Blastocyst Trophectoderm Research Articles

Trophectoderm biopsy. a simplified mechanical method

Trophectoderm biopsy is an emerging approach in preimplantaion genetic diagnosis. The most recent introduction of laser technology has facilitated this step considerably.Worldwide the majority of the IVF clinics performing trophectoderm biopsy are using the laser for zona drilling. But, the cost of laser is considered high. Blastocyst trophectoderm biopsy using micromanipulation methods was first reported by Dorkas et al. (1990); however, new improvements are still needed. The objective was to evaluate a simplified, inexpensive method to remove trophectoderm cells. Discarded day-6 expanded or hatching blastocysts of more than 140 hours post insemination from IVF/ICSI were used to evaluate a simplified mechanical method for removal of trophectoderm cells. Trophectoderm biopsies were conducted under mineral oil in Falcon 1006 Petri dishes in 10 μl drops of G-MOPS medium without protein (Vitrolife Sweden AB). An Olympus IX70 equipped with integra Ti micromanipulation system and a tool holder with an opthalmic surgery micro blade (Micro Feather Blade # K-722) were used to perform biopsies. Blastocysts were placed individually in G-MOPS medium without holding pipette. The protruding trophectoderm was at 12 o'clock position. Excision of the trophectoderm cells was achieved with the assistance of the micro blade. Ten microliters of blastocyst medium with 20% of protein was added to the biopsy drops. Embryos and cells were rinsed and moved to the culture medium for a short evaluation. A total of 42 hatching blastocysts were biopsed. All the blastocysts survived the biopsy procedure. Trophectoderm cells were removed from the biopsy dish to the rinse dish with no problem. Further development of manipulated embryos or cells were not apparently impaired. All biopsies were performed in less than 90 seconds. These results demonstrate the feasibility of the trophectoderm biopsy using a practical mechanical method.

X-chromosome inactivation in monkey embryos and pluripotent stem cells

Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.

Blastocyst preimplantation genetic diagnosis (PGD) of a mitochondrial DNA disorder

To evaluate the utility of trophectoderm biopsy for preimplantation genetic diagnosis (PGD) of mitochondrial (mt) DNA mutation load. A PGD case and analysis of blastocyst mosaicism. Academic center for reproductive medicine. A 30-year-old carrier of 35% 3243A>G mtDNA mutation load with a daughter affected by mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. Blastocyst biopsy for PGD of mutation load and gender. Variation in mutation load within and among embryos, and newborn mutation load after PGD-based selection. Oocytes and embryos were found to possess a variety of 3243A>G mutation loads from 9% to 90% in oocytes and 7% to 91% in embryos, demonstrating that PGD would be a relevant procedure. Highly consistent results were obtained within multiple biopsies of both cleavage- and blastocyst-stage embryos. Importantly, mutation loads observed in trophectoderm were predictive of the inner cell mass (r(2) = 0.97). Transfer of a male embryo, predicted to possess 12% mutation load by analysis of a trophectoderm biopsy, resulted in the delivery of a boy with tissue-specific mutation loads ranging from undetectable to 15%. This study represents the first successful clinical application of PGD to reduce the transgenerational risk of transmitting an mtDNA disorder and supports the applicability of blastocyst trophectoderm PGD for carriers of mtDNA mutations attempting reproduction.

Viral Titer Determines the Placental Expression of a Transgene Following Lentivirus-Mediated Transgenesis of Mouse Blastocysts.

Lentiviral mediated transgenesis of trophectoderm in mouse blastocysts is a powerful means for exploring the roles of a candidate gene in placenta. When zona-denuded blastocysts are transduced with lentiviral vectors carrying a transgene, the tight junctions of the epithelial trophectoderm impede access to the inner cell mass and restrict transgenesis to trophoblast derivatives. However, the transgene may insert in genomic regions that eventually become heterochromatic, resulting in transgene silencing. In this study, we sought to determine the optimal dose of viral particles to overcome subsequent silencing, thereby providing uniform and consistent transgenesis of placental tissue. In an initial experiment, in vivo derived, zona-free, mouse blastocysts were co-incubated for 7 h with a lentivirus designed to express EGFP under the regulation of a constitutive EF1a promoter at three different viral concentrations (7, 70 and 350 ng/ml), with 40 blastocyst per group in three independent replicates. Uniform EGFP could be detected by fluorescence evaluation 24 h after viral transduction in the trophectoderm of all embryos exposed to 70 and 350 ng/ml virus but not after infection at 7 ng/ml. Quantitative RT-PCR confirmed expression of the transgene encoding EGFP expression in all experimental groups and was correlated with the viral concentration employed (ANOVA P<0.05; mean±SEM; 7 ng/ml 1±0.3 vs 70 ng/ml 4.59±1.1 vs 350 ng/ml 9.88±0.8). In a separate experiment, blastocysts were incubated with lentiviral vectors at the two higher concentrations and transferred to surrogate dams (30 blastocysts/group, 10 blastocysts/recipient). Recipients were sacrificed on D12 of pregnancy and conceptuses evaluated for EGFP expression. The number of viable fetuses did not differ between the two groups (24/30 in 70 ng/ml vs 20/30 in 350 ng/ml), but less than half of the placentas obtained from blastocysts incubated in 70 ng/ml (10/24) expressed EGFP, whereas all 20 placentas from the 350 ng/ml group showed green fluorescence. In conclusion, efficient transgene expression assessed at the blastocyst stage does not ensure later expression in placenta, and higher viral concentrations can overcome transgene silencing. Supported by a fellowship from the Lalor Foundation (PBA), and grants from the Preeclampsia Foundation (BT) and NIH HD067759 (RMR).

Transcriptome of the Inner Cell Mass and Trophectoderm in Bovine Blastocyst as Determined by Next Generation Sequencing.

The first distinct differentiation in mammals occurs at the blastocyst stage. Here we determined global gene expressions patterns in the inner cell mass (ICM) and trophectoderm (TE) isolated from bovine blastocysts using next generation sequencing and evaluated how CSF2, which increases embryo competence to complete development to term, alters gene expression. Bovine embryos were produced in vitro and were treated with 0 or 10 ng/ml CSF2 from Day 6 to 8 after insemination. Blastocysts were harvested at Day 8, ICM and TE isolated, and cDNA sequenced using SOLiD 4.0 (n=3 separate pools of cDNA per treatment-cell type). A total of 870 genes were differentially expressed between ICM and TE (411 upregulated in ICM and 459 upregulated in TE). CSF2 changed expression of 25 genes in ICM and 23 in TE. Numerous genes not considered as ICM or TE markers in the mouse or human were identified. In addition, genes characteristic of ICM (NANOG, SOX2, and STAT3) and TE (ATP1B3, ELF5, and KRT18) in mouse and human showed similar patterns in bovine. Upregulated genes in ICM were consistent with maintenance of pluripotency and activation of cell proliferation. Enriched gene ontology (GO) terms included regulation of transcription, transcription regulator activity and Jak-STAT signaling pathway, pathways in cancer and chemokine signaling pathway. The last term indicates possible involvement of cytokines for communication with TE or regulation of ICM. Upregulated genes in TE were consistent with increased metabolic activity as well as interactions with the maternal environment with respect to endo- and exocytosis (GO terms included vesicle-mediated transport and lysosomes), secretion of signaling molecules (PAG2 and IFNT; steroidogenesis GO terms), and changes in cellular architecture (GO terms included actin cytoskeleton and cell projection). The percent of genes overexpressed in ICM that were classified as CpG positive was lower (P<0.015) than for genes overexpressed in TE. In conclusion, the transcriptome of the ICM and TE reflect their developmental fate and spatial orientation in the embryo, with overexpression of genes involved in pluripotency and cell proliferation in ICM and overexpression of genes required for interactions with the maternal system and for cellular rearrangement in the TE.

Development and validation of an accurate quantitative real-time polymerase chain reaction–based assay for human blastocyst comprehensive chromosomal aneuploidy screening

To develop and validate a quantitative real-time polymerase chain reaction (qPCR)-based method for blastocyst trophectoderm comprehensive chromosome screening (CCS) of aneuploidy. Prospective, randomized, and blinded. Academic center for reproductive medicine. Nine cell lines were obtained from a commercial cell line repository, and 71 discarded human blastocysts were obtained from 24 IVF patients that underwent preimplantation genetic screening. None. Consistency of qPCR diagnosis of aneuploidy compared with either conventional karyotyping of cell lines or microarray-based diagnoses of human blastocysts. Samples from nine cell lines with well characterized karyotypes were diagnosed by qPCR with 97.6% (41/42) consistency. After applying a minimum threshold for concurrence, 100% consistency was achieved. Developmentally normal blastocysts designated as aneuploid or arrested blastocysts designated as euploid by single-nucleotide polymorphism microarray analyses were assigned identical 24 chromosome diagnoses by qPCR in 98.6% of cases (70/71). Overall euploidy (n = 37) and aneuploidy (n = 34) were assigned with 100% consistency. Data was obtained for both sample types in 4 hours. These data demonstrate the first qPCR technology capable of accurate aneuploidy screening of all 24 chromosomes in 4 hours. This methodology provides an opportunity to evaluate trophectoderm biopsies with subsequent fresh euploid blastocyst transfer. Randomized controlled trials to investigate the clinical efficacy of qPCR-based CCS are currently underway.

Trophoblast-specific DNA methylation occurs after the segregation of the trophectoderm and inner cell mass in the mouse periimplantation embryo

The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages, resulting in the formation of the trophectoderm (TE) and inner cell mass (ICM) in blastocysts. Although a lower level of global DNA methylation in the genome of the TE compared with ICM has been suggested, the dynamics of the DNA methylation profile during TE/ICM differentiation has not been elucidated. To address this issue, first we identified tissue-dependent and differentially methylated regions (T-DMRs) between trophoblast stem (TS) and embryonic stem (ES) cells. Most of these TS–ES T-DMRs were also methylated differentially between trophoblast and embryonic tissues of embryonic day (E) 6.5 mouse embryos. Furthermore, we found that the human genomic regions homologous to mouse TS–ES T-DMRs were methylated differentially between human placental tissues and ES cells. Collectively, we defined them as cell-lineage-based T-DMRs between trophoblast and embryonic cell lineages (T–E T-DMRs). Then, we examined TE and ICM cells isolated from mouse E3.5 blastocysts. Interestingly, all T-DMRs examined, including the Elf5, Pou5f1 and Nanog loci, were in the nearly unmethylated status in both TE and ICM and exhibited no differences. The present results suggest that the establishment of DNA methylation profiles specific to each cell lineage follows the first morphological specification. Together with previous reports on asymmetry of histone modifications between TE and ICM, the results of the current study imply that histone modifications function as landmarks for setting up cell-lineage-specific differential DNA methylation profiles.

GnRH agonist protocol versus GnRH antagonist protocol in assisted reproduction

GnRH agonist protocol versus GnRH antagonist protocol in assisted reproduction

Establishment of Human Trophoblast Progenitor Cell Lines from the Chorion

Placental trophoblasts are key determinants of in utero development. Mouse trophoblast (TB) stem cells, which were first derived over a decade ago, are a powerful cell culture model for studying their self-renewal or differentiation. Our attempts to isolate an equivalent population from the trophectoderm of human blastocysts generated colonies that quickly differentiated in vitro. This finding suggested that the human placenta has another progenitor niche. Here, we show that the chorion is one such site. Initially, we immunolocalized pluripotency factors and TB fate determinants in the early gestation placenta, amnion, and chorion. Immunoreactive cells were numerous in the chorion. We isolated these cells and plated them in medium containing fibroblast growth factor which is required for human embryonic stem cell self-renewal, and an inhibitor of activin/nodal signaling. Colonies of polarized cells with a limited lifespan emerged. Trypsin dissociation yielded continuously self-replicating monolayers. Colonies and monolayers formed the two major human TB lineages-multinucleate syncytiotrophoblasts and invasive cytotrophoblasts (CTBs). Transcriptional profiling experiments revealed the factors associated with the self-renewal or differentiation of human chorionic TB progenitor cells (TBPCs). They included imprinted genes, NR2F1/2, HMGA2, and adhesion molecules that were required for TBPC differentiation. Together, the results of these experiments suggested that the chorion is one source of epithelial CTB progenitors. These findings explain why CTBs of fully formed chorionic villi have a modest mitotic index and identify the chorionic mesoderm as a niche for TBPCs that support placental growth.

DNA methylation profile in human placental tissues

For the first time using genome-wide Infinium HumanMethylation27 BeadChip Array (Illumina, USA) DNA methylation pattern was determined in the human cytotrophoblast and extraembryonic mesoderm. These tissues are the derivatives of trophectoderm and inner cell mass of blastocyst and have substantial differences in dynamics of epigenetic genome reprogramming during early stages of differentiation. The genome of the extraembryonic mesoderm cells has been shown to be more methylated compared to the cytotrophoblast similarly to other mammalian species. Differences in methylation pattern of single CpG-dinucleotides and dinucleotides localized within promoter CpG-islands have been found. It has been shown that the majority of single CpG-dinucleotides in both tissues were methylated whereas promoter CpG-island sites were not. Comparative analysis revealed 202 differentially methylated genes in extraembryonic mesoderm and 40 genes in cytotrophoblast. These genes are responsible for diverse biological processes. However, in the extraembryonic mesoderm the main functional groups included genes responsible for DNA binding and transcriptional factors' activity whereas in the cytotrophoblast--for transport and protein and cytokine secretion.

Association of Rex-1 to target genes supports its interaction with Polycomb function

Rex-1/Zfp42 displays a remarkably restricted pattern of expression in preimplantation embryos, primary spermatocytes, and undifferentiated mouse embryonic stem (ES) cells and is frequently used as a marker gene for pluripotent stem cells. To understand the role of Rex-1 in selfrenewal and pluripotency, we used Rex-1 association as a measure to identify potential target genes, and carried out chromatin-immunoprecipitation assays in combination with gene specific primers to identify genomic targets Rex-1 associates with. We find association of Rex-1 to several genes described previously as bivalently marked regulators of differentiation and development, whose repression in mouse embryonic stem (ES) cells is Polycomb Group-mediated, and controlled directly by Ring1A/B. To substantiate the hypothesis that Rex-1 contributes to gene regulation by PcG, we demonstrate interactions of Rex-1 and YY2 (a close relative of YY1) with Ring1 proteins and the PcG-associated proteins RYBP and YAF2, in line with interactions reported previously for YY1. We also demonstrate the presence of Rex-1 protein in both trophectoderm and Inner Cell Mass of the mouse blastocyst and in both ES and in trophectoderm stem (TS) cells. In TS cells, we were unable to demonstrate association of Rex-1 to the genes it associates with in ES cells, suggesting that association may be cell-type specific. Rex-1 might fine-tune pluripotency in ES cells by modulating Polycomb-mediated gene regulation.

Oct4-Enhanced Green Fluorescent Protein Transgenic Pigs: A New Large Animal Model for Reprogramming Studies

The domesticated pig has emerged as an important tool for development of surgical techniques, advancement of xenotransplantation, creation of important disease models, and preclinical testing of novel cell therapies. However, germ line-competent pluripotent porcine stem cells have not yet been derived. This has been a major obstacle to genetic modification of pigs. The transcription factor Oct4 is essential for the maintenance of pluripotency and for reprogramming somatic cells to a pluripotent state. Here, we report the production of transgenic pigs carrying an 18 kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct) to allow identification of pluripotent cells by monitoring Oct4 expression by EGFP fluorescence. Eleven viable transgenic piglets were produced by somatic cell nuclear transfer. Expression of the EGFP reporter construct was confined to germ line cells, the inner cell mass and trophectoderm of blastocysts, and testicular germ cells. Reprogramming of fibroblasts from these animals by fusion with pluripotent murine embryonic stem cells or viral transduction with human OCT4, SOX2, KLF4, and c-MYC cDNAs resulted in Oct4-EGFP reactivation. The OG2 pigs have thus proved useful for monitoring reprogramming and the induction and maintenance of pluripotency in porcine cells. In conclusion, the OG2 transgenic pigs are a new large animal model for studying the derivation and maintenance of pluripotent cells, and will be valuable for the development of cell therapy.

Single-gene testing combined with single nucleotide polymorphism microarray preimplantation genetic diagnosis for aneuploidy: a novel approach in optimizing pregnancy outcome

To describe a method of amplifying DNA from blastocyst trophectoderm cells (two or three cells) and simultaneously performing 23-chromosome single nucleotide polymorphism microarrays and single-gene preimplantation genetic diagnosis. Case report. IVF clinic and preimplantation genetic diagnostic centers. A 36-year-old woman, gravida 2, para 1011, and her husband who both were carriers of GM(1) gangliosidosis. The couple wished to proceed with microarray analysis for aneuploidy detection coupled with DNA sequencing for GM(1) gangliosidosis. An IVF cycle was performed. Ten blastocyst-stage embryos underwent trophectoderm biopsy. Twenty-three-chromosome microarray analysis for aneuploidy and specific DNA sequencing for GM(1) gangliosidosis mutations were performed. Viable pregnancy. After testing, elective single embryo transfer was performed followed by an intrauterine pregnancy with documented fetal cardiac activity by ultrasound. Twenty-three-chromosome microarray analysis for aneuploidy detection and single-gene evaluation via specific DNA sequencing and linkage analysis are used for preimplantation diagnosis for single-gene disorders and aneuploidy. Because of the minimal amount of genetic material obtained from the day 3 to 5 embryos (up to 6 pg), these modalities have been used in isolation of each other. The use of preimplantation genetic diagnosis for aneuploidy coupled with testing for single-gene disorders via trophectoderm biopsy is a novel approach to maximize pregnancy outcomes. Although further investigation is warranted, preimplantation genetic diagnosis for aneuploidy and single-gene testing seem destined to be used increasingly to optimize ultimate pregnancy success.

Single nucleotide polymorphism microarray–based concurrent screening of 24-chromosome aneuploidy and unbalanced translocations in preimplantation human embryos

To develop, validate, and apply a single nucleotide polymorphism (SNP) microarray-based method for simultaneous preimplantation genetic diagnosis (PGD) of unbalanced inheritance of rearranged chromosomes and 24-chromosome aneuploidy screening. Prospective clinical research study. Academic reproductive medicine center. Eighteen couples carrying a balanced reciprocal or Robertsonian chromosomal rearrangement. PGD on blastocyst trophectoderm biopsy specimens. Aneuploidy, implantation, pregnancy, and delivery rates after SNP microarray-based aneuploidy and translocation screening. Single nucleotide polymorphism microarray was capable of detecting translocation-associated imbalances as small as 9.0 megabases. In the 12 transfers performed, sustained implantation occurred for 9 (45%) of 20 balanced-normal and euploid embryos replaced. The clinical pregnancy rate in patients receiving a transfer was 75% with six singleton deliveries and three ongoing singleton pregnancies thus far. Significantly fewer embryos were eligible for transfer with the incorporation of simultaneous 24-chromosome aneuploidy screening. Arrested embryos were also significantly more likely to possess unbalanced chromosomes when compared with developmentally competent blastocysts. This SNP microarray-based method provides the first opportunity to improve outcomes through comprehensive identification of euploid embryos from translocation carrier couples.

Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O2 compared to 5% O2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O2 tension, whereas IVD101 supported blastocyst formation only under low O2 levels but enhanced the proliferation of ICM cells.

BRG1 Is Required for CDX2-Mediated Repression of Oct4 Expression in Mouse Blastocysts.

During blastocyst formation the segregation of the inner cell mass (ICM) and trophectoderm is governed by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2. Evidence indicates that suppression of Oct4 expression in the trophectoderm is mediated by Cdx2. Nonetheless, the underlying epigenetic modifiers required for Cdx2-dependent repression of Oct4 are largely unknown. Here we show that the chromatin remodeling protein Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. By employing a combination of RNA interference (RNAi) and gene expression analysis we found that both Brg1 Knockdown (KD) and Cdx2 KD blastocysts exhibit widespread expression of Oct4 in the trophectoderm. Interestingly, in Brg1 KD blastocysts and Cdx2 KD blastocysts, the expression of Cdx2 and Brg1 is unchanged, respectively. To address whether Brg1 cooperates with Cdx2 to repress Oct4 transcription in the developing trophectoderm, we utilized preimplantation embryos, trophoblast stem (TS) cells and Cdx2-inducible embryonic stem (ES) cells as model systems. We found that: (1) combined knockdown (KD) of Brg1 and Cdx2 levels in blastocysts resulted in increased levels of Oct4 transcripts compared to KD of Brg1 or Cdx2 alone, (2) endogenous Brg1 co-immunoprecipitated with Cdx2 in TS cell extracts, (3) in blastocysts Brg1 and Cdx2 co-localize in trophectoderm nuclei and (4) in Cdx2-induced ES cells Brg1 and Cdx2 are recruited to the Oct4 promoter. Lastly, to determine how Brg1 may induce epigenetic silencing of the Oct4 gene, we evaluated CpG methylation at the Oct4 promoter in the trophectoderm of Brg1 KD blastocysts. This analysis revealed that Brg1-dependent repression of Oct4 expression is independent of DNA methylation at the blastocyst stage. In toto, these results demonstrate that Brg1 cooperates with Cdx2 to repress Oct4 expression in the developing trophectoderm to ensure normal development.

Establishment of Cell Lineage-Specific DNA Methylation Profile Follows the Morphological Differentiation of Trophoblast and Embryonic Cell Lineages.

There are numerous tissue-dependent and differentially methylated regions (T-DMRs) in the mammalian genome. Cell type-specific DNA methylation status of T-DMRs constitutes DNA methylation profile of the cell, which ought to be reconstructed as cells differentiate. The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages resulting in the formation of trophectoderm (TE) and inner cell mass (ICM) in blastocyst. Although lower level of global DNA methylation in TE genome compared to ICM has been suggested, dynamics of DNA methylation profile during the TE/ICM differentiation has not been studied. In this study, we aimed to identify T-DMRs between trophoblast and embryonic cell lineages and analyzed their DNA methylation status in pre- and post-implantation embryos. Oct4 and Nanog are differentially methylated between trophoblast stem (TS) and embryonic stem (ES) cell lines (TS-ES T-DMRs). These two loci are hyper-methylated in TS cells and hypo-methylated in ES cells. Elf5 is another known TS-ES T-DMR but exhibits the opposite methylation pattern. A number of other TS-ES T-DMRs have been found so far in our laboratory. First, we addressed whether these TS-ES T-DMRs are indeed differentially methylated between trophoblast and embryonic cell lineages in vivo. Bisulfite sequencing analysis of 11 TS-ES T-DMRs revealed that the DNA methylation status of them in E6.5 extraembryonic ectoderm and epiblast coincide with those in TS and ES cells, respectively, showing that the TS-ES T-DMRs are surely cell lineage-based T-DMRs in vivo. We then collected TE and ICM cells separately from E3.5 blastocysts and subjected to bisulfite sequencing. Interestingly, genomes of TE and ICM exhibited no difference in the methylation status of all T-DMRs examined. They were in extremely hypomethylated status. Here we identified valuable T-DMRs for delineation of trophoblast/embryonic cell lineages. DNA methylation analysis of these T-DMRs suggested that the DNA methylation profiles specific to each cell lineage are established after the morphological delineation of trophoblast and embryonic cell lineages. DNA methylation may thus contribute to stabilize or fix the cell fate and the differentiation capacity. Supported by Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (20062003). (platform)

Clinical results on single cells from 470 embryos using 23-chromosome single nucleotide polymorphism (SNP) microarray preimplantation genetic screening (PGS) from 45 patients

OBJECTIVE: We amplified DNA from a single blastomere or trophectoderm cells and performed complex SNP microarray genetic analyses. DESIGN: Prospective study. MATERIALS AND METHODS: PGS patients between 12/1/09 and 4/25/10 were analyzed. 45 patients underwent embryo biopsy by laser and SNP microarray PGS primarily due to > 2 spontaneous miscarriages. 3 of these patients were tested for translocation abnormalities and aneuploidy. The maternal ages ranged from 34 to 44yrs. 398 embryos from 37 patients underwent day-3 biopsies, whereas 72 embryos from 8 patients underwent blastocyst trophectoderm biopsies. We amplified the DNA using a modified whole genome amplification (WGA) protocol. We used the Illumina Cyto-12 microarray to determine chromosome aberrations and to obtain genotype data for ∼300K SNPs. A high-resolution copy-number profile was used to identify copy number variations (CNVs). Parental DNA was simultaneously analyzed with embryo DNA for CNVs. Data was analyzed with Illumina GenomeStudio software. RESULTS: 45 patients and 470 embryos were tested. 37 patients undergoing a day-3 biopsy were considered for embryo transfer along with 1 patient undergoing a blastocyst biopsy. All embryos from an additional 7 patients undergoing blastocyst biopsies and PGS were frozen. 59% (277/470) of the embryos were abnormal. Many embryos with CNVs were considered normal because of parental CNV analysis. 3.1 ± 2 euploid embryos were identified per patient/cycle. The overall clinical pregnancy rate was 62%. The blastocyst biopsy transferred patient is pregnant.Tabled 1Clinical Pregnancy by Age GroupAgeClinical PregnancyMiscarriageNo TransferFrozen〈3556% (5/9)4% (1/23)0235-3763% (5/8)01038-4075% (9/12)004〉4044% (4/9)041 Open table in a new tab CONCLUSION: 23-chromosome SNP microarray PGS is a valuable technology in an effort to optimize the transfer of euploid embryos and to increase the delivery rates of couples undergoing in vitro fertilization (IVF) due to repeat miscarriages.

Progesterone receptor requires a co-chaperone for signalling in uterine biology and implantation.

Embryo implantation is absolutely dependent on the preparation of the uterus to the receptive stage and attainment by the blastocyst of implantation competency. Co-ordinated effects of progesterone and oestrogen are essential for these processes and determine the window of implantation. In rodents, a generalized stromal edema occurs before blastocyst attachment followed by uterine luminal closure. This leads to apposition of the blastocyst trophectoderm against the luminal epithelium and ultimately attachment. Progesterone is essential for luminal closure, which must occur for successful implantation. More importantly, progesterone is critical for almost every stage of pregnancy, including ovulation, fertilization, implantation, decidualization and pregnancy maintenance. Progesterone exerts its effects on target tissues primarily via nuclear progesterone receptor (PR) whose optimal activity is potentiated by an immunophilin co-chaperone, FK-506 binding protein 4 (FKBP52). While mice lacking PR are infertile due to complete failure of ovulation, fertilization, and implantation, female mice with targeted deletion of the Fkbp52 gene are infertile specifically because of implantation failure resulting from compromised uterine receptivity. This review highlights the evolution of knowledge about progesterone signalling during early pregnancy. Future studies are likely to provide a better understanding of FKBP52-PR signalling in promoting uterine receptivity for implantation and may reveal new targets for improving infertility.

Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

During blastocyst formation the segregation of the inner cell mass (ICM) and trophectoderm is governed by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2. Evidence indicates that suppression of Oct4 expression in the trophectoderm is mediated by Cdx2. Nonetheless, the underlying epigenetic modifiers required for Cdx2-dependent repression of Oct4 are largely unknown. Here we show that the chromatin remodeling protein Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. By employing a combination of RNA interference (RNAi) and gene expression analysis we found that both Brg1 Knockdown (KD) and Cdx2 KD blastocysts exhibit widespread expression of Oct4 in the trophectoderm. Interestingly, in Brg1 KD blastocysts and Cdx2 KD blastocysts, the expression of Cdx2 and Brg1 is unchanged, respectively. To address whether Brg1 cooperates with Cdx2 to repress Oct4 transcription in the developing trophectoderm, we utilized preimplantation embryos, trophoblast stem (TS) cells and Cdx2-inducible embryonic stem (ES) cells as model systems. We found that: (1) combined knockdown (KD) of Brg1 and Cdx2 levels in blastocysts resulted in increased levels of Oct4 transcripts compared to KD of Brg1 or Cdx2 alone, (2) endogenous Brg1 co-immunoprecipitated with Cdx2 in TS cell extracts, (3) in blastocysts Brg1 and Cdx2 co-localize in trophectoderm nuclei and (4) in Cdx2-induced ES cells Brg1 and Cdx2 are recruited to the Oct4 promoter. Lastly, to determine how Brg1 may induce epigenetic silencing of the Oct4 gene, we evaluated CpG methylation at the Oct4 promoter in the trophectoderm of Brg1 KD blastocysts. This analysis revealed that Brg1-dependent repression of Oct4 expression is independent of DNA methylation at the blastocyst stage. In toto, these results demonstrate that Brg1 cooperates with Cdx2 to repress Oct4 expression in the developing trophectoderm to ensure normal development.