Deoxyribonucleic acid methylation profiling of single human blastocysts by methylated CpG-island amplification coupled with CpG-island microarray

Objective To study whether methylated CpG-island (CGI) amplification coupled with microarray (MCAM) can be used to generate DNA (deoxyribonucleic acid) methylation profiles from single human blastocysts. Design A pilot microarray study with methylated CpG-island amplification applied to human blastocyst genomic DNA and hybridized on CpG-island microarrays. Setting University research laboratory. Patient(s) Five cryopreserved sibling 2-pronuclear zygotes that were surplus to requirements for clinical treatment by in vitro fertilization were donated with informed consent from a patient attending Bourn Hall Clinic, Cambridge, United Kingdom. Intervention(s) None. Main Outcome Measure(s) Successful generation of genome-wide DNA methylation profiles at CpG islands from individual human blastocysts, with common genomic regions of DNA methylation identified between embryos. Result(s) Between 472 and 734 CpG islands were methylated in each blastocyst, with 121 CpG islands being commonly methylated in all 5 blastocysts. A further 159 CGIs were commonly methylated in 4 of the 5 tested blastocysts. Methylation was observed at a number of CGIs within imprinted-gene, differentially methylated regions (DMRs), including placental and preimplantation-specific DMRs. Conclusion(s) The MCAM method is capable of providing comprehensive DNA methylation data in individual human blastocysts

Early primary-rather than primordial follicles constitute the main follicular reserve in the African elephant (Loxodonta africana)

Information on the ovarian follicle reserve in the African elephant (Loxodonta africana) is lacking. This study set out to determine the ratios of early preantral follicles and their relative dimensions in the ovaries of 16 African elephant aged 10–34 years. The ovaries were sectioned histologically. Follicles were counted and classified according to expansion of the pre-granulosa cells. Early primary follicles were the most common (75.8% ± 11.8%), followed by true primary follicles (23.8% ± 11.8%), whereas primordial follicles were the most rare (<2%). Measurements made on at least 100 early preantral follicles from each animal (n = 1464) indicate that growth in oocyte and nuclear diameters started with transition to the true primary stage P < 0.01. This, together with the observed ratios between the three types of early preantral follicles suggest that both classical primordial and early primary follicles contribute to the ovarian reserve in the African elephant

Gene expression regulating epithelial intercellular junction biogenesis during human blastocyst development in vitro

We investigated gene expression associated with trophectoderm epithelial intercellular junction formation in single human embryos at different stages of cleavage using RT–PCR methods based upon magnetic bead separation of polyA+ RNA. Trophectoderm tight junction (TJ) and desmosome biogenesis contribute to intercellular sealing and tissue integrity, critical for vectorial transport and blastocoel cavity formation. Expression of the various genes throughout human preimplantation development showed differing levels of sensitivity of detection; these genes included claudin-1, occludin (TM4+ and TM4 isoforms), ZO-1 (ZO-1+ and ZO-1– isoforms), ZO-2 and JAM (junction adhesion molecule), and the desmosome junction gene, DSC2 (desmocollin 2). Some transcripts appeared to be expressed throughout preimplantation development (claudin-1, JAM, occludin TM4+ and TM4, ZO-1- isoform) while others tended to be expressed preferentially in later cleavage and associated with blastocyst formation (ZO-2, ZO-1+ isoform, DSC-2), illustrating an expression pattern broadly similar to mouse cleavage stages. Human embryo transcript detection was significantly decreased when reverse transcription was performed in solid phase to generate a bead/cDNA transient library rather than after mRNA elution from beads. Transcript detection tended to be positively correlated with embryo morphological grade using the solid phase method. In blastocysts, occludin TM4–, ZO-1+ and DSC2 transcripts were the most susceptible to failure of detection, indicative of low levels of expression which may impact on trophectoderm differentiation competence. Immunoconfocal microscopy analysis of selected adhesion and TJ proteins in human embryos indicated poor membrane assembly compared with mouse blastocysts, which may further affect embryo viability

Variable imprinting of the MEST gene in human preimplantation embryos

There is evidence that expression and methylation of the imprinted paternally expressed gene 1/mesoderm-specific transcript homologue (PEG1/MEST) gene may be affected by assisted reproductive technologies (ARTs) and infertility. In this study, we sought to assess the imprinting status of the MEST gene in a large cohort of in vitro-derived human preimplantation embryos, in order to characterise potentially adverse effects of ART and infertility on this locus in early human development. Embryonic genomic DNA from morula or blastocyst stage embryos was screened for a transcribed AflIII polymorphism in MEST and imprinting analysis was then performed in cDNA libraries derived from these embryos. In 10 heterozygous embryos, MEST expression was monoallelic in seven embryos, predominantly monoallelic in two embryos, and biallelic in one embryo. Screening of cDNA derived from 61 additional human preimplantation embryos, for which DNA for genotyping was unavailable, identified eight embryos with expression originating from both alleles (biallelic or predominantly monoallelic). In some embryos, therefore, the onset of imprinted MEST expression occurs during late preimplantation development. Variability in MEST imprinting was observed in both in vitro fertilization and intracytoplasmic sperm injection-derived embryos. Biallelic or predominantly monoallelic MEST expression was not associated with any one cause of infertility. Characterisation of the main MEST isoforms revealed that isoform 2 was detected in early development and was itself variably imprinted between embryos. To our knowledge, this report constitutes the largest expression study to date of genomic imprinting in human preimplantation embryos and reveals that for some imprinted genes, contrasting imprinting states exist between embryos

Metabolism of human embryos following cryopreservation: implications for the safety and selection of embryos for transfer in clinical IVF

BACKGROUND: Cryopreservation of supernumerary embryos is routinely performed in human-assisted reproduction, providing a source of embryos which can be thawed for use in subsequent treatment cycles. However, the viability of cryopreserved embryos has traditionally relied on morphological assessment, which is a poor predictor of embryo health since freezing leads to a significant overall reduction in implantation potential, and its long-term efficacy is unknown. This study describes how the post-thaw metabolism of human embryos can be used to predict future development to the blastocyst stage. METHODS: HPLC was used to analyse the post-thaw amino acid metabolism of human embryos from day 2 to day 3 of development. RESULTS: It was possible to predict with 87% accuracy which frozen-thawed embryo would develop to the blastocyst stage. Developmentally competent embryos were more metabolically quiescent than their arresting counterparts. Amino acid turnover was also capable of distinguishing between the developmental potential of the best, Grade I embryos P &lt; 0.05. CONCLUSIONS: The data suggests that cryopreservation in IVF is a safe procedure and that amino acid turnover can be used to select which cryopreserved embryo will develop to the blastocyst stage, irrespective of their post-thaw grade