Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency.

Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here, we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PAPERCLIP.

Identification of transcriptionally-active human papillomavirus integrants through nanopore sequencing reveals viable targets for gene therapy against cervical cancer.

Integration of the human papillomavirus (HPV) genome into the cellular genome is a key event that leads to constitutive expression of viral oncoprotein E6/E7 and drives the progression of cervical cancer. However, HPV integration patterns differ on a case-by-case basis among related malignancies. Next-generation sequencing technologies still face challenges for interrogating HPV integration sites. In this study, utilizing Nanopore long-read sequencing, we identified 452 and 108 potential integration sites from the cervical cancer cell lines (CaSki and HeLa) and five tissue samples, respectively. Based on long Nanopore chimeric reads, we were able to analyze the methylation status of the HPV long control region (LCR), which controls oncogene E6/E7 expression, and to identify transcriptionally-active integrants among the numerous integrants. As a proof of concept, we identified an active HPV integrant in between RUNX2 and CLIC5 on chromosome 6 in the CaSki cell line, which was supported by ATAC-seq, H3K27Ac ChIP-seq, and RNA-seq analysis. Knockout of the active HPV integrant, by the CRISPR/Cas9 system, dramatically crippled cell proliferation and induced cell senescence. In conclusion, identifying transcriptionally-active HPV integrants with Nanopore sequencing can provide viable targets for gene therapy against HPV-associated cancers.

Effective protection: the embryonic development and clinical outcomes of emergency vitrification of 1246 oocytes and Day 0-Day 5 embryos in a natural disaster.

STUDY QUESTION: Can emergency vitrification protect embryos and oocytes during natural disasters or other events that prevent normal practice to achieve satisfactory embryonic development and clinical outcomes at a later time? SUMMARY ANSWER: Emergency vitrification of oocytes and Day 0-Day 5 (D0-D5) embryos during disasters is a safe and effective protective measure. WHAT IS KNOWN ALREADY: When some destructive events such as floods, earthquakes, tsunamis, and other accidents occur, emergency vitrification in embryo laboratories to protect human embryos, oocytes, and sperm is one of the important measures of an IVF emergency plan. However, there are few detailed reports on emergency vitrification in a state of disaster, especially about oocytes and D0 zygotes. Therefore, the effectiveness and safety of emergency vitrification of oocytes and D0-D5 embryos in disaster states are still unclear. STUDY DESIGN, SIZE, DURATION: A retrospective study was made in the Reproductive Medicine Center of the First Affiliated Hospital of Zhengzhou University from January 2018 to November 2022. The record rainstorms in Zhengzhou, China, caused severe flooding, traffic disruptions, and power outages. From 17:30, 20 July 2021 to 17:30, 21 July 2021, 1246 oocytes and D0-D5 embryos of 155 patients were vitrified whilst the laboratory had only an emergency power supply. PARTICIPANTS/MATERIALS, SETTING, METHODS: As of 21 December 2021, 1149 emergency vitrified oocytes and D0-D5 embryos of 124 patients underwent frozen-thawed embryo transfer (FET). They were divided into the following four groups according to the days of embryo culture in vitro: oocyte group, Day 0-Day 1 (D0-D1) group, Day 2-Day 3 (D2-D3) group, and Day 4-Day 5 (D4-D5) group. Control groups for each were selected from fresh cycle patients who underwent IVF/ICSI from January 2018 to October 2021. Control and emergency vitrification patients were matched on criteria that included age, fertilization method, days of embryonic development, and number and grade of transferred embryos. A total of 493 control patients were randomly selected from the eligible patients and matched with the emergency vitrification groups in a ratio of 4:1. The results of assisted reproduction and follow-up of pregnancy were analyzed. The embryonic development, clinical outcomes, and birth outcomes in each group were statistically analyzed. MAIN RESULTS AND THE ROLE OF CHANCE: A significant difference was observed in fertilization rate (81% versus 72%, P = 0.022) between the oocyte group and the control group. Significant differences were also observed in the monozygotic twin pregnancy rate (10% versus 0%, P = 0.038) and ectopic pregnancy rate (5% versus 0%, P = 0.039) between the D0-D1 group and the control group. No significant differences (P > 0.05) were observed between vitrified oocytes/D0-D1 embryos/D2-D3 embryos and the control group on the number of high-quality embryos (3.17 ± 3.00 versus 3.84 ± 3.01, P = 0.346; 5.04 ± 3.66 versus 4.56 ± 2.87, P = 0.346; 4.85 ± 5.36 versus 5.04 ± 4.64, P = 0.839), the number of usable blastocysts (1.22 ± 1.78 versus 1.21 ± 2.03, P = 0.981; 2.16 ± 2.26 versus 1.55 ± 2.08, P = 0.090; 2.82 ± 3.23 versus 2.58 ± 3.32, P = 0.706), clinical pregnancy rate (56% versus 57%, P = 0.915; 55% versus 55%, P = 1.000; 40% versus 50%, P = 0.488), miscarriage rate (30% versus 15%, P = 0.496; 5% versus 11%, P = 0.678; 17% versus 20%, P = 1.000), and live birth rate (39% versus 49%, P = 0.460; 53% versus 50%, P = 0.772; 33% versus 40%, P = 0.635). No significant differences (P > 0.05) were observed between the D4-D5 group and the control group on clinical pregnancy rate (40% versus 55%, P = 0.645), miscarriage rate (0% versus 18%, P = 1.000), and live birth rate (40% versus 45%, P = 1.000). LIMITATIONS, REASONS FOR CAUTION: The retrospective study design is a limitation. The timing and extent of natural disasters are unpredictable, so the sample size of vitrified oocytes, zygotes, and embryos is beyond experimental control. WIDER IMPLICATIONS OF THE FINDINGS: This study is the first study analyzing embryonic development, clinical outcomes, and birth outcomes of large samples of oocytes, D0 zygotes, and D1-D5 embryos after emergency vitrification under the disaster conditions. The results show that emergency vitrification is a safe and effective protective measure applicable to oocytes and D0-D5 embryos. The embryology laboratories need to be equipped with an emergency uninterrupted power supply capable of delivering for 6-8 h at full load. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (grant 81871206). The authors declare that they have no conflicts of interest. All authors have completed the ICMJE Disclosure form. TRIAL REGISTRATION NUMBER: N/A.

Adverse maternal and neonatal outcomes of preimplantation genetic testing with trophectoderm biopsy: a retrospective cohort study of 3373 intracytoplasmic sperm injection single frozen-thawed blastocyst transfer cycles.

PURPOSE: To investigate whether trophectoderm biopsy increases the risk of adverse maternal and neonatal outcomes in intracytoplasmic sperm injection (ICSI) single frozen-thawed blastocyst transfer cycles. METHODS: This respective cohort study enrolled 3373 ICSI single frozen-thawed blastocyst transfer cycles with and without trophectoderm biopsy. Statistical methods including univariate logistic regression analysis, multivariate logistic regression analysis, and stratified analyses were performed to explore the impact of trophectoderm biopsy on adverse maternal and neonatal outcomes. RESULTS: The rates of adverse maternal and neonatal outcomes were comparable between the two groups. Univariate analysis showed that the live birth rate (45.15% vs. 40.75%; P = 0.010) in the biopsied group was statistically higher than that in the unbiopsied group, and the rates of miscarriage (15.40% vs. 20.00%; P = 0.011) and birth defects (0.58% vs. 2.16%; P = 0.007) were statistically lower in the biopsied group. After adjusting for confounding factors, the rates of miscarriage (aOR = 0.74; 95% CI = 0.57-0.96; P = 0.022) and birth defects (aOR = 0.24, 95% CI = 0.08-0.70, P = 0.009) in the biopsied group were significantly lower than those in the unbiopsied group. Stratified analyses showed that the birth defects rate after biopsy was significantly reduced in the subgroups of age < 35 years old, BMI ≥ 24 kg/m2, artificial cycle with downregulation, poor-quality blastocysts, and Day 5 poor-quality blastocysts. CONCLUSION: Preimplantation genetic testing (PGT) with trophectoderm biopsy does not increase the risk of adverse maternal and neonatal outcomes in ICSI single frozen-thawed blastocyst transfer cycles, and PGT can effectively reduce the rates of miscarriage and birth defects.

Atypical PKC, regulated by Rho GTPases and Mek/Erk, phosphorylates Ezrin during eight-cell embryocompaction.

Phosphorylation of Ezrin T567 plays an important role in eight-cell embryo compaction. Yet, it is not clear how Ezrin phosphorylation is regulated during embryo compaction. Here, we demonstrated that inhibition of Mek/Erk or protein kinase C (PKC) signaling reduced the phosphorylation level of Ezrin T567 in eight-cell compacted embryos. Interestingly, the Rho GTPase inhibitor C3-transferase caused basolateral enrichment of atypical PKC (aPKC), as well as basolateral shift of phosphorylated Ezrin, suggesting aPKC may be a key regulator of Ezrin phosphorylation. Moreover, inhibition of PKC, but not Mek/Erk or Rho GTPases, affected the maintenance of Ezrin phosphorylation in compacted embryos. We further identified that aPKC is indeed required for Ezrin phosphorylation in eight-cell embryos. Taken together, Rho GTPases facilitate the apical distribution of aPKC and Ezrin. Subsequently, aPKC and Mek/Erk work together to promote Ezrin phosphorylation at the apical region, which in turn mediates the apical enrichment of filamentous actin, stabilizing the polarized apical region and allowing embryo compaction. Our data also suggested that aPKC might be the Ezrin kinase during eight-cell embryo compaction.

NCOA3 knockdown delays human embryo development.

Embryonic development is a precisely controlled sequential process influenced by complex external and internal factors; therefore, this process holds paramount significance in the context of in vitro fertilization and embryo transfer (IVF-ET), with internal oocyte and embryo quality being pivotal in determining its success. Nuclear receptor coactivator 3 (NCOA3), a member of the p160 nuclear receptor coactivators family, has been extensively studied in tumorigenesis and reportedly plays a crucial role in maintaining pluripotency in mouse embryonic stem cells (ESCs). However, its functions in human embryo development remain largely unexplored. In this study, we collected human samples, including oocytes, zygotes, and embryos, from patients at the First Affiliated Hospital of Zhengzhou University to investigate whether NCOA3 regulates human embryonic development. To this end, we employed various assays, including immunofluorescence, quantitative real-time PCR (qPCR), microinjection, and RNA sequencing. Our findings suggested that NCOA3 expression level was low in inferior embryos (with >50 % fragmentation), and its presence is closely related to the expression of the pluripotency factor NANOG. Deletion of NCOA3 delays human embryonic development. Single-oocyte RNA sequencing revealed that NCOA3 primarily participates in metabolic alterations in oocytes. In sum, these findings elucidate the pivotal roles of NCOA3 in human embryonic development-NCOA3 deletion compromise the developmental potential of embryos. These mechanistic insights into the role of NCOA3 in human embryonic development not only advances our understanding of the intricate molecular mechanisms involved but also holds potential implications for improving assisted reproductive technologies (ART) and addressing developmental disorders in human embryos.

Follicular Thyroid Carcinoma Arising from the Struma Ovarii Coexisting with Papillary Thyroid Carcinoma, Hashimoto's Thyroiditis and Polycystic Ovarian Syndrome-a Case Report and Literature Review.

Purpose: Struma ovarii is a highly specialized teratoma consisting primarily of mature thyroid tissue. However, malignant struma ovarii coexisting with thyroid carcinoma, not to mention autoimmune disease, is uncommon. Malignant struma ovarii complicated with papillary thyroid carcinoma, Hashimoto's thyroiditis and polycystic ovarian syndrome has never been reported in literature. Patients and Methods: A 32-year-old female was admitted to our hospital due to a history of abdominal distension and menolipsis over the past half a year. Physical examination touched a 6 × 6 cm mass with a clear boundary, normal movement, and no pressing pain in the right adnexal area, Imaging revealed a cystic solid mass of 6 × 7 cm in the right ovary and the level of tumor markers including CA125, CA199, CA153, CEA, AFP were normal, but with low TSH and increased TPOAb, TGAb, TRAb. Laparoscopic right ovary tumor resection was performed, followed by comprehensive staging surgery, as well as thyroidectomy after pathologic diagnosis. The patient was diagnosed with a combination of follicular thyroid cancer from struma ovarii, papillary thyroid carcinoma and Hashimoto's thyroiditis, along with polycystic ovarian syndrome. Immunohistochemical staining showed positivity for Ag, CK-pan, CK7, PAX8 and TTF-1 in the right ovarian mass, and the left thyroid was positive for the BRAF V600E mutation. Results: The patient underwent thyroxine suppression therapy and radioactive iodine 131I therapy after operation. Serum thyroglobulin was undetectable, and no signs of recurrence or metastasis were detected in the imaging examination at the 2-year follow-up. Conclusion: Malignant struma ovarii coexisting with thyroid carcinoma is rare. No report has been identified in literature review on the rare malignant struma ovarii coexisting with thyroid carcinoma, Hashimoto's thyroiditis and polycystic ovarian syndrome. Our case can offer experience of diagnosis and treatment to some extent for such rare case. Therefore, it is essential to consider the association between ovarian tumors and the endocrine system. This case is valuable in understanding the diagnosis and management of such an unusual complicated disease.

YTHDF2 regulates MSS51 expression contributing to mitochondria dysfunction of granulosa cells in polycystic ovarian syndrome patients.

RESEARCH QUESTION: Granulosa cells (GCs) dysfunction plays a crucial role in the pathogenesis of polycystic ovary syndrome (PCOS). It is reported that YTH domain-containing family protein 2 (YTHDF2) is upregulated in mural GCs of PCOS patients. What effect does the differential expression of YTHDF2 have in PCOS patients? DESIGN: Mural GCs and cumulus GCs from 15 patients with PCOS and 15 ovulatory controls and 4 cases of pathological sections in each group were collected. Real-time PCR, Western Blot, immunohistochemistry, and immunofluorescence experiments were conducted to detect gene and protein expression. RNA immunoprecipitation assay was performed to evaluate the binding relationship between YTHDF2 and MSS51. Mitochondrial morphology, cellular ATP and ROS levels and glycolysis-related gene expression were detected after YTHDF2 overexpression or MSS51 inhibition. RESULTS: In the present study, we found that YTHDF2 was upregulated in GCs of PCOS patients while MSS51 was downregulated. YTHDF2 protein can bind to MSS51 mRNA and affect MSS51 expression. The reduction of MSS51 expression or the increase in YTHDF2 expression can lead to mitochondrial damage, reduced ATP levels, increased ROS levels and reduced expression of LDHA, PFKP and PKM. CONCLUSIONS: YTHDF2 may regulate the expression of MSS51, affecting the structure and function of mitochondria in GCs and interfering with cellular glycolysis, which may disturb the normal biological processes of GCs and follicle development in PCOS patients.

Possible involvement of a MEG3-miR-21-SPRY1-NF-κB feedback loop in spermatogenic cells proliferation, autophagy, and apoptosis.

Non-obstructive azoospermia (NOA) is the most incurable form of male infertility with a complex etiology. Long non-cording RNAs (lncRNAs) were associated with regulating spermatogenesis. Herein, differentially expressed lncRNAs between NOA and control male were screened by RNA-seq analysis. MEG3 was upregulated in NOA tissues and inhibited cell proliferation and promoted cell autophagy and apoptosis in vitro. Through RNA immunoprecipitation (RIP), biotin pull-down assays, and dual-luciferase reporter assays, MEG3 was proved to act as a competing endogenous RNA of microRNA (miR)-21 and thus influenced the SPRY1/ERK/mTOR signaling pathway. Additionally, bioinformatic prediction and chip assay revealed that MEG3 was possibly regulated by nuclear factor κB (NF-κB) and SPRY1/NF-κB/MEG3 formed a feedback loop. Seminiferous tubule microinjection further investigated the effects of MEG3 on testes in vivo. These findings demonstrated that MEG3-miR-21-SPRY1-NF-κB probably acted as a feedback loop leading to azoospermia. Our study might provide a target and theoretical basis for diagnosing and treating NOA.

Role of nuclear receptor coactivator 3 (Ncoa3) in pluripotency maintenance.

Nuclear receptors, including Esrrb, Dax1, and Nr5a2, have been shown to be involved in pluripotency maintenance. Yet, the role of their coactivators in mouse embryonic stem cells remains unexplored. Here, we demonstrated that the nuclear receptor coactivator 3 (Ncoa3) is essential for pluripotency maintenance. Knockdown of Ncoa3 not only compromises the expression of pluripotency markers but also impairs in vitro and in vivo differentiation potential of mouse ESCs. Ncoa3 binds to the Nanog promoter and recruits the histone acetyltransferase CREB binding protein (CBP) and the histone arginine methyltransferase CARM1 to activate Nanog expression. Moreover, glycogen synthase kinase 3 GSK3 signaling down-regulates the Ncoa3 protein level to suppress Nanog expression. Thus, Ncoa3 not only contributes to self-renewal by activating Nanog but also facilitates ESC differentiation as a break point to disrupt the core transcriptional circuitry of pluripotency.

Whole Genome Assembly of Human Papillomavirus by Nanopore Long-Read Sequencing.

Human papillomavirus (HPV) is a causal agent for most cervical cancers. The physical status of the HPV genome in these cancers could be episomal, integrated, or both. HPV integration could serve as a biomarker for clinical diagnosis, treatment, and prognosis. Although whole-genome sequencing by next-generation sequencing (NGS) technologies, such as the Illumina sequencing platform, have been used for detecting integrated HPV genome in cervical cancer, it faces challenges of analyzing long repeats and translocated sequences. In contrast, Oxford nanopore sequencing technology can generate ultra-long reads, which could be a very useful tool for determining HPV genome sequence and its physical status in cervical cancer. As a proof of concept, in this study, we completed whole genome sequencing from a cervical cancer tissue and a CaSki cell line with Oxford Nanopore Technologies. From the cervical cancer tissue, a 7,894 bp-long HPV35 genomic sequence was assembled from 678 reads at 97-fold coverage of HPV genome, sharing 99.96% identity with the HPV sequence obtained by Sanger sequencing. A 7904 bp-long HPV16 genomic sequence was assembled from data generated from the CaSki cell line at 3857-fold coverage, sharing 99.99% identity with the reference genome (NCBI: U89348). Intriguingly, long reads generated by nanopore sequencing directly revealed chimeric cellular-viral sequences and concatemeric genomic sequences, leading to the discovery of 448 unique integration breakpoints in the CaSki cell line and 60 breakpoints in the cervical cancer sample. Taken together, nanopore sequencing is a unique tool to identify HPV sequences and would shed light on the physical status of HPV genome in its associated cancers.

Comparative study on the pregnancy outcomes of in vitro fertilization-embryo transfer between patients with different ovarian responses (a STROBE-compliant article).

There have been few studies on large-sample data of cleavage-stage embryo and blastocyst transfers. We compared the pregnancy outcomes of patients with different ovarian responses after the transfer of different numbers of embryos in different developmental stages.Patients were divided into 3 groups including low response group, medium response group, and high response group according to different ovarian responses. Patients in each group were further divided into 4 subgroups including group A: transfer of 1 D3 embryo, group B: transfer of 2 D3 embryos; group C: transfer of 1 D5 blastocyst; and group D: transfer of 2 D5 blastocysts.In low ovarian responders, the implantation rate, clinical pregnancy rate and live birth rate were significantly lower in the group A than in the groups B and C. In medium ovarian responders, the implantation rate was significantly higher, but the multiple pregnancy rate was significantly lower in the group C than in the group B. The multiple pregnancy rate was significantly higher in the group D than in the group C. In high ovarian responders, the implantation rate was significantly lower, but the multiple pregnancy rate was significantly higher in the group B than in group C.Based on the above results, the single blastocyst transfer is preferable for the patients with different ovarian responses.

Enhancement of porcine intramuscular fat content by overexpression of the cytosolic form of phosphoenolpyruvate carboxykinase in skeletal muscle.

Intramuscular fat (IMF) content has been generally recognized as a desirable trait in pork meat because of its positive effect on eating quality. An effective approach to enhance IMF content in pork is the generation of transgenic pigs. In this study, we used somatic cell nuclear transfer (SCNT) to generate cloned pigs exhibiting ectopic expression of phosphoenolpyruvate carboxykinase (PEPCK-C) driven by an α-skeletal-actin gene promoter, which was specifically expressed in skeletal muscle. Using qRT-PCR and Western blot analysis, we demonstrated that PEPCK-C was functionally expressed and had a significant effect on total fatty acid content in the skeletal muscle of the transgenic pigs, while the n-6/n-3 polyunsaturated fatty acid (PUFA) ratio showed no difference between transgenic and control pigs. Thus, genetically engineered PEPCK-Cmus pigs may be an effective solution for the production of IMF-enriched pork.

LIN28 phosphorylation by MAPK/ERK couples signalling to the post-transcriptional control of pluripotency.

Signalling and post-transcriptional gene control are both critical for the regulation of pluripotency, yet how they are integrated to influence cell identity remains poorly understood. LIN28 (also known as LIN28A), a highly conserved RNA-binding protein, has emerged as a central post-transcriptional regulator of cell fate through blockade of let-7 microRNA biogenesis and direct modulation of mRNA translation. Here we show that LIN28 is phosphorylated by MAPK/ERK in pluripotent stem cells, which increases its levels via post-translational stabilization. LIN28 phosphorylation had little impact on let-7 but enhanced the effect of LIN28 on its direct mRNA targets, revealing a mechanism that uncouples LIN28's let-7-dependent and -independent activities. We have linked this mechanism to the induction of pluripotency by somatic cell reprogramming and the transition from naive to primed pluripotency. Collectively, our findings indicate that MAPK/ERK directly impacts LIN28, defining an axis that connects signalling, post-transcriptional gene control, and cell fate regulation.

LIN28 Regulates Stem Cell Metabolism and Conversion to Primed Pluripotency.

The RNA-binding proteins LIN28A and LIN28B play critical roles in embryonic development, tumorigenesis, and pluripotency, but their exact functions are poorly understood. Here, we show that, like LIN28A, LIN28B can function effectively with NANOG, OCT4, and SOX2 in reprogramming to pluripotency and that reactivation of both endogenous LIN28A and LIN28B loci are required for maximal reprogramming efficiency. In human fibroblasts, LIN28B is activated early during reprogramming, while LIN28A is activated later during the transition to bona fide induced pluripotent stem cells (iPSCs). In murine cells, LIN28A and LIN28B facilitate conversion from naive to primed pluripotency. Proteomic and metabolomic analysis highlighted roles for LIN28 in maintaining the low mitochondrial function associated with primed pluripotency and in regulating one-carbon metabolism, nucleotide metabolism, and histone methylation. LIN28 binds to mRNAs of proteins important for oxidative phosphorylation and modulates protein abundance. Thus, LIN28A and LIN28B play cooperative roles in regulating reprogramming, naive/primed pluripotency, and stem cell metabolism.

Expression and function of natural antisense transcripts in mouse embryonic stem cells.

Non-coding RNAs (ncRNAs), such as microRNAs and large intergenic non-coding RNAs, have been shown to play essential roles in regulating pluripotency. Yet, it is not clear the role of natural antisense transcripts (NATs), also belonging to ncRNAs, in embryonic stem cells. However, the role of NATs in embryonic stem cells remains unknown. We further confirmed the expression of the NATs of three key pluripotency genes, Oct4, Nanog and Sox2. Moreover, overexpression of Sox2-NAT reduces the expression of Sox2 protein, and slightly enhances the Sox2 mRNA level. Altogether, our data indicated that like other ncRNAs, NATs might be involved in pluripotency maintenance.

Molecular basis of the first cell fate determination in mouse embryogenesis.

Through proliferation and differentiation, a single cell, the zygote, can give rise to a complex organism composed of many types of cells. Up to the eight-cell embryo stage, the blastomeres are morphologically identical and distributed symmetrically in the mammalian embryo. Functionally, in some species, they are all totipotent. However, due to the compaction of blastomeres and the asymmetrical cell division at the late phase of the eight-cell embryo, the blastomeres of the morula are no longer identical. During the transition from morula to blastocyst, blastomeres differentiate, resulting in the first cell fate decision in embryogenesis, namely, the segregation of the inner cell mass and the trophectoderm. In this review, we will discuss the regulatory mechanisms essential for the cell fate choice during blastocyst development, including transcriptional regulation, epigenetic regulation, microRNAs, and signal transduction.