Search

Blastocyst vitrification has significantly improved embryo transfer methods, leading to higher implantation success rates and better pregnancy outcomes in subsequent frozen embryo transfer cycles. This study aimed to simulate the transcriptional changes caused by vitrifying human blastocysts using mouse blastocysts as a model and to further investigate these changes' effects. Utilizing a human vitrification protocol, we implanted both vitrified and fresh embryos into mice. We observed the implantation success rates and performed transcriptomic analysis on the blastocysts. To validate the results from messenger RNA sequencing, we conducted reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) to measure the expression levels of specific genes. Based on mRNA profiling, we predicted the microRNAs responsible for the regulation and used qPCR basic microRNA assays for validation. Our observations revealed a higher implantation success rate for vitrified embryos than fresh embryos. Transcriptomic analysis showed that vitrified-warmed blastocysts exhibited differentially expressed genes (DEGs) primarily associated with thermogenesis, chemical carcinogenesis-reactive oxygen species, oxidative phosphorylation, immune response, and MAPK-related signaling pathways. RT-qPCR confirmed increased expression of genes such as Cdk6 and Nfat2 , and decreased expression of genes such as Dkk3 and Mapk10 . Additionally, gene-microRNA interaction predictions and microRNA expression analysis identified twelve microRNAs with expression patterns consistent with the predicted results, suggesting potential roles in uterine epithelial cell adhesion, trophectoderm development, invasive capacity, and immune responses. Our findings suggest that vitrification induces transcriptomic changes in mouse blastocysts, and even small changes in gene expression can enhance implantation success. These results highlight the importance of understanding the molecular mechanisms underlying vitrification to optimize embryo transfer techniques and improve pregnancy outcomes.

The present study examined whether male resveratrol intake affected mitochondrial DNA copy number (mt-cn) and telomere length (TL) in blastocysts fathered by young and aged male mice. C57BL/6N male mice supplied with water or water containing 0.1 mM resveratrol were used for embryo production at 14-23 and 48-58 weeks of age. Two-cell-stage embryos were collected from the oviducts of superovulated female mice (8-15 weeks old) and cultured for 3 days until the blastocyst stage. Mt-cn and TL levels were measured by real-time polymerase chain reaction. Resveratrol intake did not affect body weight or water consumption. Resveratrol intake increased the expression levels of SIRT1 in the liver, the antioxidative ability of serum, and extended TL in the heart, whereas there was no significant difference in mt-cn in the heart or TL in sperm. The rate of blastocyst development was significantly lower in aged male mice than in younger mice, and resveratrol intake increased the total number of blastocysts derived from both young and aged males. Resveratrol intake did not affect mt-cn or TL in blastomeres of blastocyst-stage embryos derived from young mice, but significantly increased both mt-cn and TL in blastomeres of blastocysts derived from aged fathers. In conclusion, resveratrol intake increased mt-cn and TL levels in blastocysts derived from aged male mice.

Background: The establishment of stable porcine embryonic stem cells (pESCs) can contribute to basic and biomedical research, including comparative developmental biology, as well as assessing the safety of stem cell-based therapies. Despite these advantages, most pESCs obtained from in vitro blastocysts require complex media and feeder layers, making routine use, genetic modification, and differentiation into specific cell types difficult. We aimed to establish pESCs with a single cell-passage ability, high proliferative potency, and stable in long-term culture from in vitro-derived blastocysts using a simplified serum-free medium., Methods: We evaluated the establishment efficiency of pESCs from in vitro blastocysts using various basal media (DMEM/F10 (1:1), DMEM/F12, and a-MEM) and factors (FGF2, IWR-1, CHIR99021, and WH-4-023). The pluripotency and self-renewal capacity of the established pESCs were analyzed under feeder or feeder-free conditions. Ultimately, we developed a simplified culture medium (FIW) composed of FGF2, IWR-1, and WH-4-023 under serum-free conditions., Results: The pESC-FIW lines were capable of single-cell passaging with short cell doubling times and expressed the pluripotency markers POU5F1, SOX2, and NANOG, as well as cell surface markers SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed a stable proliferation rate and normal karyotype, even after 50 passages. Transcriptome analysis revealed that pESC-FIW were similar to reported pESC maintained in complex media and showed gastrulating epiblast cell characteristics. pESC-FIW were maintained for multiple passages under feeder-free conditions on fibronectin-coated plates using mTeSR™, a commercial medium used for feeder-free culture, exhibiting characteristics similar to those observed under feeder conditions., Conclusions: These results indicated that inhibition of WNT and SRC was sufficient to establish pESCs capable of single-cell passaging and feeder-free expansion under serum-free conditions. The easy maintenance of pESCs facilitates their application in gene editing technology for agriculture and biomedicine, as well as lineage commitment studies., (© 2024. The Author(s).)

This study aimed to develop a method to evaluate the quality of bovine in vitro fertilized (IVF) embryos based on gene expression profiling via whole-transcriptome amplification. The expression of 11 developmentally important genes in individual bovine in vivo-derived (IVD) and IVF embryos were examined. Gene expression profiling was conducted by classifying the expression level of each gene in individual embryos as low, medium, or high. The IVF group had a higher (P < 0.01) proportion of embryos with low expression of SOX2, NANOG, and FGF4. In addition, a correlation analysis between the expression levels of each gene in individual embryos demonstrated that the relationship between gene expression differed with respect to IVD and IVF embryos. Our results suggest that the expression profiling of developmentally important genes using IVD embryos as normal controls could be a useful indicator for evaluating the quality of bovine IVF embryos.

For the intracytoplasmic sperm injection (ICSI) procedure in pigs, an electrical pulse (EP) has been used as an effective method for oocyte stimulation, but unlike sperm, EP is unable to induce Ca 2+ oscillations. In this study, we investigated the effects of generating artificial Ca 2+ oscillations with phospholipase Cζ (PLCζ) mRNA, a candidate sperm factor, on fertilization, embryonic development, and gene expression after ICSI. Firstly, the concentration of PLCζ mRNA of a fixed volume (1.0 pl) that would induce a pattern of Ca 2+ rise similar to that of in vitro fertilized (IVF) sperm was examined and determined to be 300 ng/μl. Secondly, the effects of oocyte stimulation methods on fertilization and embryonic development were investigated. ICSI-oocytes were activated by EP (EP group) or by PLCζ mRNA (PLCζ group). Furthermore, IVF-oocytes (IVF group) and ICSI-oocytes with and without an injection of buffer (buffer and untreated groups, respectively) were used as controls. It was found that the rates of normal fertilization in the PLCζ and EP groups were significantly higher than those in the buffer and untreated groups. The blastocyst formation rates did not differ among the groups. The embryo quality in the EP group was inferior to those in the PLCζ and IVF groups. Additionally, the expression level of a proapoptosis-related gene (Caspase-3) in the EP group was significantly higher than those in the PLCζ and IVF groups. Our data suggest that oocyte activation by PLCζ mRNA has the effect of improving embryo quality.

Background: Extensive research has been conducted on embryonic developmental disorders linked to Polycystic Ovary Syndrome (PCOS), a pathological condition that affects 5-10% of women and is characterized by irregularities in the menstrual cycle and infertility. By employing RNA sequencing (RNA-seq), we performed an in-depth investigation of PCOS-related changes in gene expression patterns at the mouse blastocyst stage., Methods: The zygotes of female B6D2 mice were obtained and then differentiated into blastocysts in K + Simplex Optimised Medium (KSOM) cultures containing exo-NC (negative control for exosomes) or exo-LIPE-AS1 (a novel exosomal marker of PCOS). Subsequently, blastocysts were collected for RNA-seq. The bioinformatics was performed to analyze and compare the differences of gene expression profile between blastocysts of control and PCOS group., Results: There were 1150 differentially expressed genes (DEGs) between the two groups of mouse blastocysts; 243 genes were upregulated and 907 downregulated in the blastocysts of the exo-LIPE-AS1 group compared to those of the exo-NC group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the genes involved in amino acid synthesis and glutathione metabolic pathways were down-regulated in exo-LIPE-AS1 group., Conclusion: This study has revealed that blastocyst developmental retardation may be associated with the downregulation of amino acid synthesis and glutathione metabolism, which may affect energy metabolism, biosynthesis, cellular osmotic pressure, antioxidant synthesis, ROS clearance or mitochondrial function, and ultimately cause blastocyst cell development abnormalities. Our research offers encouraging data on the mechanisms underlying aberrant embryonic development in patients with PCOS as well as potential treatment strategies., (© 2024. The Author(s).)

In Brief: In the present study the sustainable effect of L-carnitine during the culture period on the post-transfer development was investigated. Taken together, we uncovered direct effects of L-carnitine on the bioenergetic profile of day 7 blastocysts along with sustainable effects on mtDNA copy numbers and transcriptome profile of bovine day 14 embryos., Abstract: L-Carnitine (LC) is known to play key roles in lipid metabolism and antioxidative activity, implicating enhanced cryotolerance of bovine blastocysts. However, sustainability of LC supplementation during culture period on preimplantation development beyond the blastocyst stage has not been investigated so far. Therefore, all embryos were cultured under fatty acid-free conditions, one group with LC (LC embryos) and the control group without LC (control) supplementation. Transfer to recipients was conducted on day 6. Elongation-stage embryos were recovered on day 14; metrics of embryo recollection, developmental rates as regards early elongation-stage as well as mean embryo length did not differ between the groups. Gene expression analyses via NGS revealed 341 genes to be differentially regulated between elongation-stage embryos derived from LC supplementation compared to controls. These played mainly a role in molecular functions and biological processes like oxidoreductase activity, ATP-dependent activity, cellular stress, and respiration. Pathways like oxidative phosphorylation and thermogenesis, extracellular matrix receptor signaling, PI3K-Akt, and focal adhesion were affected by differentially regulated genes. Moreover, all DEGs located on the mitochondria were significantly downregulated in LC embryos, being in line with lower mitochondrial copy number and mtDNA integrity compared to the control group. Finally, we uncovered alterations of the bioenergetic profile on day 7 as a consequence of LC supplementation for the first time, revealing significantly higher oxygen consumption rates, ATP linked respiration and spare capacity for LC embryos. In summary, we uncovered direct effects of LC supplementation during the culture period on the bioenergetic profile along with sustainable effects on mtDNA copy numbers and transcriptome profile of bovine day 14 embryos.

We have established trophoblast cell lines, from parthenogenesis-derived buffalo blastocysts. The buffalo trophoblast cells were cultured continuously over 200 days and 21 passages. These cells were observed by phase-contrast microscopy for their morphology and characterized by reverse transcriptase polymerase chain reaction and immunofluorescence against trophoblast-specific markers and cytoskeletal proteins. Trophoblast cells showed positive staining for CDX2, a marker of these cells at both blastocyst and cell line levels. Epithelial morphology of these cells was revealed by positive staining against cytokeratins and tubulin but not against vimentin and dolichos biflorus agglutinin. Gene expression profiles of many important placenta-specific genes were studied in the primary trophectoderm outgrowths, which were collected on days 0, 5, 9, 12 and 15 of culture and trophoblast cell line at passages 12-15. Therefore, the trophoblast cell line derived can potentially be used for in vitro studies on buffalo embryonic development.

Preimplantation genetic testing (PGT) can minimize the risk of birth defects. However, the accuracy and applicability of routine PGT is confounded by uneven genome coverage and high allele drop-out rate from existing single-cell whole genome amplification methods. Here, a method to diagnose genetic mutations and concurrently evaluate embryo competence by leveraging the abundant mRNA transcript copies present in trophectoderm cells is developed. The feasibility of the method is confirmed with 19 donated blastocysts. Next, the method is applied to 82 embryos from 26 families with monogenic defects for simultaneous mutation detection and competence assessment. The accuracy rate of direct mutation detection is up to 95%, which is significantly higher than DNA-based method. Meanwhile, this approach correctly predicted seven out of eight (87.5%) embryos that failed to implant. Of six embryos that are predicted to implant successfully, four met such expectations (66.7%). Notably, this method is superior at conditions for mutation detection that are challenging when using DNA-based PGT, such as when detecting pathogenic genes with a high de novo rate, multiple pseudogenes, or an abnormal expansion of CAG trinucleotide repeats. Taken together, this study establishes the feasibility of an RNA-based PGT that is also informative for assessing implantation competence., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Genetic mosaicism, characterized by multiple genotypes within an individual, is considered an obstacle to CRISPR/Cas9 genome editing in animal models. Despite the various strategies for minimizing mosaic mutations, no definitive methods exist to eliminate them. This study aimed to enhance gene editing efficiency in porcine zygotes using CRISPR/Cas9, which targets specific genes through centrifugation and zona pellucida removal before electroporation. Centrifugation at 2000 × g did not adversely affect blastocyst formation rates in zygotes electroporated with gRNA targeting the GGTA1 gene; instead, it led to increased total and monoallelic mutation rates compared with control zygotes without centrifugation. However, the groups had no significant differences in biallelic mutation rates. In zygotes electroporated with gRNA targeting the CMAH gene, centrifugation treatments exceeding 1000 × g significantly increased both biallelic mutation rates and mutation efficiency. The combination of centrifugation and zona pellucida removal did not have a detrimental effect on blastocyst formation rates. It led to a higher rate of double biallelic mutations in embryos targeting both GGTA1 and CMAH compared to embryos without centrifugation treatment. In summary, our results demonstrate that pre-electroporation treatments, including centrifugation and zona pellucida removal, positively influenced the reduction of mosaic mutations, with the effectiveness of centrifugation depending on the specific gRNA used., (© 2024. The Society for In Vitro Biology.)

Cloning by somatic cell nuclear transfer (SCNT) remained challenging for Rhesus monkeys, mostly due to its low efficiency and neonatal death. Genome-scale analyses revealed that monkey SCNT embryos displayed widespread DNA methylation and transcriptional alterations, thus including loss of genomic imprinting that correlated with placental dysfunction. The transfer of inner cell masses (ICM) from cloned blastocysts into ICM-depleted fertilized embryos rescued placental insufficiency and gave rise to a cloned Rhesus monkey that reached adulthood without noticeable abnormalities.

The generation of genetically engineered pig models that develop pancreas-specific tumors has the potential to advance studies and our understanding of pancreatic cancer in humans. TP53 mutation causes organ-nonspecific cancers, and PDX1-knockout results in the loss of pancreas development. The aim of the present study was to generate a PDX1-knockout pig chimera carrying pancreas complemented by TP53 mutant cells via phytohemagglutinin (PHA)-mediated blastomere aggregation using PDX1 and TP53 mutant blastomeres, as a pig model for developing tumors in the pancreas with high frequency. First, the concentration and exposure time to PHA to achieve efficient blastomere aggregation were optimized. The results showed that using 300 µg/mL PHA for 10 min yielded the highest rates of chimeric blastocyst formation. Genotyping analysis of chimeric blastocysts derived from aggregated embryos using PDX1- and TP53-edited blastomere indicated that approximately 28.6% carried mutations in both target regions, while 14.3-21.4% carried mutations in one target. After the transfer of the chimeric blastocysts into one recipient, the recipient became pregnant with three fetuses. Deep sequencing analysis of the PDX1 and TP53 regions using ear and pancreas samples showed that one fetus carried mutations in both target genes, suggesting that the fetus was a chimera derived from embryo-aggregated PDX1 and TP53 mutant blastomeres. Two out of three fetuses carried only the PDX1 mutation, indicating that the fetuses developed from embryos not carrying TP53-edited blastomeres. The results of the present study could facilitate the further improvement and design of high-frequency developing pancreatic tumor models in pigs., (© 2024. The Society for In Vitro Biology.)

Purpose: There are no clinical treatments to prevent/revert age-related alterations associated with oocyte competence decline in the context of advanced maternal age. Those alterations have been attributed to oxidative stress and mitochondrial dysfunction. Our study aimed to test the hypothesis that in vitro maturation (IVM) medium supplementation with antioxidants (resveratrol or phloretin) may revert age-related oocyte competence decline., Methods: Bovine immature oocytes were matured in vitro for 23 h (young) and 30 h (aged). Postovulatory aged oocytes (control group) and embryos obtained after fertilization were examined and compared with oocytes supplemented with either 2 μM of resveratrol or 6 μM phloretin (treatment groups) during IVM., Results: Aged oocytes had a significantly lower mitochondrial mass and proportion of mitochondrial clustered pattern, lower ooplasmic volume, higher ROS, lower sirtuin-1 protein level, and a lower blastocyst rate in comparison to young oocytes, indicating that postovulatory oocytes have a lower quality and developmental competence, thus validating our experimental model. Supplementation of IVM medium with antioxidants prevented the generation of ROS and restored the active mitochondrial mass and pattern characteristic of younger oocytes. Moreover, sirtuin-1 protein levels were also restored but only following incubation with resveratrol. Despite these findings, the blastocyst rate of treatment groups was not significantly different from the control group, indicating that resveratrol and phloretin could not restore the oocyte competence of postovulatory aged oocytes., Conclusion: Resveratrol and phloretin can both revert the age-related oxidative stress and mitochondrial dysfunction during postovulatory aging but were insufficient to enhance embryo developmental rates under our experimental conditions., (© 2024. The Author(s).)

Success of animal cloning is limited by oocyte quality, which is closely linked to reprogramming ability. The number of layers of cumulus cells is typically used to assess the quality of oocyte; a minimum of one-third of collected cumulus-oocyte complexes (COCs) are discarded as inferior oocytes because they have less cumulus cells. Melatonin, which has been recognised for its ability to sequester free radicals and perform multiple functions, has emerged as a potentially effective candidate for enhancing inferior oocytes quality and, consequently, embryo development competency. The current study investigates to improve the quality of inferior oocytes by supplementation of melatonin (10 -9 M) during in vitro maturation (IVM) and subsequent cloned embryo production and its mechanism. The results indicate that melatonin supplementation significantly (p<0.05) enhances inferior oocytes maturation, reduces oxidative stress by reducing ROS levels, and improves mitochondrial function by boosting GSH levels. The melatonin treatment (10 -9 M) enhances the expression of SOD, GPx1, GDF 9, BMP 15, ATPase 6, and ATPase 8 in inferior oocytes. Furthermore, melatonin treatment increases the total cell number in the treated groups, promoting cloned blastocyst formation rates derived from inferior oocytes. Furthermore, compared to the control, 10 -9 M melatonin supplementation enhances H3K9ac acetylation and lowers H3K27me3 methylation in cloned blastocysts derived from inferior oocytes. In conclusion, 10 -9 M melatonin supplementation during IVM increased inferior oocyte maturation and promoted cloned buffalo embryo development by lowering oxidative stress and promoting epigenetic alterations. These studies show that melatonin may improve the quality of poor oocytes and buffalo cloning., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: To evaluate if single serum human chorionic gonadotropin (hCG) level measurements are sufficient for pregnancy monitoring after single embryo transfer (sET) and to compare the hCG levels between fresh (FRET) and frozen embryo transfers (FET) in medically assisted reproduction., Methods: This was a retrospective exploratory cohort study including all patients who met the inclusion criteria, who received a single FRET (n = 249) or FET (n = 410) of a day five blastocyst at the IVF clinic at the Johannes Kepler University Linz between 2011 and 2020. hCG levels were measured on day 14 after embryo transfer. Threshold values for the viability of pregnancies were determined using receiver operating characteristic (ROC) curves., Results: Significantly higher hCG levels were found in those who received FET than in those who received FRET (1222.8 ± 946.7 mU/ml vs. 862.7 ± 572.9 mU/ml; p < 0.001). Optimal threshold values predicting a viable pregnancy were 368.5 mU/ml and 523 mU/ml in the FRET and FET groups, respectively., Conclusions: After FET, higher hCG values after 14 days of embryo transfer must be considered in pregnancy monitoring. Additionally, a single threshold hCG value seems to be sufficient for determining pregnancy viability. To exclude ectopic pregnancies, subsequent ultrasound examination is a mandatory requirement., (© 2024. The Author(s).)

Histone lysine crotonylation, an evolutionarily conserved modification differing from acetylation, exerts pivotal control over diverse biological processes. Among these are gene transcriptional regulation, spermatogenesis, and cell cycle processes. However, the dynamic changes and functions of histone crotonylation in preimplantation embryonic development in mammals remain unclear. Here, we show that the transcription coactivator P300 functions as a writer of histone crotonylation during embryonic development. Depletion of P300 results in significant developmental defects and dysregulation of the transcriptome of embryos. Importantly, we demonstrate that P300 catalyzes the crotonylation of histone, directly stimulating transcription and regulating gene expression, thereby ensuring successful progression of embryo development up to the blastocyst stage. Moreover, the modification of histone H3 lysine 18 crotonylation (H3K18cr) is primarily localized to active promoter regions. This modification serves as a distinctive epigenetic indicator of crucial transcriptional regulators, facilitating the activation of gene transcription. Together, our results propose a model wherein P300-mediated histone crotonylation plays a crucial role in regulating the fate of embryonic development., (© 2024. The Author(s).)

Mammalian blastocyst formation involves the specification of the trophectoderm followed by the differentiation of the inner cell mass into embryonic epiblast and extra-embryonic primitive endoderm (PrE). During this time, the embryo maintains a window of plasticity and can redirect its cellular fate when challenged experimentally. In this context, we found that the PrE alone was sufficient to regenerate a complete blastocyst and continue post-implantation development. We identify an in vitro population similar to the early PrE in vivo that exhibits the same embryonic and extra-embryonic potency and can form complete stem cell-based embryo models, termed blastoids. Commitment in the PrE is suppressed by JAK/STAT signaling, collaborating with OCT4 and the sustained expression of a subset of pluripotency-related transcription factors that safeguard an enhancer landscape permissive for multi-lineage differentiation. Our observations support the notion that transcription factor persistence underlies plasticity in regulative development and highlight the importance of the PrE in perturbed development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Background: Balanced insertional translocations (BITs) can increase the risk of infertility, recurrent miscarriages or neonatal birth defects due to chromosomal imbalances in gametes. However, studies on preimplantation genetic testing (PGT) for patients carrying BITs are inadequate., Methods: A preimplantation genetic genotyping and haplotype analysis approach was developed and implemented in this study. Genome-wide SNP genotyping was performed, followed by core family-based haplotype analysis. The balanced insertion segments in euploid embryos were inferred from the haplotypes inherited from the carrier parent., Results: A total of 10 BIT carrier couples were enrolled in our study. 15 in vitro fertilisation cycles were conducted, resulting in 73 blastocysts biopsied and subjected to PGT analysis. Among these, 20 blastocysts displayed rearrangement-related imbalances, 13 exhibited de novo aneuploidies, 15 presented a complex anomaly involving both imbalances and additional aneuploidies, while 25 were euploid. Within the euploid embryos, 12 were balanced carrier embryos and 13 were non-carrier embryos. To date, eight non-carrier and one carrier embryos have been transferred, resulting in seven clinical pregnancies. All pregnancies were recommended to perform prenatal diagnosis, our date revealed complete concordance between fetal genetic testing results and PGT results. Presently, five infants have been born from these pregnancies, and two pregnancies are still ongoing., Conclusion: The proposed method facilitates comprehensive chromosome screening and the concurrent identification of balanced insertions or normal karyotypes in embryos. This study offers an effective and universally applicable strategy for BIT carriers to achieve a healthy pregnancy and prevent the transmission of BITs to their offspring., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

A high incidence of pregnancy failures occurs in cattle during the second week of pregnancy as blastocysts transition into an elongated conceptus. This work explored whether interleukin-6 supplementation during in vitro embryo production would improve subsequent conceptus development. Bovine embryos were treated with 0 or 100 ng/mL recombinant bovine interleukin-6 beginning on day 5 post-fertilization. At day 7.5 post-fertilization, blastocysts were transferred into estrus synchronized beef cows (n = 5 recipients/treatment, 10 embryos/recipient). Seven days after transfer (day 14.5), cows were euthanized to harvest reproductive tracts and collect conceptuses. Individual conceptus lengths and stages were recorded before processing for RNA sequencing. Increases in conceptus recovery, length, and the proportion of tubular and filamentous conceptuses were detected in conceptuses derived from interleukin-6-treated embryos. The interleukin-6 treatment generated 591 differentially expressed genes in conceptuses (n = 9-10/treatment). Gene ontology enrichment analyses revealed changes in transcriptional regulation, DNA-binding, and antiviral actions. Only a few differentially expressed genes were associated with extraembryonic development, but several differentially expressed genes were associated with embryonic regulation of transcription, mesoderm and ectoderm development, organogenesis, limb formation, and somatogenesis. To conclude, this work provides evidence that interleukin-6 treatment before embryo transfer promotes pre-implantation conceptus development and gene expression in ways that resemble the generation of a robust conceptus containing favorable abilities to survive this critical period of pregnancy., (© 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.)

Betaine has important roles in preimplantation mouse embryos, including as an organic osmolyte that functions in cell volume regulation in the early preimplantation stages and as a donor to the methyl pool in blastocysts. The origin of betaine in oocytes and embryos was largely unknown. Here, we found that betaine was present from the earliest stage of growing oocytes. Neither growing oocytes nor early preantral follicles could take up betaine, but antral follicles were able to transport betaine and supply the enclosed oocyte. Betaine is synthesized by choline dehydrogenase, and female mice lacking Chdh did not have detectable betaine in their oocytes or early embryos. Supplementing betaine in their drinking water restored betaine in the oocyte only when supplied during the final stages of antral follicle development but not earlier in folliculogenesis. Together with the transport results, this implies that betaine can only be exogenously supplied during the final stages of oocyte growth. Previous work showed that the amount of betaine in the oocyte increases sharply during meiotic maturation due to upregulated activity of choline dehydrogenase within the oocyte. This betaine present in mature eggs was retained after fertilization until the morula stage. There was no apparent role for betaine uptake via the SIT1 (SLC6A20) betaine transporter that is active at the 1- and 2-cell stages. Instead, betaine was apparently retained because its major route of efflux, the volume-sensitive organic osmolyte - anion channel, remained inactive, even though it is expressed and capable of being activated by a cell volume increase., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Preimplantation embryos undergo a series of important biological events, including epigenetic reprogramming and lineage differentiation, and the key genes and specific mechanisms that regulate these events are critical to reproductive success. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase involved in the regulation of a variety of cellular functions, yet its precise function and mechanism in preimplantation embryonic development remain unknown. Our results showed that RNAi-mediated silencing of USP7 in mouse embryos or treatment with P5091, a small molecule inhibitor of USP7, significantly reduced blastocyst rate and blastocyst quality, and decreased total and trophectoderm cell numbers per blastocyst, as well as destroyed normal lineage differentiation. The results of single-cell RNA-seq, reverse transcription-quantitative polymerase chain reaction, western blot, and immunofluorescence staining indicated that interference with USP7 caused failure of the morula-to-blastocyst transition and was accompanied by abnormal expression of key genes (Cdx2, Oct4, Nanog, Sox2) for lineage differentiation, decreased transcript levels, increased global DNA methylation, elevated repressive histone marks (H3K27me3), and decreased active histone marks (H3K4me3 and H3K27ac). Notably, USP7 may regulate the transition from the morula to blastocyst by stabilizing the target protein YAP through the ubiquitin-proteasome pathway. In conclusion, our results suggest that USP7 may play a crucial role in preimplantation embryonic development by regulating lineage differentiation and key epigenetic modifications., (© 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.)

Background: In recent years, with benefits from the continuous improvement of clinical technology and the advantage of fertility preservation, the application of embryo cryopreservation has been growing rapidly worldwide. However, amidst this growth, concerns about its safety persist. Numerous studies have highlighted the elevated risk of perinatal complications linked to frozen embryo transfer (FET), such as large for gestational age (LGA) and hypertensive disorders during pregnancy. Thus, it is imperative to explore the potential risk of embryo cryopreservation and its related mechanisms., Methods: Given the strict ethical constraints on clinical samples, we employed mouse models in this study. Three experimental groups were established: the naturally conceived (NC) group, the fresh embryo transfer (Fresh-ET) group, and the FET group. Blastocyst formation rates and implantation rates were calculated post-embryo cryopreservation. The impact of FET on fetal growth was evaluated upon fetal and placental weight. Placental RNA-seq was conducted, encompassing comprehensive analyses of various comparisons (Fresh-ET vs. NC, FET vs. NC, and FET vs. Fresh-ET)., Results: Reduced rates of blastocyst formation and implantation were observed post-embryo cryopreservation. Fresh-ET resulted in a significant decrease in fetal weight compared to NC group, whereas FET reversed this decline. RNA-seq analysis indicated that the majority of the expression changes in FET were inherited from Fresh-ET, and alterations solely attributed to embryo cryopreservation were moderate. Unexpectedly, certain genes that showed alterations in Fresh-ET tended to be restored in FET. Further analysis suggested that this regression may underlie the improvement of fetal growth restriction in FET. The expression of imprinted genes was disrupted in both FET and Fresh-ET groups., Conclusion: Based on our experimental data on mouse models, the impact of embryo cryopreservation is less pronounced than other in vitro manipulations in Fresh-ET. However, the impairment of the embryonic developmental potential and the gene alterations in placenta still suggested it to be a risky operation., (© 2024. The Author(s).)

Profiling bovine blastocyst transcriptome at the single-cell level has enabled us to reveal the first cell lineage segregation, during which the inner cell mass (ICM), trophectoderm (TE), and an undefined population of transitional cells were identified. By comparing the transcriptome of blastocysts derived in vivo (IVV), in vitro from a conventional culture medium (IVC), and in vitro from an optimized reduced nutrient culture medium (IVR), we found a delay of the cell fate commitment to ICM in the IVC and IVR embryos. Developmental potential differences between IVV, IVC, and IVR embryos were mainly contributed by ICM and transitional cells. Pathway analysis of these non-TE cells between groups revealed highly active metabolic and biosynthetic processes, reduced cellular signaling, and reduced transmembrane transport activities in IVC embryos that may lead to reduced developmental potential. IVR embryos had lower activities in metabolic and biosynthetic processes but increased cellular signaling and transmembrane transport, suggesting these cellular mechanisms may contribute to improved blastocyst development compared to IVC embryos. However, the IVR embryos had compromised development compared to IVV embryos with notably over-active transmembrane transport activities that impaired ion homeostasis., (© 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.)

Muse cells, identified as cells positive for the pluripotent surface marker SSEA-3, are pluripotent-like endogenous stem cells located in the bone marrow (BM), peripheral blood, and organ connective tissues. The detailed characteristics of SSEA-3(+) cells in extraembryonic tissue, however, are unknown. Here, we demonstrated that similar to human-adult tissue-Muse cells collected from the BM, adipose tissue, and dermis as SSEA-3(+), human-umbilical cord (UC)-SSEA-3(+) cells express pluripotency markers, differentiate into triploblastic-lineage cells at a single cell level, migrate to damaged tissue, and exhibit low telomerase activity and non-tumorigenicity. Notably, ~ 20% of human-UC-SSEA-3(+) cells were negative for X-inactive specific transcript (XIST), a naïve pluripotent stem cell characteristic, whereas all human adult tissue-Muse cells are XIST-positive. Single-cell RNA sequencing revealed that the gene expression profile of human-UC-SSEA-3(+) cells was more similar to that of human post-implantation blastocysts than human-adult tissue-Muse cells. The DNA methylation level showed the same trend, and notably, the methylation levels in genes particularly related to differentiation were lower in human-UC-SSEA-3(+) cells than in human-adult tissue-Muse cells. Furthermore, human-UC-SSEA-3(+) cells newly express markers specific to extraembryonic-, germline-, and hematopoietic-lineages after differentiation induction in vitro whereas human-adult tissue-Muse cells respond only partially to the induction. Among various stem/progenitor cells in living bodies, those that exhibit properties similar to post-implantation blastocysts in a naïve state have not yet been found in humans. Easily accessible human-UC-SSEA-3(+) cells may be a valuable tool for studying early-stage human development and human reproductive medicine., (© 2024. The Author(s).)

Only 30% of embryos from in vitro fertilized oocytes successfully implant and develop to term, leading to repeated transfer cycles. To reduce time-to-pregnancy and stress for patients, there is a need for a diagnostic tool to better select embryos and oocytes based on their physiology. The current standard employs brightfield imaging, which provides limited physiological information. Here, we introduce METAPHOR: Metabolic Evaluation through Phasor-based Hyperspectral Imaging and Organelle Recognition. This non-invasive, label-free imaging method combines two-photon illumination and AI to deliver the metabolic profile of embryos and oocytes based on intrinsic autofluorescence signals. We used it to classify i) mouse blastocysts cultured under standard conditions or with depletion of selected metabolites (glucose, pyruvate, lactate); and ii) oocytes from young and old mouse females, or in vitro-aged oocytes. The imaging process was safe for blastocysts and oocytes. The METAPHOR classification of control vs. metabolites-depleted embryos reached an area under the ROC curve (AUC) of 93.7%, compared to 51% achieved for human grading using brightfield imaging. The binary classification of young vs. old/in vitro-aged oocytes and their blastulation prediction using METAPHOR reached an AUC of 96.2% and 82.2%, respectively. Finally, organelle recognition and segmentation based on the flavin adenine dinucleotide signal revealed that quantification of mitochondria size and distribution can be used as a biomarker to classify oocytes and embryos. The performance and safety of the method highlight the accuracy of noninvasive metabolic imaging as a complementary approach to evaluate oocytes and embryos based on their physiology., Competing Interests: Competing interests statement:The lead laboratory in this work has received private funding from the Venture Capital (VC) Scranton Enterprises. The authors declare that this funder has no role in the conceptualization, design, data collection, analysis, decision to publish, or preparation of the manuscript.

The receptive phase of the uterus is marked by structural and functional maturation of the endometrium. During this limited time span, the blastocyst competency is superimposed on the receptive endometrium. It is a well-known fact that lipid signalling in early-stage pregnancy has a crucial role in successful embryogenesis. In our study, CD-1 mouse uteri after normal and in vitro fertilization (IVF) were investigated at 6.5, 8.5, and 10.5 days of pregnancy. Matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry and liquid chromatography coupled tandem mass spectrometry were used for identification of phosphatidylcholine (PC) lipid structures. In the embryonal tissues, PC 32:0 and PC 34:0 were increased, while in the antemesometrial (AM) decidua the two 20:4-containing PCs, PC 36:4 and PC 38:4 were increased. In transferred uterus samples, higher expressions of PC 34:0, PC 34:1, PC 34:2, PC 36:1, and PC 36:2 in mesometrial decidua were seen, whereas the two 20:4-containing PCs, PC 36:4 and PC 38:4 showed increased expression in the AM and lateral decidua. This paper shows a significant spatio-temporal change in lipid metabolism during IVF procedures for the first time.

The hypoblast is an essential extraembryonic tissue set aside within the inner cell mass in the blastocyst. Research with human embryos is challenging. Thus, stem cell models that reproduce hypoblast differentiation provide valuable alternatives. We show here that human naive pluripotent stem cell (PSC) to hypoblast differentiation proceeds via reversion to a transitional ICM-like state from which the hypoblast emerges in concordance with the trajectory in human blastocysts. We identified a window when fibroblast growth factor (FGF) signaling is critical for hypoblast specification. Revisiting FGF signaling in human embryos revealed that inhibition in the early blastocyst suppresses hypoblast formation. In vitro, the induction of hypoblast is synergistically enhanced by limiting trophectoderm and epiblast fates. This finding revises previous reports and establishes a conservation in lineage specification between mice and humans. Overall, this study demonstrates the utility of human naive PSC-based models in elucidating the mechanistic features of early human embryogenesis., Competing Interests: Declaration of interests G.G. is an inventor on a patent relating to human naive pluripotent stem cells filed by the University of Cambridge. S.L. reports non-financial support from Pfizer outside the submitted work., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A dispersed cytoplasmic distribution of mitochondria is a hallmark of normal cellular organization. Here, we have utilized the expression of exogenous Trak2 in mouse oocytes and embryos to disrupt the dispersed distribution of mitochondria by driving them into a large cytoplasmic aggregate. Our findings reveal that aggregated mitochondria have minimal impact on asymmetric meiotic cell divisions of the oocyte. In contrast, aggregated mitochondria during the first mitotic division result in daughter cells with unequal sizes and increased micronuclei. Further, in two-cell embryos, microtubule-mediated centering properties of the mitochondrial aggregate prevent nuclear centration, distort nuclear shape, and inhibit DNA synthesis and the onset of embryonic transcription. These findings demonstrate the motor protein-mediated distribution of mitochondria throughout the cytoplasm is highly regulated and is an essential feature of cytoplasmic organization to ensure optimal cell function., Competing Interests: Competing interests statement:The authors declare no competing interest.

Adverse pregnancy outcomes have been associated with the presence of glyphosate (G) in umbilical cord, serum, and urine samples from pregnant women. Our aim was to study the effect of G on blastocyst implantation using an in vitro mouse model, and the migration and acquisition of endothelial phenotype of the human trophoblastic HTR8/SVneo (H8) cells. In mouse blastocysts, no differences in attachment time and implantation outgrowth area were observed after G exposure. H8 cell migration was stimulated by 0.625 μM G without cytotoxicity. After 6 h, the mRNA expression of vascular endothelial growth factor (VEGF) and C-C motif chemokine ligand 2 (CCL2) was upregulated in H8 cells exposed to 1.25 μM G when compared vehicle-treated cells (p ≤ 0.05). No differences were observed in interleukin 11, VEGF receptor 1, and coagulation factor II thrombin receptor in H8 cells exposed to different concentrations of G for 6 h compared to the vehicle. Interestingly, exposure to G did not alter angiogenesis as measured by a tube formation assay. Taken all together, these results suggest that G exposure may contribute as a risk factor during pregnancy, due to its ability to alter trophoblast migration and gene expression., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aneuploidy is frequently detected in early human embryos as a major cause of early pregnancy failure. However, how aneuploidy affects cellular function remains elusive. Here, we profiled the transcriptomes of 14,908 single cells from 203 human euploid and aneuploid blastocysts involving autosomal and sex chromosomes. Nearly all of the blastocysts contained four lineages. In aneuploid chromosomes, 19.5% ± 1.2% of the expressed genes showed a dosage effect, and 90 dosage-sensitive domains were identified. Aneuploidy leads to prevalent genome-wide transcriptome alterations. Common effects, including apoptosis, were identified, especially in monosomies, partially explaining the lower cell numbers in autosomal monosomies. We further identified lineage-specific effects causing unstable epiblast development in aneuploidies, which was accompanied by the downregulation of TGF-β and FGF signaling, which resulted in insufficient trophectoderm maturation. Our work provides crucial insights into the molecular basis of human aneuploid blastocysts and may shed light on the cellular interaction during blastocyst development., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Early embryonic development is a finely orchestrated process that requires precise regulation of gene expression coordinated with morphogenetic events. TATA-box binding protein-associated factors (TAFs), integral components of transcription initiation coactivators like TFIID and SAGA, play a crucial role in this intricate process. Here we show that disruptions in TAF5, TAF12 and TAF13 individually lead to embryonic lethality in the mouse, resulting in overlapping yet distinct phenotypes. Taf5 and Taf12 mutant embryos exhibited a failure to implant post-blastocyst formation, and Taf5 mutants have aberrant lineage specification within the inner cell mass. In contrast, Taf13 mutant embryos successfully implant and form egg-cylinder stages but fail to initiate gastrulation. Strikingly, we observed a depletion of pluripotency factors in TAF13-deficient embryos, including OCT4, NANOG and SOX2, highlighting an indispensable role of TAF13 in maintaining pluripotency. Transcriptomic analysis revealed distinct gene targets affected by the loss of TAF5, TAF12 and TAF13. Thus, we propose that TAF5, TAF12 and TAF13 convey locus specificity to the TFIID complex throughout the mouse genome., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Telomere length (TL) and mitochondrial DNA copy number (mt-cn) are associated with embryonic development. Here, we investigated the correlation between TL and mt-cn in bovine embryos to determine whether TL regulates mt-cn. TL and mt-cn were closely correlated in embryos derived from six bulls. Treatment of embryos with a telomerase inhibitor (TMPyP) and siTERT shortened the TL and reduced mt-cn in blastocysts. RNA-sequencing of blastocysts developed with TMPyP revealed differentially expressed genes associated with transforming growth factor-β1 signaling and inflammation. In conclusion, TL regulates mt-cn in embryos., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Problem: Lipopolysaccharide (LPS) from gram-negative bacteria has reportedly been associated with infectious diseases like metritis, which has a substantial adverse effect on animal reproductive performance and causes serious financial losses for the dairy sector. The current work aimed to establish the impact of LPS on in vitro oocyte maturation and subsequent in vitro developmental competence of oocytes, as well as to investigate the explanatory molecular mechanism underlying this effect., Method of Study: Buffalo cumulus-oocyte complexes (COCs) were challenged with 0, 5, 10 and 20 µg/mL LPS during IVM followed by IVF and IVC. Cytoplasmic and nuclear maturation, cleavage and blastocyst rate, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP, ΔΨm) and transcript abundance of genes related to inflammation, antioxidation and apoptosis were evaluated., Results: The maturation and subsequent embryonic development competency were found to be significantly (p ≤ 0.05) reduced with the addition of 10 and 20 µg/mL LPS to IVM media. ROS production accompanied by a decreased ΔΨm was recorded in LPS-treated oocytes in comparison to the control group (p ≤ 0.05). Our results were further supported by the transcriptional expression of proinflammatory (TLR4, CD14 and RPS27A) and apoptotic gene (Caspase 3) which were found to be significantly increased while antioxidant genes (SOD2 and GPX1) were decreased significantly in matured oocytes and blastocyst after LPS exposure., Conclusions: The deleterious effects of LPS are mediated through ROS generation, which triggers inflammatory processes via the TLR4 pathway and impairs oocyte maturation and subsequent embryonic development., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The present study was conducted to investigate the global proteome of 8-day-old equine blastocysts. Follicular dynamics of eight adult mares were monitored by ultrasonography and inseminated 24 h after the detection of a preovulatory follicle. Four expanded blastocysts were recovered, pooled, and subjected to protein extraction and mass spectrometry. Protein identification was conducted based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, and PepExplorer). Enrichment analysis was performed using g:Profiler, Panther, and String platforms. After the elimination of identification redundancies among search tools (at three levels, based on identifiers, peptides, and cross-database mapping), 1977 proteins were reliably identified in the samples of equine embryos. Proteomic analysis unveiled robust metabolic activity in the 8-day equine embryo, highlighted by an abundance of proteins engaged in key metabolic pathways like the TCA cycle, ATP biosynthesis, and glycolysis. The prevalence of chaperones among highly abundant proteins suggests that regulation of protein folding, and degradation is a key process during embryo development. These findings pave the way for developing new strategies to improve equine embryo media and optimize in vitro fertilization techniques., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Reactive oxygen species (ROS) are associated with oocyte maturation inhibition, and N-acetyl-l-cysteine (NAC) partially reduces their harmful effects. Mitochondrial E3 ubiquitin ligase 1 (Mul1) localizes to the mitochondrial outer membrane. We found that female Mul1-deficient mice are infertile, and their oocytes contain high ROS concentrations. After fertilization, Mul1-deficient embryos showed a DNA damage response (DDR) and abnormal preimplantation embryogenesis, which was rescued by NAC addition and ROS depletion. These observations clearly demonstrate that loss of Mul1 in oocytes increases ROS concentrations and triggers DDR, resulting in abnormal preimplantation embryogenesis. We conclude that manipulating the mitochondrial ROS levels in oocytes may be a potential therapeutic approach to target infertility., (© 2024 Federation of European Biochemical Societies.)

Transposable elements (TEs) are highly expressed in preimplantation development. Preimplantation development is the phase when the cells of the early embryo undergo the first cell fate choice and change from being totipotent to pluripotent. A range of studies have advanced our understanding of TEs in preimplantation, as well as their epigenetic regulation and functional roles. However, many questions remain about the implications of TE expression during early development. Challenges originate first due to the abundance of TEs in the genome, and second because of the limited cell numbers in preimplantation. Here we review the most recent technological advancements promising to shed light onto the role of TEs in preimplantation development. We explore novel avenues to identify genomic TE insertions and improve our understanding of the regulatory mechanisms and roles of TEs and their RNA and protein products during early development., (© 2024 The Author(s).)

Pronuclear transfer has been successfully used in human-assisted reproduction to suppress the adverse effects of a defective oocyte cytoplasm or to bypass an idiopathic developmental arrest. However, the effects of the initial parental genome remodelling in a defective cytoplasm on the subsequent development after pronucleus transfer have not been systematically studied. By performing pronuclear transfer in pre-replication and post-replication mouse embryos, we show that the timing of the procedure plays a critical role. Although apparently morphologically normal blastocysts were obtained in both pre- and post-replication pronuclear transfer groups, post-replication pronuclear transfer led to a decrease in developmental competence and profound changes in embryonic gene expression. By inhibiting the replication in the abnormal cytoplasm before pronuclear transfer into a healthy cytoplasm, the developmental potential of embryos could be largely restored. This shows that the conditions under which the first embryonic replication occurs strongly influence developmental potential. Although pronuclear transfer is the method of choice for mitigating the impact of a faulty oocyte cytoplasm on early development, our results show that the timing of this intervention should be restricted to the pre-replication phase., (© 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.)

Whole salivary gland generation and transplantation offer potential therapies for salivary gland dysfunction. However, the specific lineage required to engineer complete salivary glands has remained elusive. In this study, we identify the Foxa2 lineage as a critical lineage for salivary gland development through conditional blastocyst complementation (CBC). Foxa2 lineage marking begins at the boundary between the endodermal and ectodermal regions of the oral epithelium before the formation of the primordial salivary gland, thereby labeling the entire gland. Ablation of Fgfr2 within the Foxa2 lineage in mice leads to salivary gland agenesis. We reversed this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts, resulting in mice that survived to adulthood with salivary glands of normal size, comparable to those of their littermate controls. These findings demonstrate that CBC-based salivary gland regeneration serves as a foundational experimental approach for future advanced cell-based therapies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Emerging evidence highlights the regulatory role of paired-like (PRD-like) homeobox transcription factors (TFs) in embryonic genome activation (EGA). However, the majority of PRD-like genes are lost in rodents, thus prompting an investigation into PRD-like TFs in other mammals. Here, we showed that PRD-like TFs were transiently expressed during EGA in human, monkey, and porcine fertilized embryos, yet they exhibited inadequate expression in their cloned embryos. This study, using pig as the research model, identified LEUTX as a key PRD-like activator of porcine EGA through genomic profiling and found that LEUTX overexpression restored EGA failure and improved preimplantation development and cloning efficiency in porcine cloned embryos. Mechanistically, LEUTX opened EGA-related genomic regions and established histone acetylation via recruiting acetyltransferases p300 and KAT2A. These findings reveal the regulatory mechanism of LEUTX to govern EGA in pigs, which may provide valuable insights into the study of early embryo development for other non-rodent mammals., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Although, oocytes from prepubertal donors are known to be less developmentally competent than those from adult donors it does not restrain their ability to produce full-term pregnancies. The transcriptomic profile of embryos could be used as a predictor for embryo's individual developmental competence. The aim of the study was to compare transcriptomic profile of blastocysts derived from prepubertal and pubertal heifers oocytes. Bovine cumulus-oocyte complexes (COCs) were obtained by ovum pick- up method from prepubertal and pubertal heifers. After in vitro maturation COCs were fertilized and cultured to the blastocyst stage. Total RNA was isolated from both groups of blastocysts and RNA-seq was performed. Gene ontology analysis was performed by DAVID (Database for Annotation, Visualization and Integrated Discovery)., Results: A higher average blastocyst rate was obtained in the pubertal than in the pre-pubertal group. There were no differences in the quality of blastocysts between the examined groups. We identified 436 differentially expressed genes (DEGs) between blastocysts derived from researched groups, of which 247 DEGs were downregulated in blastocysts derived from pubertal compared to prepubertal heifers oocytes, and 189 DEGs were upregulated. The genes involved in mitochondrial function, including oxidative phosphorylation (OXPHOS) were found to be different in studied groups using Kyoto Encyclopedia of Genes (KEGG) pathway analysis and 8 of those DEGs were upregulated and 1 was downregulated in blastocysts derived from pubertal compared to prepubertal heifers oocytes. DEGs associated with mitochondrial function were found: ATP synthases (ATP5MF-ATP synthase membrane subunit f, ATP5PD- ATP synthase peripheral stalk subunit d, ATP12A- ATPase H+/K + transporting non-gastric alpha2 subunit), NADH dehydrogenases (NDUFS3- NADH: ubiquinone oxidoreductase subunit core subunit S3, NDUFA13- NADH: ubiquinone oxidoreductase subunit A13, NDUFA3- NADH: ubiquinone oxidoreductase subunit A3), cytochrome c oxidase (COX17), cytochrome c somatic (CYCS) and ubiquinol cytochrome c reductase core protein 1 (UQCRC1). We found lower number of apoptotic cells in blastocysts derived from oocytes collected from prepubertal than those obtained from pubertal donors., Conclusions: Despite decreased expression of genes associated with OXPHOS pathway in blastocysts from prepubertal heifers oocytes, the increased level of ATP12A together with the lower number of apoptotic cells in these blastocysts might support their survival after transfer., (© 2024. The Author(s).)

Generating cell types with properties of embryo cells with full developmental potential is of great biological importance. Here, we present a protocol for generating mouse morula-like cells (MLCs) resembling 8- to 16-cell stage embryo cells. We describe steps for induction, via increasing Stat3 activation, and the isolation of MLCs. We then detail procedures for segregating MLCs into blastocyst cell fates and how to create embryo-like structures from them. This system provides a stem-cell-based embryo model to study early embryo development. For complete details on the use and execution of this protocol, please refer to Li et al. 1 ., Competing Interests: Declaration of interests J.C.R.S. and H.L. submitted a patent application (202210437013.X) related to this work (filed by Guangzhou National Laboratory)., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Amphiregulin (AR) is a growth factor that resembles the epidermal growth factor (EGF) and serves various functions in different cells. However, no systematic studies or reports on the role of AR in human oocytes have currently been performed or reported. This study aimed to explore the role of AR in human immature oocytes during in vitro maturation (IVM) and in vitro fertilization (IVF) in achieving better embryonic development and to provide a basis for the development of a pre-insemination culture medium specific for cumulus oocyte complexes (COCs)., Methods: First, we examined the concentration of AR in the follicular fluid (FF) of patients who underwent routine IVF and explored the correlation between AR levels and oocyte maturation and subsequent embryonic development. Second, AR was added to the IVM medium to culture immature oocytes and investigate whether AR could improve the effects of IVM. Finally, we pioneered the use of a fertilization medium supplemented with AR for the pre-insemination culture of COCs to explore whether the involvement of AR can promote the maturation and fertilization of IVF oocytes, as well as subsequent embryonic development., Results: A total of 609 FF samples were examined, and a positive correlation between AR levels and blastocyst formation was observed. In our IVM study, the development potential and IVM rate of immature oocytes, as well as the fertilization rate of IVM oocytes in the AR-added groups, were ameliorated significantly compared to the control group (All P < 0.05). Only the IVM-50 group had a significantly higher blastocyst formation rate than the control group (P < 0.05). In the final IVF study, the maturation, fertilization, high-quality embryo, blastocyst formation, and high-quality blastocyst rates of the AR-added group were significantly higher than those of the control group (All P < 0.05)., Conclusion: AR levels in the FF positively correlated with blastocyst formation, and AR involvement in pre-insemination cultures of COCs can effectively improve laboratory outcomes in IVF. Furthermore, AR can directly promote the in vitro maturation and developmental potential of human immature oocytes at an optimal concentration of 50 ng/ml., 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 Fan, Wang, Ye, Yang, Zhang, Zhang, Yan, Wang, Ding, Chen, Zou, Ji, Zou and Zhang.)

Early trophoblast differentiation is crucial for embryo implantation, placentation and fetal development. Dynamic changes in DNA methylation occur during preimplantation development and are critical for cell fate determination. However, the underlying regulatory mechanism remains unclear. Recently, we derived morula-like expanded potential stem cells from human preimplantation embryos (hEPSC-em), providing a valuable tool for studying early trophoblast differentiation. Data analysis on published datasets showed differential expressions of DNA methylation enzymes during early trophoblast differentiation in human embryos and hEPSC-em derived trophoblastic spheroids. We demonstrated downregulation of DNA methyltransferase 3 members (DNMT3s) and upregulation of ten-eleven translocation methylcytosine dioxygenases (TETs) during trophoblast differentiation. While DNMT inhibitor promoted trophoblast differentiation, TET inhibitor hindered the process and reduced implantation potential of trophoblastic spheroids. Further integrative analysis identified that glutamyl aminopeptidase (ENPEP), a trophectoderm progenitor marker, was hypomethylated and highly expressed in trophoblast lineages. Concordantly, progressive loss of DNA methylation in ENPEP promoter and increased ENPEP expression were detected in trophoblast differentiation. Knockout of ENPEP in hEPSC-em compromised trophoblast differentiation potency, reduced adhesion and invasion of trophoblastic spheroids, and impeded trophoblastic stem cell (TSC) derivation. Importantly, TET2 was involved in the loss of DNA methylation and activation of ENPEP expression during trophoblast differentiation. TET2-null hEPSC-em failed to produce TSC properly. Collectively, our results illustrated the crucial roles of ENPEP and TET2 in trophoblast fate commitments and the unprecedented TET2-mediated loss of DNA methylation in ENPEP promoter., (© 2024. The Author(s).)

Liver disease is a major global health challenge. There is a shortage of liver donors worldwide, and hepatocyte transplantation (HT) may be an effective treatment to overcome this problem. However, the present approaches for generation of hepatocytes are associated with challenges, and interspecies chimera-derived hepatocytes produced by interspecies blastocyst complementation (IBC) may be promising donor hepatocytes because of their more comprehensive hepatic functions. In this study, we isolated mouse hepatocytes from mouse-rat chimeric livers using IBC and found that interspecies chimera-derived hepatocytes exhibited mature hepatic functions in terms of lipid accumulation, glycogen storage, and urea synthesis. Meanwhile, they were more similar to endogenous hepatocytes than hepatocytes derived in vitro. Interspecies chimera-derived hepatocytes could relieve chronic liver fibrosis and reside in the injured liver after transplantation. Our results suggest that interspecies chimera-derived hepatocytes are a potentially reliable source of hepatocytes and can be applied as a therapeutic approach for HT., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Preimplantation embryo culture, pivotal in assisted reproductive technology (ART), has lagged in innovation compared to embryo selection advancements. This review examines the persisting gap between in vivo and in vitro embryo development, emphasizing the need for improved culture conditions. While in humans this gap is hardly estimated, animal models, particularly bovines, reveal clear disparities in developmental competence, cryotolerance, pregnancy and live birth rates between in vitro-produced (IVP) and in vivo-derived (IVD) embryos. Molecular analyses unveil distinct differences in morphology, metabolism, and genomic stability, underscoring the need for refining culture conditions for better ART outcomes. To this end, a deeper comprehension of oviduct physiology and embryo transport is crucial for grasping embryo-maternal interactions' mechanisms. Research on autocrine and paracrine factors, and extracellular vesicles in embryo-maternal tract interactions, elucidates vital communication networks for successful implantation and pregnancy. In vitro, confinement, and embryo density are key factors to boost embryo development. Advanced dynamic culture systems mimicking fluid mechanical stimulation in the oviduct, through vibration, tilting, and microfluidic methods, and the use of innovative softer substrates, hold promise for optimizing in vitro embryo development.

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.)

Single-cell transcriptomic studies of differentiating systems allow meaningful understanding, especially in human embryonic development and cell fate determination. We present an innovative method aimed at modeling these intricate processes by leveraging scRNAseq data from various human developmental stages. Our implemented method identifies pseudo-perturbations, since actual perturbations are unavailable due to ethical and technical constraints. By integrating these pseudo-perturbations with prior knowledge of gene interactions, our framework generates stage-specific Boolean networks (BNs). We apply our method to medium and late trophectoderm developmental stages and identify 20 pseudo-perturbations required to infer BNs. The resulting BN families delineate distinct regulatory mechanisms, enabling the differentiation between these developmental stages. We show that our program outperforms existing pseudo-perturbation identification tool. Our framework contributes to comprehending human developmental processes and holds potential applicability to diverse developmental stages and other research scenarios.

In somatic cells, DNA repair is attenuated during mitosis to prevent the formation of anaphase bridges and facilitate the proper segregation of sister chromatids. Irradiation-induced γH2AX foci persist for hours in M phase somatic cells. However, we observed that anaphase bridges formed in a significant fraction of mouse zygotes irradiated during mitosis. Additionally, γH2AX signals in M phase zygotes peaked 30 min after irradiation and subsequently reduced with a half-life within 1-2 h. These results suggest that the DNA repair system may operate efficiently in M phase zygotes following irradiation, leading to the frequent formation of anaphase bridges. The absence of H2AX promoted the successful segregation of sister chromatids and enhanced the development of embryos to the blastocyst stage. The DNA repair system may be differentially regulated during the M phase of the first cell cycle to ensure the immediate elimination of damaged zygotes, thereby efficiently preventing transmission of mutations to subsequent generations.