Bourn Hall Clinic

Medically assisted reproduction, now considered a routine, successful treatment for infertility worldwide, has produced at least 8 million live births. However, a growing body of evidence is pointing toward an increased incidence of epigenetic/imprinting disorders in the offspring, raising concern that the techniques involved may have an impact on crucial stages of early embryo and fetal development highly vulnerable to epigenetic influence. In this paper, the key role of methylation processes in epigenesis, namely the essential biochemical/metabolic pathways involving folates and one-carbon cycles necessary for correct DNA/histone methylation, is discussed. Furthermore, potential contributors to epigenetics dysregulation during the three phases of assisted reproduction: preparation for and controlled ovarian hyperstimulation (COH); methylation processes during the preimplantation embryo culture stages; the effects of unmetabolized folic acid (UMFA) during embryogenesis on imprinting methyl “tags”, are described. Advances in technology have opened a window into developmental processes that were previously inaccessible to research: it is now clear that ART procedures have the potential to influence DNA methylation in embryonic and fetal life, with an impact on health and disease risk in future generations. Critical re-evaluation of protocols and procedures is now an urgent priority, with a focus on interventions targeted toward improving ART procedures, with special attention to in vitro culture protocols and the effects of excessive folic acid intake.

Methylation is a universal biochemical process which covalently adds methyl groups to a variety of molecular targets. It plays a critical role in two major global regulatory mechanisms, epigenetic modifications and imprinting, via methyl tagging on histones and DNA. During reproduction, the two genomes that unite to create a new individual are complementary but not equivalent. Methylation determines the complementary regulatory characteristics of male and female genomes. DNA methylation is executed by methyltransferases that transfer a methyl group from S-adenosylmethionine, the universal methyl donor, to cytosine residues of CG (also designated CpG). Histones are methylated mainly on lysine and arginine residues. The methylation processes regulate the main steps in reproductive physiology: gametogenesis, and early and late embryo development. A focus will be made on the impact of assisted reproductive technology and on the impact of endocrine disruptors (EDCs) via generation of oxidative stress.

Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac1. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human2-5 and cow6. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical-basal cell polarity, with expression of atypical protein kinaseC (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos.

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SummaryDespite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR–Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR–Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

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Maintaining consistent and reliably high success rates is a daily challenge for every IVF laboratory. This step-by-step guide is an essential aid in navigating the complex maze of physical, chemical, biological, and logistic parameters that underpin successful gamete and embryo culture: temperature, pH, osmolality, gas supplies, air quality, light exposure, infections, managing supplies, personnel, as well as overall quality control. Numerous real-life troubleshooting case reports are presented, identifying all aspects necessary for troubleshooting. Process maps and flow charts accompanying each chapter offer a logical and systematic approach to problem solving in the laboratory. This is an essential resource for scientists in assisted reproductive technology and specialists in reproductive biology and medicine, helping IVF clinics to achieve the dream of every infertile couple: the birth of a healthy child.

A primary endothelial cell dysfunction is thought to be involved in systemic sclerosis (SSc)-associated fibroproliferative vasculopathy of the microcirculation and small arterioles, even in sites not affected by fibrosis. Because the role of fibroblasts in pathologic modifications and vascular wall remodeling is relatively unclear, and because the retina provides a unique opportunity to assess microcirculation in the absence of resident fibroblasts, we systematically evaluated retinal vessels in patients with SSc. Digital retinal images were obtained from both eyes of 93 consecutive patients with fully characterized SSc and 29 healthy controls matched 1:1 for age and sex with selected patients without diabetes, hypertension history, or antihypertensive treatment. Internal microvascular calibers (erythrocyte column width in μm) by central retinal arteriolar and venular equivalents and arteriolar to venular ratio were measured using validated software. Arteriolar and venular calibers were similar in patients and their matched controls (mean ± SEM; 187 ± 2 vs 184 ± 3, p = 0.444, and 211 ± 2 vs 216 ± 3, p = 0.314, respectively). Both arteriolar and venular calibers and their ratio in patients with SSc were not associated with disease duration, extent of skin involvement, pulmonary fibrosis, digital ulcers or pitting scars, amputations, digital capillaroscopic findings, inflammatory indices, or autoantibodies. The evidence that retinal microcirculation is spared in SSc suggests that fibroproliferative vasculopathy may depend on specific cellular or soluble factors not present in the retinal environment.