve oocyte developmental competence in a mouse model of advanced maternal age. Oocytes from 12-14 month C57BL6 J × CBA mice were treated with 10 ng/ml of GM-CSF during IVM, and embryo development, mitochondrial activity, spindle formation and chromosomal alignment were examined. The addition of GM-CSF tended to increase fertilisation rates (76.19 vs. 82.03%; P = 0.07) but did not affect cumulus expansion compared with control. The addition of GM-CSF also increased blastocysts rates (51.10 vs. 61.52%; P < 0.01) and the number of good quality blastocysts (33.31 vs. 44.13%; P < 0.05) present at 96 h of culture as well as inner cell mass (12.64 vs. 15.62 ; P < 0.01) and total cell number (42.98 vs. 48.78 ; P < 0.05). GM-CSF treatment also increased mitochondrial membrane potential two to three fold in the outer (2.86 vs. 0.97; P < 0.001), intermediate (3.25 vs. 0.89; P < 0.001) and peri nuclear areas (3.62 vs. 1.08; P < 0.001). GM-CSF treatment did not influence spindle formation or chromosomal alignment. Together our results indicate that the addition of GM-CSF during IVM may improve oocyte quality in women of advanced maternal age.

Prepubertal animals are often preferred as sources of oocytes for assisted reproductive technologies (ARTs) in laboratory mice, but the normality and developmental competence of these oocytes remain controversial. This study systematically examined in vitro fertilization competence, embryo development, and fetal development after embryo transfer (ET) using oocytes from C57BL/6J mice aged 3 to 10 weeks. Oocytes were collected from superovulated females, fertilized, and cultured in vitro for 96 h or transferred at 2-cell stage to recipient females. Additionally, fetal development was compared between unfrozen and frozen-thawed in vitro-fertilized 2-cell embryos across different age groups. The number of ovulated oocytes per animal decreased with age, while the percentage of morphologically normal oocytes was highest in 3-week-old mice (99%) compared to older ages (70-86%, P < 0.05). Although fertilization percentages were consistently high (≥ 97%), blastocyst development in vitro, the nuclear counts of blastocysts and fetal development after ET were lowest for embryos from 3-week-old mice. Development of frozen-thawed embryos to fetuses was significantly reduced compared to unfrozen embryos in all age groups, except for those from 10-week-old mice. These findings suggest that oocytes from prepubertal mice, particularly from 3-week-old mice, are less developmentally competent than those from older mice. Therefore, the age of animals for oocyte source should be carefully considered based on the specific requirements of the research or ART applications.

This article reports a case of Massilia timonae, an understudied Gram-negative rod, in the culture media of an embryo produced by intracytoplasmic sperm injection into the oocyte cytoplasm. Cultivation was monitored with a time-lapse technology. Microbial contamination can cause embryo developmental arrest.

Research Question: Are there significant differences between cumulative live birth rates (CLBR) after short or extended embryo culture when comparisons are performed per cycle?, Design: This French national study included all IVF cycles performed from January 2016 to December 2019 with at least one cleaved embryo at day 2. The day 2/3 and day 5/6 groups were identified using the National Biomedicine Agency register. Only attempts involving the vitrification method were included. CLBR was assessed with 1 and 2 years of follow-up using a logistic regression model. The evolution of CLBR per cycle was compared over 1 year of follow-up., Results: In total, 133,250 cycles met the inclusion criteria (70,528 and 62,722 in the day 2/3 and day 5/6 groups). In multivariate analysis including several patient and cycle characteristics, the CLBR per cycle was significantly lower in the day 5/6 compared with the day 2/3 group. A significantly higher rate of fresh embryo transfer cancellation was observed in the day 5/6 compared with the day 2/3 group. The evolution of the CLBR was significantly different in favour of the day 2/3 group in cases with three or fewer day 2 embryos, whatever the patient's age, in their two first attempts as well as in their third or further attempts., Conclusions: Overall, the nationwide results per cycle suggest that extended embryo culture until the blastocyst stage, even when used in combination with vitrification, could not improve live birth rates. Moreover, where three or fewer day 2 embryos are obtained, it might be preferable to use the short embryo culture strategy., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.)

Background: This study aimed to analyse the expression of microRNA-223 (miR-223) in embryo culture medium and its correlation with pregnancy outcomes., Methods: Two hundred and two patients undergoing in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI) were divided into clinical pregnancy group (n = 101) and non-pregnant group (n = 101). The baseline data, clinical indicators, and the expression level of miR-223 in the embryo medium were compared between the two groups. Logistic regression analysis was used to analyse the relationship between each index and the pregnancy outcome. Receiver operator characteristic curve was carried out to evaluate the differential ability of miR-223 in pregnancy status. Bioinformatics methods were used to identify the target genes of miR-223 and elucidate their functions., Results: Compared with pregnancy group, the non-pregnancy group exhibited a reduction in miR-223 expression ( p  < 0.001). Multivariate analysis revealed that miR-223 reduction was an independent factor for pregnancy failure ( p  < 0.05). The ROC curve demonstrated the discriminative capability of miR-223 in distinguishing pregnancy and non-pregnancy. In addition, bioinformatics analysis indicated that the target genes of miR-223 were predominantly located in the endocytic vesicle membrane and were primarily enriched in adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathways., Conclusion: In this study, levels of miR-223 in the embryo culture medium predicted pregnancy outcomes in subjects undergoing IVF/ICSI. Low expression of miR-223 was a risk factor for adverse pregnancy outcomes in subjects.

Mechanisms controlling trophoblast (TB) proliferation and differentiation during embryo implantation are poorly understood. Human trophoblast stem cells (TSC) and BMP4/A83-01/PD173074-treated pluripotent stem cell-derived trophoblast cells (BAP) are two widely employed, contemporary models to study TB development and function, but how faithfully they mimic early TB cells has not been fully examined. We evaluated the transcriptomes of TB cells from BAP and TSC and directly compared them with those from peri-implantation human embryos during extended embryo culture (EEC) between embryonic days 8 to 12. The BAP and TSC grouped closely with TB cells from EEC within each TB sublineage following dimensional analysis and unsupervised hierarchical clustering. However, subtle differences in transcriptional programs existed within each TB sublineage. We also validated the presence of six genes in peri-implantation human embryos by immunolocalization. Our analysis reveals that both BAP and TSC models have features of peri-implantation TB s, while maintaining minor transcriptomic differences, and thus serve as valuable tools for studying implantation in lieu of human embryos., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Study Question: What are the implications of the presence cytoplasmic strings (Cyt-S) and their quantity and dynamics for the pre-implantation development of human blastocysts?, Summary Answer: Cyt-S are common in human embryos and are associated with faster blastocyst development, larger expansion, and better morphological quality., What Is Known Already: Cyt-S are dynamic cellular projections connecting inner cell mass and trophectoderm (TE) cells, that can be observed during blastocyst expansion. Their prevalence in human embryos has been estimated to be between 44% and 93%. Data relevant to their clinical implications and role in development are lacking, limited, or controversial., Study Design, Size, Duration: Retrospective study conducted at a single IVF center between May 2013 and November 2014 and involving 124 pre-implantation genetic testing for aneuploidy cycles in a time-lapse incubator with ≥1 blastocyst biopsied and vitrified (N = 370 embryos assessed). These cycles resulted in 87 vitrified-warmed single-euploid blastocyst transfers., Participants/materials, Setting, Methods: ICSI, continuous blastocyst culture (Days 5-7), TE biopsy of fully expanded blastocysts without Day 3 zona pellucida drilling, qPCR to assess uniform full-chromosome aneuploidies, and vitrification were all performed. Only vitrified-warmed euploid single-embryo-transfers were conducted. Blastocyst morphological quality was defined according to Gardner's criteria. The AI-based software CHLOE™ (Fairtility) automatically registered timings from time of starting blastulation (tSB) to biopsy (t-biopsy, i.e. blastocyst full-expansion) as hours-post-insemination (hpi), embryo area (including zona pellucida in µm2), and spontaneous blastocyst collapses. One senior embryologist manually annotated Cyt-S presence, quantity, timings, and type (thick cell-to-cell connections and/or threads). All significant associations were confirmed through regression analyses. All couples', cycles', and embryos' main features were also tested for associations with Cyt-S presence, quantity, and dynamics., Main Results and the Role of Chance: About 94.3% of the patients (N = 117/124) had ≥1 embryo with Cyt-S. Out of a total of 370 blastocysts, 55 degenerated between blastulation and full-expansion (N = 55/370, 14.9%). The degeneration rate among embryos with ≥1 Cyt-S was 10.8% (N = 33/304), significantly lower than that of embryos without Cyt-S (33.3%, N = 22/66, P < 0.01). Of the remaining 315 viable blastocysts analyzed, 86% (N = 271/315; P < 0.01) had ≥1 Cyt-S, on average 3.5 ± 2.1 per embryo ranging 1-13. The first Cyt-S per viable embryo appeared at 115.3 ± 12.5 hpi (85.7-157.7), corresponding to 10.5 ± 5.8 h (0.5-31) after tSB. Overall, we analyzed 937 Cyt-S showing a mean duration of 3.8 ± 2.7 h (0.3-20.9). Cyt-S were mostly threads (N = 508/937, 54.2%) or thick cell-to-cell connections becoming threads (N = 382/937, 40.8%) than thick bridges (N = 47/937, 5.0%). The presence and quantity of Cyt-S were significantly associated with developmentally faster (on average 6-12 h faster) and more expanded (on average 2700 µm2-larger blastocyst's area at t-biopsy) embryos. Also, the presence and duration of Cyt-S were associated with better morphology. Lastly, while euploidy rates were comparable between blastocysts with and without Cyt-S, all euploid blastocysts transferred from the latter group failed to implant (N = 10)., Limitations, Reasons for Caution: Cyt-S presence and dynamics were assessed manually on seven focal planes from video frames recorded every 15 min. The patients included were mostly of advanced maternal age. Only associations could be reported, but no causations/consequences. Lastly, larger datasets are required to better assess Cyt-S associations with clinical outcomes., Wider Implications of the Findings: Cyt-S are common during human blastocyst expansion, suggesting their physiological implication in this process. Their presence, quantity and dynamics mirror embryo viability, and morphological quality, yet their role is still unknown. Future basic science studies are encouraged to finally describe Cyt-S molecular nature and biophysical properties, and Artificial Intelligence tools should aid these studies by incorporating Cyt-S assessment., Study Funding/competing Interest(s): None., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Excessive consumption of fructose-sweetened foods and beverages is a growing concern worldwide. Studies have demonstrated that fructose consumption before and during pregnancy can result in adverse outcomes such as decreased decidualization, increased fetal losses, and low birth weight. The study investigated the teratogenic effects of fructose on rat embryos during organogenesis using whole embryo culture. Within the scope of the study, 4 groups were formed as control, low, medium, and high-dose fructose (HDF) with 10 embryos in each group. The 9.5-day-old rat embryos were cultured with different concentrations of fructose (1, 5 and 10 mM) for 48 h and the possible effects of fructose were examined using morphological scoring, histochemistry, immunofluorescence, and TUNEL methods. According to the analyses, protein synthesis and proliferation were decreased, vascular formation was suppressed, and apoptosis was increased in embryos exposed to fructose, especially at concentrations of 5 mM and above. According to the morphological scoring results, it was determined that heart, hind limb, and somite development were retarded in all experimental groups compared to the control group, developmental retardation increased in direct proportion to fructose concentration, and also significant malformations were observed in all parameters examined in the HDF group. In addition, analysis of yolk sac diameter, head length, crown rump length and somite numbers showed that these parameters were significantly decreased in all experimental groups. End of the study, it was concluded that fructose at concentrations of 1 mM and above may induce embryonic development retardation and other anomalies by decreasing protein synthesis and cell proliferation, suppressing vascular formation, and increasing apoptosis in embryonic tissues., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: In vitro embryo production is increasingly used for genetic improvement in cattle but bypasses the oviduct environment and exposes the embryos to oxidative stress with deleterious effects on further development. Here we aimed to examine the effect of oviduct epithelial spheroids (OES) on embryo development and quality in terms of morphology and gene expression during two co-culture times (4 days: up to embryonic genome activation at 8-16 cell stage vs. 7 days: up to blastocyst stage) and under two oxygen levels (5% vs. 20%)., Methods: Bovine presumptive zygotes produced by in vitro fertilization (day 0) using in-vitro matured oocytes were cultured in droplets of synthetic oviductal fluid (SOF) medium with or without (controls) OES for 4 or 7 days under 5% or 20% oxygen (4 treated and 2 control groups). Cleavage rates were evaluated on day 2 and blastocyst rates on days 7-8. Expanded blastocysts on days 7-8 were evaluated for total cell numbers and gene expression analysis by RNA-sequencing., Results: Under 20% oxygen, blastocyst rates and total cell numbers were significantly higher in the presence of OES for 4 and 7 days compared to controls (P < 0.05), with no difference according to the co-culture time. Under 5% oxygen, the presence of OES did not affect blastocyst rates but increased the number of cells per blastocyst after 7 days of co-culture (P < 0.05). Both oxygen level and OES co-culture had a significant impact on the embryonic transcriptome. The highest number of differentially expressed genes (DEGs) was identified after 7 days of co-culture under 20% oxygen. DEGs were involved in a wide range of functions, including lipid metabolism, membrane organization, response to external signals, early embryo development, and transport of small molecules among the most significantly impacted., Conclusion: OES had beneficial effects on embryo development and quality under both 5% and 20% oxygen, mitigating oxidative stress. Stronger effects on embryo quality and transcriptome were obtained after 7 than 4 days of co-culture. This study shows the impact of OES on embryo development and reveals potential molecular targets of OES-embryo dialog involved in response to stress and early embryonic development., (© 2024. The Author(s).)

Study Question: Could an artificial intelligence (AI) algorithm predict fetal heartbeat from images of vitrified-warmed embryos?, Summary Answer: Applying AI to vitrified-warmed blastocysts may help predict which ones will result in implantation failure early enough to thaw another., What Is Known Already: The application of AI in the field of embryology has already proven effective in assessing the quality of fresh embryos. Therefore, it could also be useful to predict the outcome of frozen embryo transfers, some of which do not recover their pre-vitrification volume, collapse, or degenerate after warming without prior evidence., Study Design, Size, Duration: This retrospective cohort study included 1109 embryos from 792 patients. Of these, 568 were vitrified blastocysts cultured in time-lapse systems in the period between warming and transfer, from February 2022 to July 2023. The other 541 were fresh-transferred blastocysts serving as controls., Participants/materials, Setting, Methods: Four types of time-lapse images were collected: last frame of development of 541 fresh-transferred blastocysts (FTi), last frame of 467 blastocysts to be vitrified (PVi), first frame post-warming of 568 vitrified embryos (PW1i), and last frame post-warming of 568 vitrified embryos (PW2i). After providing the images to the AI algorithm, the returned scores were compared with the conventional morphology and fetal heartbeat outcomes of the transferred embryos (n = 1098). The contribution of the AI score to fetal heartbeat was analyzed by multivariate logistic regression in different patient populations, and the predictive ability of the models was measured by calculating the area under the receiver-operating characteristic curve (ROC-AUC)., Main Results and the Role of Chance: Fetal heartbeat rate was related to AI score from FTi (P < 0.001), PW1i (P < 0.05), and PW2i (P < 0.001) images. The contribution of AI score to fetal heartbeat was significant in the oocyte donation program for PW2i (odds ratio (OR)=1.13; 95% CI [1.04-1.23]; P < 0.01), and in cycles with autologous oocytes for PW1i (OR = 1.18; 95% CI [1.01-1.38]; P < 0.05) and PW2i (OR = 1.15; 95% CI [1.02-1.30]; P < 0.05), but was not significantly associated with fetal heartbeat in genetically analyzed embryos. AI scores from the four groups of images varied according to morphological category (P < 0.001). The PW2i score differed in collapsed, non-re-expanded, or non-viable embryos compared to normal/viable embryos (P < 0.001). The predictability of the AI score was optimal at a post-warming incubation time of 3.3-4 h (AUC = 0.673)., Limitations, Reasons for Caution: The algorithm was designed to assess fresh embryos prior to vitrification, but not thawed ones, so this study should be considered an external trial., Wider Implications of the Findings: The application of predictive software in the management of frozen embryo transfers may be a useful tool for embryologists, reducing the cancellation rates of cycles in which the blastocyst does not recover from vitrification. Specifically, the algorithm tested in this research could be used to evaluate thawed embryos both in clinics with time-lapse systems and in those with conventional incubators only, as just a single photo is required., Study Funding/competing Interests: This study was supported by the Regional Ministry of Innovation, Universities, Science and Digital Society of the Valencian Community (CIACIF/2021/019) and by Instituto de Salud Carlos III (PI21/00283), and co-funded by European Union (ERDF, 'A way to make Europe'). M.M. received personal fees in the last 5 years as honoraria for lectures from Merck, Vitrolife, MSD, Ferring, AIVF, Theramex, Gedeon Richter, Genea Biomedx, and Life Whisperer. There are no other competing interests., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: To generate an effective embryo prediction model and identify a non-invasive evaluation method by analyzing microRNAs (miRNAs) in embryo culture medium. Design: Analysis of microRNA profiles from spent culture medium of blastocysts with good morphology that did or did not result in pregnancy. Setting: Clinical and experimental research. Patients: Sixty patients who underwent thawed embryo transfer of blastocysts after intracytoplasmic sperm injection. Intervention(s): None. Main outcome measure(s): The association of miRNA abundance levels secreted by blastocysts in culture medium and implantation success. Results: Our RNA sequencing analysis found a total of 53 differentially expressed miRNAs in the culture media of pregnancy and non-pregnancy groups. Twenty-one miRNAs were analyzed for their potential to predict implantation success. Eight miRNAs (hsa-miR-191-5p, hsa-miR-320a, hsa-miR-92a-3p, hsa-miR-509-3p, hsa-miR-378a-3p, hsa-miR-28-3p, hsa-miR-512-5p, and hsa-miR-181a-5p) were further extracted from the results of a logistic regression analysis of qPCR Ct values. A prediction model for high-quality blastocysts was generated using the eight miRNAs, with an average accuracy of 0.82 by 5-fold cross validation. Conclusion: We isolated blastocyst miRNAs that may predict implantation success and created a model to predict viable embryos. Increasing the number of investigated cases and further studying the effect of each miRNA on embryonic development is needed to refine the miRNA-based predictive model. [ABSTRACT FROM AUTHOR]

The developmental program of the embryo displays a plasticity that can result in long-acting effects that extend into postnatal life. In mammals, adult phenotype can be altered by changes in the maternal environment during the preimplantation period. One characteristic of developmental programming during this time is that the change in adult phenotype is often different for female offspring than for male offspring. In this paper, we propose the hypothesis that sexual dimorphism in preimplantation programming is mediated, at least in part, by sex-specific responses of embryos to maternal regulatory molecules whose secretion is dependent on the maternal environment. The strongest evidence for this idea comes from the study of colony-stimulating factor 2 (CSF2). Expression of CSF2 from the oviduct and endometrium is modified by environmental factors of the mother, in particular seminal plasma and obesity. Additionally, CSF2 alters several properties of the preimplantation embryo and has been shown to alleviate negative consequences of culture of mouse embryos on postnatal phenotype in a sex-dependent manner. In cattle, exposure of preimplantation bovine embryos to CSF2 causes sex-specific changes in gene expression, interferon-τ secretion and DNA methylation later in pregnancy (day 15 of gestation). It is likely that several embryokines can alter postnatal phenotype through actions directed towards the preimplantation embryo. Identification of these molecules and elucidation of the mechanisms by which sexually-disparate programming is established will lead to new insights into the control and manipulation of embryonic development.

Following blastocyst hatching, ungulate embryos undergo a prolonged preimplantation period termed conceptus elongation. Conceptus elongation constitutes a highly susceptible period for embryonic loss, and the embryonic requirements during this process are largely unknown, but multiple lipid compounds have been identified in the fluid nourishing the elongating conceptuses. Peroxisome proliferator-activated receptors mediate the signaling actions of prostaglandins and other lipids, and, between them, PPARG has been pointed out to play a relevant role in conceptus elongation by a functional study that depleted PPARG in both uterus and conceptus. The objective of this study has been to determine if embryonic PPARG is required for bovine embryo development. To that aim, we have generated bovine PPARG knock-out embryos in vitro using two independent gene ablation strategies and assessed their developmental ability. In vitro development to Day 8 blastocyst was unaffected by PPARG ablation, as total, inner cell mass, and trophectoderm cell numbers were similar between wild-type and knock-out D8 embryos. In vitro post-hatching development to D12 was also comparable between different genotypes, as embryo diameter, epiblast cell number, embryonic disk formation, and hypoblast migration rates were unaffected by the ablation. The development of tubular stages equivalent to E14 was assessed in vivo, following a heterologous embryo transfer experiment, observing that the development of extra-embryonic membranes and of the embryonic disk was not altered by PPARG ablation. In conclusion, PPARG ablation did not impaired bovine embryo development up to tubular stages., (© The Author(s) 2024. Published by Oxford University Press behalf of Society for the Study of Reproduction.)

Diosmetin (DIOS), a natural flavonoid monomer derived from lemons and present in various plants such as spearmint and spider moss, exhibits antioxidant, anti-inflammatory, and antiaging properties. Nonetheless, its impact on early embryonic development in pigs remains unexplored. This study aimed to determine the influence of DIOS supplementation in an in vitro culture (IVC) medium on porcine embryo development and to elucidate the underlying mechanisms. Findings revealed that embryos cultured in IVC medium with 0.1 μM DIOS demonstrated an increased blastocyst formation rate, higher total cell number, reduced LC3B and CASPASE3 levels, elevated Nrf2 levels, decreased ROS, and enhanced GSH and mitochondrial membrane potential at the 4-cell embryonic stage. Additionally, the expression of proapoptotic genes (CAS3, CAS8, and BAX) and autophagy-related genes (BECLIN1, ATG5, LC3B, and P62) was downregulated, whereas the expression of embryonic development-related genes (CDK1 and CDK2), antioxidant-related genes (SOD1 and SOD2), and mitochondrial biogenesis-related genes (NRF2) was upregulated. These findings suggest that DIOS promotes early embryonic development in pigs by mitigating oxidative stress and enhancing mitochondrial function, thereby reducing autophagy and apoptosis levels., (© 2024 Wiley Periodicals LLC.)

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.)

Objective: To investigate the ideal time in culture to optimize embryo cell-free deoxyribonucleic acid (cfDNA) analysis in frozen-thawed blastocysts undergoing noninvasive preimplantation genetic testing for aneuploidy (PGT-A). Cell-free DNA is released into the spent blastocyst media (spent media) by the embryo. However, the optimal timing to determine maximal cfDNA in the case of frozen-thawed blastocysts undergoing noninvasive PGT-A remains to be elucidated., Design: In this prospective observational study, 135 spent media and corresponding whole blastocysts were collected from January 2021 through March 2022., Setting: Private fertility clinics., Patients: Day-5 frozen-thawed blastocysts were cultured for 8 hours (Day-5 Short) or 24 hours (Day-5 Long), whereas day-6 frozen-thawed blastocysts were cultured for 8 hours (Day-6 Short). The spent media and whole blastocysts were then collected for further analysis. Spent media and whole blastocysts were amplified using whole genome amplification and sequenced using next-generation sequencing., Main Outcome Measures: Informativity and concordance rates between cfDNA in spent media and whole blastocyst DNA were compared according to the different times in culture., Results: When comparing time in culture, informativity rates for spent media were significantly higher for Day-5 Long and Day-6 Short (>91%) compared with the Day-5 Short group (<60%). A similar trend was observed for cases with and without a previous PGT-A. Regarding blastocyst expansion grade, informativity rates were lower on Day-5 Short compared with Day-5 Long and Day-6 Short, regardless of expansion degree. This decrease was significant for Gardner-grade expansion grades 3, 4, and 5-6. In addition, for a similar time in culture, the grade of expansion did not have an impact on the informativity rates. For concordance rates, no significant differences were observed among the 3 groups. In all cases, concordance rates were 90.5% for Day-5 Short, 93.6% for Day-5 Long, and 92.3% for Day-6 Short. No impact of the expansion grade was observed on concordance rates., Conclusion: Noninvasive PGT-A in frozen-thawed blastocysts yields very high concordance rates with whole blastocysts, possibly limiting the need for invasive PGT-A and making it available for a wider range of patients., Competing Interests: Declaration of Interests G.A. has nothing to disclose. M.B. has nothing to disclose. C.M.G.-P. has nothing to disclose. L.N.-S. is an employee of Igenomix. E.V.Z. has nothing to disclose. J.A.C. is an employee of Igenomix. C.S. has nothing to disclose. D.S. reports support from Igenomix who ran niPGT samples for the submitted work; speakers bureau for Igenomix and EMD Serono; Igenomix Scientific Advisory Board. C.R. is an employee of Igenomix., (Copyright © 2024 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Despite the cytotoxicity and embryotoxicity previously reported artesunate is a recommended drug to treat malaria for adults, children, and women in the first trimester of pregnancy. To address the putative effects of artesunate on female fertility and preimplantation embryo development, when the pregnancy is not detectable yet, artesunate was added to the oocyte in vitro maturation and in vitro embryo development of bovine. Briefly, in experiment 1 the cumulus-oocyte complexes (COCs) were in vitro matured for 18 h with 0.5, 1, or 2 µg/mL of artesunate or not (negative control) and then checked for nuclear maturation and subsequent embryo development. In experiment 2, the COCs were in vitro matured and fertilized without artesunate, which was added (0.5, 1, or 2 µg/mL) from the 1st to the 7th day of embryo culture along with a negative and a positive control group with doxorubicin. As a result, the use of artesunate on oocyte in vitro maturation did not differ from the negative control ( p  > 0.05) regarding nuclear maturation, cleavage, and blastocyst formation. Also, artesunate on in vitro embryo culture did not differ from negative control ( p  > 0.05) regarding cleavage and blastocyst formation, except for positive control, with doxorubicin ( p  < 0.05). In conclusion, under the conditions investigated, there was no evidence of artesunate toxicity on oocyte competence and the preimplantation period of in vitro embryo development in the bovine model, however, artesunate use still should be taken carefully as the outcome of implantation after oocytes and blastocysts exposure to artesunate remains unknown.

Research Question: Does artificial shrinkage before fresh blastocyst transfer improve clinical pregnancy rates in IVF?, Design: In this monocentric prospective, randomized, double-blind, controlled pilot study, 150 couples undergoing fresh single-blastocyst transfer were randomized between 20 May 2018 and 22 February 2022. In the artificial shrinkage group (AS group), a single laser pulse was directed to the cellular junction of the trophectoderm on the opposite side of the inner cell mass in each blastocyst. IVF outcomes were clinical pregnancy, multiple pregnancy and live birth rates. Cell-free DNA (cfDNA) concentration was also measured by quantitative real-time PCR in the blastocyst culture medium., Results: In total, 142 couples underwent fresh single-blastocyst transfer: control group, no artificial shrinkage, n = 47; and AS group, artificial shrinkage, n = 95; An intention-to-treat (ITT) analysis was employed. After a reassessment and the exclusion of patients with major protocol deviations, 139 couples underwent fresh single-blastocyst transfer under optimal conditions: control group, n = 47; and AS group, n = 92; a per-protocol analysis was used here. The clinical and laboratory characteristics were not significantly different between the groups. The clinical pregnancy rate was similar in the control and AS groups (ITT: 48.9% versus 49.5%, P = 0.97; per protocol: 48.94% versus 51.1%, P = 0.89). The multiple pregnancy rate and the live birth rate were also similar between the groups. No significant differences in gestational age, birthweight or proportion of male/female newborns were observed. The concentration of cfDNA in the blastocyst culture medium was not associated with IVF outcome., Conclusions: Large-scale randomized controlled trials are required to confirm these preliminary results., (Copyright © 2024. Published by Elsevier Ltd.)

Research Question: Can microbes vertically transmit from semen and follicular fluid to embryo culture media during assisted reproductive technology (ART) treatment?, Design: Spent embryo culture media (SECM), seminal fluid and follicular fluid samples were collected from 61 couples with infertility undergoing ART treatment at the Prince of Wales Hospital, Hong Kong SAR, China. Metagenomic analysis was conducted using 16s rRNA sequencing to identify the source of microbes in SECM, correlation between the semen microbiome and male infertility, and correlation between the follicular fluid microbiome and female infertility., Results: Microbial vertical transmission into SECM was reported in 82.5% of cases, and semen was the main source of contamination in conventional IVF cases. The increased abundances of Staphylococcus spp. and Streptococcus anginosus in semen had negative impacts on total motility and sperm count, respectively (P < 0.001). Significant increases in abundance of the genera Prophyromonas, Neisseria and Facklamia were observed in follicular fluid in women with anovulation, uterine factor infertility and unexplained infertility, respectively (P < 0.01). No significant correlation was found between the bacteria identified in all sample types and ART outcomes, including fertilization rate, embryo development, number of available embryos, and clinical pregnancy rate., Conclusion: Embryo culture media can be contaminated during ART treatment, not only by seminal microbes but also by follicular fluid and other sources of microbes. Strong correlations were found between specific microbial taxa in semen and sperm quality, and between the follicular fluid microbiome and the aetiology of female infertility. However, no significant association was found between the microbiomes of SECM, semen and follicular fluid and ART outcomes., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Study Question: Does the morphological quality on Day 3 influence the pregnancy outcomes of euploid blastocysts?, Summary Answer: The morphological quality on Day 3 affects the clinical pregnancy rate (CPR) and live birth rate (LBR) of low-quality euploid blastocysts., What Is Known Already: The morphological grading of Day 3 embryos affects the pregnancy outcome of cleavage-stage embryos and is an excellent indicator to predict embryo development potential. However, it is still unclear whether morphological quality on Day 3 is associated with pregnancy outcomes of the euploid blastocyst., Study Design, Size, Duration: This retrospective cohort study comprised 1275 patients who received single euploid blastocyst transfer between January 2016 and August 2021 at a tertiary teaching hospital., Participants/materials, Setting, Methods: Patients were grouped into two groups according to the morphological grading on Day 3 of transferred blastocysts: high-quality (HQ, including Grades I and II) Day 3 embryos and low-quality (LQ, Grade III) Day 3 embryos. The primary outcomes were CPR and LBR. Interactions of development days (Day 5 and Day 6) and morphological quality (high- and low-quality) of blastocysts with morphological quality of Day 3 embryos on pregnancy outcomes were tested in the stratified analysis and logistic regression models. The multivariate logistic regression analysis was conducted to investigate the independent effect of the morphological quality of Day 3 embryos on pregnancy outcomes after adjusting for potentially confounding factors., Main Results and the Role of Chance: The CPR and LBR of the HQ Day 3 embryos group were statistically higher than those of the LQ Day 3 embryos group (CPR: 59.73% versus 49.70%, respectively, P = 0.015; LBR: 49.73% versus 41.21%, respectively, P = 0.041). The development days of blastocysts did not exhibit a multiplicative interaction with the morphological quality of Day 3 embryos on the CPR (P for interaction = 0.648) and LBR (P for interaction = 0.925). The morphological quality of blastocysts exhibits a multiplicative interaction with the morphological quality of Day 3 embryos on the CPR (P for interaction = 0.020) and LBR (P for interaction = 0.012). After adjusting for potential confounders, the HQ Day 3 embryo group was positively associated with the CPR (adjusted odds ratio (aOR): 2.10, 95% CI: 1.31-3.36, P = 0.002) and LBR (aOR: 1.97, 95% CI: 1.20-3.25, P = 0.008) of LQ blastocysts. However, the morphological quality on Day 3 was not significantly associated with the CPR (aOR: 0.95, 95% CI: 0.58-1.55, P = 0.835) and LBR (aOR: 0.86, 95% CI: 0.53-1.40, P = 0.550) of HQ blastocysts., Limitations, Reasons for Caution: Selection and confounding bias introduced by the retrospective design cannot be completely eliminated in this study, although multivariable logistic analysis was conducted to adjust for potential confounders. Also, some subgroups had small sample sizes, which may reduce statistical power. Moreover, participants in our study only received single euploid blastocyst transfer, and whether the results could apply to blastocysts with unknown ploidy status is unclear., Wider Implications of the Findings: This study found that the morphological quality on Day 3 was significantly associated with the CPR and LBR of LQ blastocysts; Therefore, when only LQ euploid blastocysts are available for transfer, blastocysts derived from HQ Day 3 embryos are recommended., Study Funding/competing Interest(s): No external funding was obtained. The authors have no conflicts of interest to declare., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© 2024. The Author(s).)

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.

Research Question: Is the total duration of spontaneous blastocyst collapse to re-expansion before biopsy related to ploidy and live birth rates after single euploid blastocyst transfer?, Design: This was a retrospective cohort study of 600 preimplantation genetic testing cycles for aneuploidy (PGT-A) cycles, involving 2203 biopsied blastocysts, at a large reproductive medicine centre. Features of spontaneous blastocyst collapse from full to expanded stage, before biopsy, were observed using an embryoscope viewer for embryos cultured in a time-lapse incubator. In total, 568 cycles of frozen blastocyst transfers, either single euploid or mosaic, were performed. Correlations between collapse features and PGT-A outcomes were evaluated, as well as live birth rate, following euploid embryo transfer., Results: Blastocysts with lower morphological quality or delayed development had significantly higher rates of collapse, multiple collapses, and a longer duration of collapse to re-expansion. After controlling for confounders, such as oocyte age, morphological quality of blastocyst, and day of biopsy, multivariate logistic regression revealed that the total duration of collapse to re-expansion was an independent predictor of lower euploidy rate; the multivariate OR was 0.85 (95% CI 0.77-0.95; P = 0.00). Furthermore, even with euploid embryo transfer, the probability of a live birth decreased as the total duration of collapse to re-expansion increased; the multivariate OR was 0.79 (95% CI 0.64-0.98; P = 0.033)., Conclusion: The total duration of blastocyst collapse to re-expansion could be used as a predictor of lower euploidy and live birth rate. When developing blastocyst algorithms for pregnancy prediction, the duration of spontaneous blastocyst collapse should be included as a significant variable., (Copyright © 2024 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

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.)

Objective: To evaluate whether laser-mediated assisted hatching (AH) performed on vitrified/warmed blastocysts before embryo transfer can improve live birth rate., Design: The "pArtiaL zonA pelluciDa removal by assisteD hatchINg of blastocysts (ALADDIN)" is a 2-center comparative study with a parallel randomized controlled design., Setting: University hospital., Patients: Participants were recruited between September 2018 and November 2021. They were aged 18-39 years, underwent nondonor in vitro fertilization cycles, and were scheduled for elective single embryo transfer with vitrified/warmed blastocysts. Those with uterine abnormalities, body mass index of >35 kg/m 2 , severe male factor infertility, or performing preimplantation genetic testing were excluded., Intervention: Assisted hatching was performed using a 1,480 nm diode laser, removing approximately one-third of the zona pellucida with continuous 0.2 ms pulses applied from the 1-5 o'clock positions., Main Outcome Measures: The primary outcome was the live birth rate. Secondary end points included clinical pregnancy, miscarriage, multiple pregnancies, preterm births, obstetric and neonatal complications, and congenital anomalies., Results: Overall, 698 participants met the inclusion criteria and were randomized: 352 patients were assigned to the AH arm and 346 to the control arm. Of the participants, 105 (29.8%) and 101 (29.2%), respectively, achieved a live birth after treatment. The relative risk of live birth in patients with vitrified/warmed blastocysts treated with AH was 1.02 (95% confidence interval, 0.86-1.19). Exploratory subgroup analyses for women's age, recruiting centers, indications for in vitro fertilization, method of insemination, blastocyst quality, and days of blastocyst development failed to highlight any clinical situation that could benefit from AH in thawed blastocysts., Conclusion: In patients undergoing frozen embryo transfer with vitrified/warmed blastocysts, laser AH does not improve the live birth rate. Further studies are required to rule out milder but potentially interesting benefits in specific subgroups of patients., Trial Registration: ClinicalTrials.gov: NCT03623659., Competing Interests: Declaration of Interests E.P. reported grants and personal fees from MSD, grants from Ferring, IBSA, TEVA, and Gedeon Richter, grants and personal fees from Merck. P.V. received honoraria for editorial engagements. E.S. received honoraria for presentations at meetings from IBSA and Gedeon Richter, and also is handling 2 research grants from Ferring and IBSA. A.A. has nothing to disclose. M.R. has nothing to disclose. C.G. has nothing to disclose. V.B. has nothing to disclose. G.B. has nothing to disclose. M.P. has nothing to disclose. E.R. has nothing to disclose. S.F. has nothing to disclose. A.P. has nothing to disclose., (Copyright © 2024 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the positive predictive value and false positive risk of copy number variations (CNV's) detected in cell free deoxyribonucleic acid (DNA) from spent culture media for nonviable or aneuploid embryos., Design: Diagnostic/prognostic accuracy study., Patient(s): Patients aged 35 and younger with an indication for IVF-ICSI and elective single frozen embryo transfer at a single, private IVF center., Intervention: Embryo selection was performed according to the conventional grading, blinded to noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) results. After clinical outcomes were established, spent culture media samples were analyzed., Main Outcome Measures: Prognostic accuracy of CNVs according to niPGT-A results to predict nonviability or clinical aneuploidy., Results: One hundred twenty patients completed the study. Interpretations of next-generation sequencing (NGS) profiles were as follows: 7.5% (n = 9) failed quality control; 62.5% (n = 75) no CNVs detected; and 30% (n = 36) abnormal copy number detected. Stratification of abnormal NGS profiles was as follows: 15% (n = 18) whole chromosome and 15% (n = 18) uncertain reproductive potential. An intermediate CNV was evident in 27.8% (n = 5) of the whole chromosome abnormalities. The negative predictive value for samples with no detected abnormality was 57.3% (43/75). Whole chromosome abnormality was associated with a positive predictive value of 94.4% (17/18), lower sustained implantation rate (5.6%, 1/18), and higher relative risk (RR) for nonviability compared with no detected abnormalities (RR 2.21, 95% CI: 1.66-2.94). No other CNVs were associated with significant differences in the sustained implantation or RRs for nonviability. Unequal sex chromosome proportions suggested that maternal contamination was not uncommon. A secondary descriptive analysis of 705 supernumerary embryos revealed proportions of NGS profile interpretations similar to the transferred cohort. Significant median absolute pairwise differences between certain subcategories of CNV abnormalities were apparent., Conclusion: Whole chromosome abnormalities were associated with a high positive predictive value and significant RR for nonviability. Embryos associated with other CNVs had sustained implantation rates similar to those with no abnormalities detected. Further studies are required to validate the clinical applicability of niPGT-A., Clinical Trial Registration Number: clinicaltrials.gov (NCT04732013)., Competing Interests: Declaration of Interests G.N. is a shareholder in The Fertility Partners (TFP), the parent company of Olive Fertility Centre. S.R. has minority ownership interests in Sequence46. S.T. has minority ownership interests in Sequence46; C.W. has minority ownership interests in Sequence46. Thermo Fisher Scientific is a vendor to Sequence46 but does not have any other affiliations with the authors. Thermo Fisher provided consumables for the NGS methods required for the study but no direct financial support., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mechanisms of initial cell fate decisions differ among species. To gain insights into lineage allocation in humans, we derived ten human embryonic stem cell lines (designated UCSFB1-10) from single blastomeres of four 8-cell embryos and one 12-cell embryo from a single couple. Compared with numerous conventional lines from blastocysts, they had unique gene expression and DNA methylation patterns that were, in part, indicative of trophoblast competence. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by the transcriptomic data, immunolocalization of EOMES, T brachyury, GDF15 and active β-catenin revealed differential expression among blastomeres of 8- to 10-cell human embryos. The UCSFB lines formed derivatives of the three germ layers and CDX2-positive progeny, from which we derived the first human trophoblast stem cell line. Our data suggest heterogeneity among early-stage blastomeres and that the UCSFB lines have unique properties, indicative of a more immature state than conventional lines.

Preimplantation culture of mouse embryos has been suggested to result in reduced anxiety-like behavior in adulthood. Here, we investigated the effects of in vitro fertilization (IVF), embryo culture, and different diets on anxiety-like behavior using the elevated plus maze (EPM). We hypothesized that exposure to suboptimal conditions during the preimplantation stage would interact with the suboptimal diet to alter behavior. The expression of genes related to anxiety was then assessed by quantitative real-time polymerase chain reaction in various brain regions. When fed a normal diet during gestation and a moderately high-fat Western diet (WD) postnatally, naturally conceived (NC) and IVF mice showed similar anxiety-like behavior on the EPM. However, when fed a low-protein diet prenatally and a high-fat diet postnatally (LP/HF), NC mice showed a modest increase in anxiety-like behavior, whereas IVF mice showed the opposite: a strongly reduced anxiety-like behavior on the EPM. The robust reduction in anxiety-like behavior in IVF males fed the LP/HF diets was, intriguingly, associated with reduced expression of MAO-A, CRFR2, and GABA markers in the hypothalamus and cortex. These findings are discussed in relation to the developmental origin of health and disease hypothesis and the 2-hit model, which suggests that 2 events, occurring at different times in development, can act synergistically with long-term consequences observed during adulthood.

The proepicardium (PE) is an embryonic tissue that gives rise to multipotent vascular progenitors. Most notably the PE gives rise to the epicardium, cardiac fibroblasts, myocardium, and coronary vessels including both vascular smooth muscle and vascular endothelium. Much attention has been given to epicardial-derived cells that show the capacity to differentiate into a wide variety of vascular progenitors including cardiomyocytes. However, it is the PE itself that possesses the greatest potential as a source of multipotent vascular progenitors. We show here a simple method to manually isolate mouse PE at the ninth day of mouse embryonic development and culture highly pure PE tissue in serum-free conditions. This PE culture method allows for the ex vivo analysis of specific growth factors on PE and epicardial development with greater efficiency and precision than existing epicardial culture methods.

The preimplantation period is a time of reprogramming that may be vulnerable to disruption. This question has wide clinical relevance since the number of children conceived by in vitro fertilization (IVF) is rising. To examine this question, outbred mice (CF1 × B6D2F1) conceived by IVF and cultured using Whitten medium and 20% O2 (IVFWM group, less optimal) or K simplex optimized medium with amino acids and 5% O2 (IVFKAA group, more optimal and similar to conditions used in human IVF) were studied postnatally. We found that flushed blastocysts transferred to recipient mice provided the best control group (FB group), as this accounted for the effects of superovulation, embryo transfer, and litter size. We observed that many physiological parameters were normal. Reassuringly, IVFKAA offspring did not differ significantly from FB offspring. However, male IVFWM mice (but not females) were larger during the first 19 wk of life and exhibited glucose intolerance. Male IVFWM mice also showed enlarged left heart despite normal blood pressure. Expression of candidate imprinted genes (H19, Igf2, and Slc38a4) in multiple adult tissues did not show differences among the groups; only Slc38a4 was down-regulated following IVF (in both culture conditions) in female adipose tissue. These studies demonstrate that adult metabolism is affected by the type of conditions encountered during the preimplantation stage. Further, the postnatal growth trajectory and glucose homeostasis following ex vivo manipulation may be sexual dimorphic. Future work on the long-term effects of IVF offspring should focus on glucose metabolism and the cardiovascular system.

Borders are important as they demarcate developing tissue into distinct functional units. A key challenge is the discovery of mechanisms that can convert morphogen gradients into tissue borders. While mechanisms that produce ultrasensitive cellular responses provide a solution, how extracellular morphogens drive such mechanisms remains poorly understood. Here, we show how Bone Morphogenetic Protein (BMP) and Fibroblast Growth Factor (FGF) pathways interact to generate ultrasensitivity and borders in the dorsal telencephalon. BMP and FGF signaling manipulations in explants produced border defects suggestive of cross inhibition within single cells, which was confirmed in dissociated cultures. Using mathematical modeling, we designed experiments that ruled out alternative cross inhibition mechanisms and identified a cross-inhibitory positive feedback (CIPF) mechanism, or "toggle switch", which acts upstream of transcriptional targets in dorsal telencephalic cells. CIPF explained several cellular phenomena important for border formation such as threshold tuning, ultrasensitivity, and hysteresis. CIPF explicitly links graded morphogen signaling in the telencephalon to switch-like cellular responses and has the ability to form multiple borders and scale pattern to size. These benefits may apply to other developmental systems.

Objective: The aim of this study was to compare the pregnancy outcomes of in vitro fertilization with embryo transfer between embryos cultured in a time-lapse monitoring system (TLS) and those cultured in a conventional incubator (CI). Methods: The medical records of 250 fertilized embryos from 141 patients undergoing infertility treatment with assisted reproductive technology at a tertiary hospital from June 2018 to May 2020 were reviewed. The study population was divided into TLS and CI groups at a 1 to 1 ratio (125 embryos per group). The primary outcome was the live birth rate. Results: The TLS group had a significantly higher clinical pregnancy rate (46.4% vs. 27.2%, p=0.002), implantation rate (27.1% vs. 12.0%, p=0.004), and live birth rate (32.0% vs. 18.4%, p=0.013) than the CI group. Furthermore, subgroup analyses of the clinical pregnancy rate and live birth rate in the different age groups favored the TLS group. However, this difference only reached statistical significance in the live birth rate in women aged over 40 years and the clinical pregnancy rate in women aged 35-40 years (p=0.048 and p=0.031, respectively). The miscarriage rate, cleavage rate, and blastocyst rate were comparable. Conclusion: TLS application improved the live birth rate, implantation rate, and clinical pregnancy rate, particularly in the advanced age group in this study, while the other reproductive outcomes were comparable. Large randomized controlled trials are needed to further explore the ramifications of these findings, especially in different age groups. [ABSTRACT FROM AUTHOR]

Study Question: What factors associated with embryo culture techniques contribute to the rate of medium osmolality change over time in an embryo culture incubator without added humidity?Summary Answer: The surface area-to-volume ratio of culture medium (surface area of the medium exposed to an oil overlay), as well as the density and height of the overlaying oil, all interact in a quantitative way to affect the osmolality rise over time.What Is Known Already: Factors such as medium volume, different oil types, and associated properties, individually, can affect osmolality change during non-humidified incubation.Study Design, Size, Duration: Several experimental designs were used, including simple single-factor completely randomized designs, as well as a multi-factor response surface design. Randomization was performed at one or more levels for each experiment. Osmolality measurements were performed over 7 days, with up to 8 independent osmolality measurements performed per treatment group over that time. For the multi-factor study, 107 independent combinations of factor levels were assessed to develop the mathematical model.Participants/materials, Setting, Methods: This study was conducted in a research laboratory setting. Commercially available embryo culture medium and oil was used. A MINC incubator without water for humidification was used for the incubation. Osmolality was measured with a vapor pressure osmometer after calibration. Viscometry and density were conducted using a rheometer, and volumetric flasks with an analytical balance, respectively. Data analyses were conducted with several commercially available software programs.Main Results and the Role Of Chance: Preliminary experiments showed that the surface area-to-volume ratio of the culture medium, oil density, and oil thickness above the medium all contributed significantly (P < 0.05) to the rise in osmolality. A multi-factor experiment showed that a combination of these variables, in the form of a truncated cubic polynomial, was able to predict the rise in osmolality, with these three variables interacting in the model (P < 0.05). Repeatability, as measured by the response of identical treatments performed independently, was high, with osmolality values being ± 2 of the average in most instances. In the final mathematical model, the terms of the equation were significant predictors of the outcome, with all P-values being significant, and only one P-value > 0.0001.Limitations, Reasons For Caution: Although the range of values for the variables were selected to encompass values that are expected to be encountered in usual embryo culture conditions, variables outside of the range used may not result in accurate model predictions. Although the use of a single incubator type and medium type is not expected to affect the conclusions, that remains an uncertainty.Wider Implications Of the Findings: Using this predictive model will help to determine if one should be cautious in using a specific system and will provide guidance on how a system may be modified to provide improved stability during embryo culture.Study Funding/competing Interest(s): This study was funded by Cook Medical. The author is a Team Lead and Senior Scientist at Cook Medical. The author has no other conflicts of interest to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Retinol (ROL) and its biologically active metabolite, all-trans retinoic acid (ATRA), are essential for a number of reproductive processes. However, there is a paucity of information regarding their roles in ovarian folliculogenesis, oocyte maturation, and early embryogenesis. The objectives of this study were to quantify and compare peripheral plasma (PP) and follicular fluid (FF) retinoid levels, including ATRA in women undergoing in vitro fertilization (IVF) and to investigate the relationship between retinoid levels and embryo quality. Retinoid levels were evaluated in PP and FF from 79 women undergoing IVF at the time of oocyte retrieval and corresponding embryo quality assessed on a daily basis after retrieval for 3 days until uterine transfer. Analysis compared the retinoid levels with day 3 embryo grades and between endometriosis versus control patients. Results demonstrated distinctive levels of retinoid metabolites and isomers in FF versus PP. There was a significantly larger percentage of high-quality grade I embryos derived from the largest versus smallest follicles. An increase in follicle size also correlated with a >50% increase in FF ROL and ATRA concentrations. Independent of follicle size, FF yielding grade I versus nongrade I embryos showed higher mean levels of ATRA but not ROL. In a nested case-control analysis, control participants had 50% higher mean levels of ATRA in their FF and PP than women with endometriosis. These findings strongly support the proposition that ATRA plays a fundamental role in oocyte development and quality, and that reduced ATRA synthesis may contribute to decreased fecundity of participants with endometriosis.

Since the birth of the first IVF-conceived child in 1978, the use of assisted reproductive technologies (ART) has grown dramatically, contributing to the successful birth of 5 million individuals worldwide. However, there are several reported associations of ART with pregnancy complications, such as low birthweight (LBW), preterm birth, birth defects, epigenetic disorders, cancer and poor metabolic health. Whether this is attributed to ART procedures or to the subset of the population seeking ART remains a controversy, but the most relevant question today concerns the potential long-term implications of assisted conception. Recent evidence has emerged suggesting that ART-conceived children have distinct metabolic profiles that may predispose to cardiovascular pathologies in adulthood. Because the eldest IVF individuals are still too young to exhibit components of chronic middle-aged syndromes, the use of animal models has become particularly useful in describing the effects of unusual or stressful preimplantation experiences on adult fitness. Elucidating the molecular mechanisms by which embryos integrate environmental signals into development and metabolic gene expression programs will be essential for optimizing ART procedures such as in vitro culture conditions, embryo selection and transfer. In the future, additional animal studies to identify mechanisms underlying unfavorable ART outcomes, as well as more epidemiological reviews to monitor the long-term health of ART children are required, given that ART procedures have become routine medical practice.

Objectives: To explore the correlation between mitochondrial quantity and the blastocyst development timeline as well as their respective contributions to early pregnancy., Methods: A retrospective study was conducted using a dataset comprising 2,633 embryos that underwent preimplantation genetic testing for aneuploidy (PGT-A) between January 2016 and December 2023. The study was divided into three subsets to address distinct aspects: the representativeness of a single trophectoderm (TE) biopsy for mitochondrial quantity (n=43), the correlation between morphokinetic features and mitochondrial quantity (n=307), and the association analysis among mitochondrial quantity, blastocyst timeline factor, and reproductive outcomes (n=2,283). Distribution assessment of mitochondrial quantity across an individual blastocyst involved the identification within multiple biopsies and spent culture media. Timeline evaluation included correlating mitochondrial quantity with time-lapse datasets. Finally, multivariate logistic regression models, incorporating potential effectors alongside mitochondrial quantity, were employed to analyze their respective contributions to early pregnancy endpoints., Results: Of distribution assessment, mitochondrial quantity exhibited an even distribution across the entire trophectoderm (Spearman's ρ=0.82), while no detectable mtDNAs in the corresponding spent culture media. Then the timeline correlation study revealed significant association between mitochondrial quantity and blastocyst features of both the day of expanded blastocyst formation (95% Confidence intervals, CIs: 0.27~4.89, p=0.03) and the timing of expanded blastocyst formation (tEB) (95% CIs: -0.24~-0.01, p=0.04) in the regression model, indicating a strong dependency between mitochondrial quantity and the blastocyst development timeline. For the contribution to early pregnancy, multivariate logistic regression models showed that the day of expanded blastocyst formation contributed to four endpoints persistently: positive for HCG (odd ratio, OR: 0.71, p=0.006), gestational sac (OR: 0.78, p=0.04), fetal heartbeat (OR: 0.71, p=0.004), and progression to 14 weeks (OR: 0.69, p=0.002). Contrastingly, no notable correlation was observed between the mitochondrial quantity and these endpoints., Conclusions: Strong interaction was observed between mitochondrial quantity and the blastocyst timeline, particularly the timing of expanded blastocyst formation. It suggests that the primary determinant influencing pregnancy outcomes lies in the time-dependent parameter of blastocyst rather than in the specific mitochondrial quantity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chuang, Chou, Kuan, Liu, Kao, Wu, Lai, Hsieh, Liang, Chen and Chen.)

This study examines the expression of three microRNAs (hsa-miR-661, hsa-miR-21-5p, hsa-miR-372-5p) in spent pre-implantation embryos culture media to identify possible new non-invasive biomarkers of embryo competence, predictive of development to the blastocyst stage. A preliminary analysis on 16 patients undergoing IVF cycles was performed by collecting and stored spent culture media on the fifth/sixth day of embryo culture. Expression of miRNAs was evaluated according to the embryos' fate: 1) NE/DG: non-evolved or degenerate embryos; 2) BLOK: embryos developed to the blastocyst stage. Preliminary results revealed a higher miRNAs expression in NE/DG spent media. To elucidate the roles of these miRNAs, we employed a robust bioinformatics pipeline involving: 1) in-silico miRNA Target Prediction using RNAHybrid, which identified the most-likely gene targets; 2) Construction of a Protein-Protein Interaction network via GeneMania, linking genes with significant biological correlations; 3) application of modularity-based clustering with the gLay app in Cytoscape, resulting in three size-adapted subnets for focused analysis; 4) Enrichment Analysis to discern the biological pathways influenced by the miRNAs. Our bioinformatics analysis revealed that hsa-miR-661 was closely associated with pathways regulating cell shape and morphogenesis of the epithelial sheet. These data suggest the potential use of certain miRNAs to identify embryos with a higher likelihood of developing to the blastocyst stage. Further analysis will be necessary to explore the reproducibility of these findings and to understand if miRNAs here investigated can be used as biomarkers for embryo selection before implantation into the uterus or if they may be reliable predictors of IVF outcome., Competing Interests: Declaration of Competing Interest No potential conflict of interest was reported by the authors., (Copyright © 2024 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

The quality of the recipient cytoplasm was reported as a crucial factor in maintaining the vitality of SCNT embryos and SCNT efficiency for dairy cows. Compared with oocytes matured in vivo, oocytes matured in vitro showed abnormal accumulation and metabolism of cytoplasmic lipids. L-carnitine treatment was found to control fatty acid transport into the mitochondrial β-oxidation pathway, which improved the process of lipid metabolism. The results of this study show that 0.5 mg/ml L-carnitine significantly reduced the cytoplasmic lipid content relative to control. No significant difference was observed in the rate of oocyte nuclear maturation, but the in vitro developmental competence of SCNT embryos was improved in terms of increased blastocyst production and lower apoptotic index in the L-carnitine treatment group. In addition, the pregnancy rate with SCNT embryos in the treatment group was significantly higher than in the control group. In conclusion, the present study demonstrated that adding L-carnitine to the maturation culture medium could improve the developmental competence of SCNT embryos both in vitro and in vivo by reducing the lipid content of the recipient cytoplasm., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest; financial; personal or other relationship with other people and organization., (Copyright © 2024 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Objective: Cryopreservation has some adverse effects on embryos including cell metabolism reduction, mitochondria and plasma membrane damage, excess production of 'Reactive Oxygen Species' and damage to DNA. In the present study. In this study we assessed the effect of coenzyme Q10 as an exogenous antioxidant on mouse embryos following cryopreservation., Methods: We collected mice embryos at the morula stage from uterine horns on the third day of gestation. The morulae were divided into 9 groups (1 control, 2 vehicles and 6 experimental), then vitrified. The culture and/or vitrification media of the experimental groups were supplemented by 10 or 30 μM of CoQ10. After one week, the embryos were warmed and then cultured. After 48 hours of embryo culture, the blastocyst rate, total cell number, viability; and after 72 hours of embryo culture, we assessed the hatching rate., Results: Blastocyst rate and hatching rate were significantly reduced in the groups containing 30 μM CoQ10 supplemented culture media compared to other groups (p<0.05). The hatching rate in the groups containing 10 μM CoQ10 supplemented in both culture and vitrification media was significantly higher than in the other groups (p<0.05). In groups containing 10 μM CoQ10 supplemented culture media, the viability was higher than that in the other groups (p<0.05)., Conclusions: It seems that CoQ10 in a dose-dependent manner is able to improve hatching rate and viability following cryopreservation through its antioxidant and anti-apoptotic properties, and through the production of ATP.

Hydroxychloroquine (HCQ) is a safe antimalarial drug but its overdosage or inappropriate use, such as during the pandemic, may cause adverse effects once this drug is considered a potent inhibitor of autophagy. Information about HCQ's effects on the reproductive field, including gametes and initial embryos, is limited. In this study, we evaluated the effect of HCQ (1, 6, 12, and 24 μM) on pre-implantation embryo development, autophagy, and apoptosis of bovine embryos produced in vitro. A dose-response experiment showed a reduction (p < 0.05) in cleavage only at the highest concentration. Blastocyst rate was gradually reduced (p < 0.05) with the increase of HCQ dosage starting at 6 μM, with no embryo formation occurring at 24 μM. Further analysis showed that embryos treated with 12 μM of HCQ had a higher (p < 0.05) accumulation of acidic autophagic vesicles on Days 5 and 7 of development and a higher (p < 0.01) apoptotic index on Day 7. To our knowledge, this is the first study to evaluate the effects of HCQ on embryo pre-implantation development in mammals. The results contribute with more information related to the study of autophagy in embryology as well as add some discussion on HCQ toxicology and its effects on reproductive cells., Competing Interests: Declaration of competing interest Authors have no competing interests to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: Before blastocyst development, embryos undergo morphological and metabolic changes crucial for their subsequent growth. This study aimed to investigate the relationship between morula compaction and blastocyst formation and the subsequent chromosomal status of the embryos., Methods: This retrospective cohort study evaluated embryo development (n = 371) using time-lapse imaging; 94 blastocysts underwent preimplantation genetic testing for aneuploidy (PGT-A)., Results: The embryos were classified as fully (Group 1, n = 194) or partially (Group 2, n = 177) compacted. Group 1 had significantly higher proportions of good- and average-quality blastocysts than Group 2 (21.6% vs. 3.4%, p = 0.001; 47.9% vs. 26.6%, p = 0.001, respectively). The time from the morula stage to the beginning and completion of compaction and blastocyst formation was significantly shorter in Group 1 than in Group 2 (78.6 vs. 82.4 h, p = 0.001; 87.0 vs. 92.2 h, p = 0.001; 100.2 vs. 103.7 h, p = 0.017, respectively). Group 1 embryos had larger surface areas than Group 2 embryos at various time points following blastocyst formation. Group 1 blastocysts had significantly higher average expansion rates than Group 2 blastocysts (653.6 vs. 499.2 μm 2 /h, p = 0.001). PGT-A revealed a higher proportion of euploid embryos in Group 1 than in Group 2 (47.2% vs. 36.6%, p = 0.303)., Conclusion: Time-lapse microscopy uncovered a positive relationship between compaction and blastocyst quality and its association with embryo ploidy. Hence, compaction evaluation should be prioritized before blastocyst selection for transfer or cryopreservation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Study Question: Do embryos displaying abnormal cleavage (ABNCL) up to Day 3 have compromised live birth rates and neonatal outcomes if full blastulation has been achieved prior to transfer?, Summary Answer: ABNCL is associated with reduced full blastulation rates but does not impact live birth rates and neonatal outcomes once full blastulation has been achieved., What Is Known Already?: It is widely accepted that ABNCL is associated with reduced implantation rates of embryos when transferred at the cleavage stage. However, evidence is scarce in the literature reporting birth outcomes from blastocysts arising from ABNCL embryos, likely because they are ranked low priority for transfer., Study Design, Size, Duration: This retrospective cohort study included 1562 consecutive autologous in vitro fertilization cycles (maternal age 35.1 ± 4.7 years) performed at Fertility North, Australia between January 2017 and June 2022. Fresh transfers were performed on Day 3 or 5, with remaining embryos cultured up to Day 6 before vitrification. A total of 6019 embryos were subject to blastocyst culture, and a subset of 664 resulting frozen blastocysts was included for live birth and neonatal outcome analyses following single transfers., Participants/materials, Setting, Methods: ABNCL events were annotated from the first mitotic division up to Day 3, including direct cleavage (DC), reverse cleavage (RC) and <6 intercellular contact points at the 4-cell stage (<6ICCP). For DC and RC in combination, the ratios of affected blastomeres over the total number of all blastomeres up to Day 3 were also recorded. All pregnancies were followed up until birth with gestational age, birthweight, and sex of the baby being recorded., Main Results and the Role of Chance: Full blastulation rates for embryos showing DC (19.5%), RC (41.7%), <6ICCP (58.8%), and mixed (≥2) ABNCL types (26.4%) were lower than the rates for those without ABNCL (67.2%, P < 0.01 respectively). Subgroup analysis showed declining full blastulation rates with increasing ratios of combined DC/RC affected blastomeres over all blastomeres up to the 8-cell stage (66.2% when 0 affected, 47.0% when 0.25 affected, 27.4% when 0.5 affected, 14.5% when 0.75 affected, and 7.7% when all affected, P < 0.01). However, once full blastulation had been achieved, no difference was detected between DC, RC, <6ICCP, and no ABNCL blastocysts following single frozen transfers in subsequent live birth rates (25.9%, 33.0%, 36.0% versus 30.8%, P > 0.05, respectively), gestational age (38.7 ± 1.6, 38.5 ± 1.2, 38.3 ± 3.5 versus 38.5 ± 1.8 weeks, P > 0.05, respectively) and birthweight (3343.0 ± 649.1, 3378.2 ± 538.4, 3352.6 ± 841.3 versus 3313.9 ± 509.6 g, P > 0.05, respectively). Multiple regression (logistic or linear as appropriate) confirmed no differences in all of the above measures after accounting for potential confounders., Limitations, Reasons for Caution: Our study is limited by its retrospective nature, making it impossible to control every known or unknown confounder. Embryos in our dataset, being surplus after selection for fresh transfer, may not represent the general embryo population., Wider Implications of the Findings: Our findings highlight the incremental impact of ABNCL, depending on the ratio of affected blastomeres up to Day 3, on subsequent full blastulation. The reassuring live birth and neonatal outcomes of ABNCL blastocysts imply a potential self-correction mechanism among those embryos reaching the blastocyst stage, which provides valuable guidance for clinical practice and patient counseling., Study Funding/competting Interest(s): This research is supported by an Australian Government Research Training Program (RTP) Scholarship. All authors report no conflict of interest., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)