Transcriptomic signatures in trophectoderm and inner cell mass of human blastocysts classified according to developmental potential, maternal age and morphology.

Selection of high-quality embryos is important to achieve successful pregnancy in assisted reproductive technology (ART). Recently, it has been debated whether RNA-sequencing (RNA-Seq) should be applied to ART to predict embryo quality. However, information on genes that can serve as markers for pregnant expectancy is limited. Furthermore, there is no information on which transcriptome of trophectoderm (TE) or inner cell mass (ICM) is more highly correlated with pregnant expectancy. Here, we performed RNA-Seq analysis of TE and ICM of human blastocysts, the pregnancy expectation of which was retrospectively determined using the clinical outcomes of 1,890 cases of frozen-thawed blastocyst transfer. We identified genes that were correlated with the expected pregnancy rate in ICM and TE, respectively, with a larger number of genes identified in TE than in ICM. Downregulated genes in the TE of blastocysts that were estimated to have lower expectation of pregnancy included tight junction-related genes such as CXADR and ATP1B1, which have been implicated in peri-implantation development. Moreover, we identified dozens of differentially expressed genes by regrouping the blastocysts based on the maternal age and the Gardner score. Additionally, we showed that aneuploidy estimation using RNA-Seq datasets does not correlate with pregnancy expectation. Thus, our study provides an expanded list of candidate genes for the prediction of pregnancy in human blastocyst embryos.

Chromosomal analyses of human giant diploid oocytes by next-generation sequencing.

PURPOSE: Although giant oocytes (GOs) having about twice cytoplasmic volume compared with general oocytes in mammals including the human are rarely recovered, it is thought that GOs have potentially chromosomal abnormalities. The aim of the present study was to assess chromosome numbers in chromosome-spindle complexes (CSCs) and polar bodies of human GOs by using micromanipulation for sampling and next-generation sequencing (NGS) for analyses of the chromosome numbers. METHODS: When recovered oocytes whose cytoplasm has lager than 140 µm or above, the oocytes were defined as GOs, and recovered GOs were vitrified. After warming, the CSCs, polar bodies, and enucleated cytoplasm were collected by micromanipulation from 3 GOs. The collected samples were analyzed by NGS. RESULTS: Chromosomal aneuploidy in the GOs was confirmed in all the three GOs. Comparing the CSCs with the chromosomes from polar bodies, the deletion and overlapping chromosome numbers were complementary in each GO. CONCLUSIONS: The authors could collect the CSCs and the polar bodies from human GOs by micromanipulation, and then could analyze the chromosome numbers of the GOs by NGS method. As our data suggest that human GOs have chromosomal abnormalities, GOs should be excluded from clinical purpose as gamete sources for embryo transfer in the human.

First mitotic spindle formation is led by sperm centrosome-dependent MTOCs in humans.

Unlike in mice, multinucleated blastomeres appear at a high frequency in the two-cell-stage embryos in humans. In this Point of View article, we demonstrate that the first mitotic spindle formation led by sperm centrosome-dependent microtubule organizing centers may cause a high incidence of zygotic division errors using human tripronuclear zygotes.

Ovarian stimulation using human chorionic gonadotrophin impairs blastocyst implantation and decidualization by altering ovarian hormone levels and downstream signaling in mice.

Ovarian stimulation induced by follicle-stimulating hormone and human chorionic gonadotrophin (hCG) is commonly used in assisted reproductive technology to increase embryo production. However, recent clinical and animal studies have shown that ovarian stimulation disrupts endometrial function and embryo development and adversely affects pregnancy outcomes. How ovarian stimulation impairs pregnancy establishment and the precise mechanisms by which this stimulation reduces the chances of conception remain unclear. In this study, we first demonstrated that ovarian stimulation using hCG alone impairs implantation, decidualization and fetal development of mice by generating abnormal ovarian hormone levels. We also showed that ovarian hormone levels were altered because of changes in the levels of the enzymes involved in their synthesis in the follicles and corpora lutea. Furthermore, we determined that anomalous ovarian hormone secretion induced by ovarian stimulation alters the spatiotemporal expression of progesterone receptors and their downstream genes, especially in the uterine epithelium. Epithelial estrogenic signaling and cell proliferation were promoted on the day of implantation in stimulated mice and these changes led to the failure of uterine transition from the prereceptive to the receptive state. Collectively, our findings indicate that ovarian stimulation using hCG induces an imbalance in steroid hormone secretion, which causes a failure of the development of uterine receptivity and subsequent implantation and decidualization by altering the expression of steroid receptors and their downstream signaling associated with embryo implantation.

The production of a diabetic mouse using constructs encoding porcine insulin promoter-driven mutant human hepatocyte nuclear factor-1alpha.

A diabetic mouse model was produced using a mutant human hepatocyte nuclear factor-1alpha gene (HNF1alphaP291fsinsC) regulated by the porcine insulin promoter. The functionality of two different constructs containing HNF1alphaP291fsinsC, termed PD1 and PD2 (cytomegalovirus enhancer minus and plus), were examined in transgenic mice. The blood glucose levels and body weights of the PD1 transgenic mice did not differ from their non-transgenic littermates over the period from 3 to 8 weeks of age. Conversely, the PD2 transgenic mice exhibited hyperglycemia and decreased body weight. Western blot analysis demonstrated that mutant HNF-1alpha protein (HNF1alphaP291), derived from the PD2 transgene, was expressed in the PD2 mice. Morphometric studies of the pancreas of a PD2 mouse revealed that the number of pancreatic islets present was less than that in the non-transgenic mice, indicating disturbed islet neogenesis. These results suggest that impaired insulin secretion in disrupted islets causes hyperglycemia. In addition, the phenotype of PD2 transgenic mice similar to that of the HNF-1alpha gene-deficient mouse, which displays growth retardation and impaired viability. These results indicate that HNF1alphaP291 expression driven by the porcine insulin promoter, together with the cytomegalovirus enhancer, induces a diabetic phenotype in transgenic mice.

Production of a transgenic pig expressing human albumin and enhanced green fluorescent protein.

We introduced a fusion gene of human albumin and enhanced green fluorescent protein (EGFP) into porcine oocytes using the sperm vector method, and produced a piglet that showed clear expression of GFP in the hooves and skin. PCR and Southern blotting analysis of genomic DNA extracted from the piglet's tissues, including the liver, showed that the tissues carried the transgene. RT-PCR analysis demonstrated that both the human albumin and EGFP genes were expressed in the tissues. The fact that human albumin gene was integrated and expressed in the liver of the transgenic pig opened a way for us to achieve our goal, which was the use of transgenic pigs for the bioartificial liver support system.