Establishing a Method to Detect Fragile X CGG Repeats within the FMR1 Gene of Embryo Trophectoderm Biopsies

Fragile X Syndrome (FXS) is an X-linked disorder characterized by a CGG trinucleotide repeat located within the 5’ untranslated region of the Fragile X Mental Retardation 1 gene (FMR1). The FMR1 gene is further categorized into classifications of protein function such as normal, intermediate, premutation or full mutation depending on the number of CGG repeats present23. A normal FMR1 gene exhibits anywhere between 5 and 44 repeats. An allele in intermediate range displays 45-54 repeats. A premutation allele occurs when 55-200 CGG repeats are present and levels of the FMR1 gene product, Fragile X Mental Retardation Protein (FMRP) start to deplete. When alleles fall into this range, they are subject to expansion when transmitted into subsequent generations. Additionally, female premutation carriers are at risk for inheriting Fragile X-associated Primary Ovarian Insufficiency (FXPOI), which causes the ovaries to not work correctly. A full mutation displays complete methylation of the FMR1 gene, no FMRP production, and greater than 200 repeats. An individual diagnosed with a full mutation for FXS exhibits developmental and behavioral issues that include, but are not limited to, intellectual impairment, failure to meet milestones, and lack of impulse control20. Due to the severity of a full mutation of FXS, and risk of FXPOI and expansion, female patients seek reproductive assistance from infertility specialists. A current method of testing for FXS involves the use of quantitative Polymerase Chain Reaction (qPCR)-based single nucleotide polymorphism (SNP) genotyping for linkage analysis. The inheritance pattern of the affected allele is tracked throughout this process; however, the number of repeats present is not observed. This project aims to establish a method for detecting Fragile X CGG repeats within embryo trophectoderm (TE) biopsies. The determination of CGG repeats is a crucial addition to current methods of testing to avoid the risk of expansion and to assist patients in obtaining a healthy pregnancy12. This study was broken down into the completion of four phases. It began with the validation of Asuragen’s Amplidex PCR/CE FMR1 reagent kit to detect CGG repeats within the FMR1 gene of genomic DNA (gDNA) samples. The use of gDNA with this kit served as a gold standard for comparisons throughout this project. The next phase included, cell lines with known CGG repeat sizes to mimic TE biopsies. They were then tested on four different amplification methods. Those methods included GenomePlex WGA4, SurePlex DNA, REPLI-g Single Cell Kit, and targeted pre-amplification. Next, arrested whole embryos were used to test the ability of the chosen amplification method on embryonic samples. Lastly, TE biopsies from discarded aneuploid whole embryos to observe the developed methodology’s accuracy on clinical samples. The gDNA results were as expected and provided a source for the validation of this methodology. Although three out of the four amplification methods being tested did not provide usable results, the REPLI-g Single Cell Kit showed very promising results. It showed 100% consistency with the expected results from the cell lines used. This amplification method was then used on arrested whole embryos and showed CGG repeat sizes as expected from parental genotypes. The TE biopsies showed encouraging results, suggesting that this procedure has potential for clinically implementation. CGG repeat sizes and expansion can be observed using this new methodology. It has the potential application to assess the amount of expansion for patients with limited numbers of usable embryos. Transferring embryos with FMR1 premutation alleles will encompass more diligent genetic counseling and detailed consents

DNA methylation-based age prediction and telomere length in white blood cells and cumulus cells of infertile women with normal or poor response to ovarian stimulation.

An algorithm assessing the methylation levels of 353 informative CpG sites in the human genome permits accurate prediction of the chronologic age of a subject. Interestingly, when there is discrepancy between the predicted age and chronologic age (age acceleration or AgeAccel ), patients are at risk for morbidity and mortality. Identification of infertile patients at risk for accelerated reproductive senescence may permit preventative action. This study aimed to assess the accuracy of the epigenetic clock concept in reproductive age women undergoing fertility treatment by applying the age prediction algorithm in peripheral (white blood cells [WBCs]) and follicular somatic cells (cumulus cells [CCs]), and to identify whether women with premature reproductive aging (diminished ovarian reserve) were at risk of AgeAccel in their age prediction. Results indicated that the epigenetic algorithm accurately predicts age when applied to WBCs but not to CCs. The age prediction of CCs was substantially younger than chronologic age regardless of the patient\u27s age or response to stimulation. In addition, telomeres of CCs were significantly longer than that of WBCs. Our findings suggest that CCs do not demonstrate changes in methylome-predicted age or telomere-length in association with increasing female age or ovarian response to stimulation

Comparison of histological delineations of medial temporal lobe cortices by four independent neuroanatomy laboratories

The medial temporal lobe (MTL) cortex, located adjacent to the hippocampus, is crucial for memory and prone to the accumulation of certain neuropathologies such as Alzheimer's disease neurofibrillary tau tangles. The MTL cortex is composed of several subregions which differ in their functional and cytoarchitectonic features. As neuroanatomical schools rely on different cytoarchitectonic definitions of these subregions, it is unclear to what extent their delineations of MTL cortex subregions overlap. Here, we provide an overview of cytoarchitectonic definitions of the entorhinal and parahippocampal cortices as well as Brodmann areas (BA) 35 and 36, as provided by four neuroanatomists from different laboratories, aiming to identify the rationale for overlapping and diverging delineations. Nissl-stained series were acquired from the temporal lobes of three human specimens (two right and one left hemisphere). Slices (50 μm thick) were prepared perpendicular to the long axis of the hippocampus spanning the entire longitudinal extent of the MTL cortex. Four neuroanatomists annotated MTL cortex subregions on digitized slices spaced 5 mm apart (pixel size 0.4 μm at 20× magnification). Parcellations, terminology, and border placement were compared among neuroanatomists. Cytoarchitectonic features of each subregion are described in detail. Qualitative analysis of the annotations showed higher agreement in the definitions of the entorhinal cortex and BA35, while the definitions of BA36 and the parahippocampal cortex exhibited less overlap among neuroanatomists. The degree of overlap of cytoarchitectonic definitions was partially reflected in the neuroanatomists' agreement on the respective delineations. Lower agreement in annotations was observed in transitional zones between structures where seminal cytoarchitectonic features are expressed less saliently. The results highlight that definitions and parcellations of the MTL cortex differ among neuroanatomical schools and thereby increase understanding of why these differences may arise. This work sets a crucial foundation to further advance anatomically-informed neuroimaging research on the human MTL cortex

Correction to: Cluster identification, selection, and description in Cluster randomized crossover trials: the PREP-IT trials

An amendment to this paper has been published and can be accessed via the original article

Comparison of histological delineations of medial temporal lobe cortices by four independent neuroanatomy laboratories

The medial temporal lobe (MTL) cortex, located adjacent to the hippocampus, is crucial for memory and prone to the accumulation of certain neuropathologies such as Alzheimer's disease neurofibrillary tau tangles. The MTL cortex is composed of several subregions which differ in their functional and cytoarchitectonic features. As neuroanatomical schools rely on different cytoarchitectonic definitions of these subregions, it is unclear to what extent their delineations of MTL cortex subregions overlap. Here, we provide an overview of cytoarchitectonic definitions of the cortices that make up the parahippocampal gyrus (entorhinal and parahippocampal cortices) and the adjacent Brodmann areas (BA) 35 and 36, as provided by four neuroanatomists from different laboratories, aiming to identify the rationale for overlapping and diverging delineations. Nissl-stained series were acquired from the temporal lobes of three human specimens (two right and one left hemisphere). Slices (50 µm thick) were prepared perpendicular to the long axis of the hippocampus spanning the entire longitudinal extent of the MTL cortex. Four neuroanatomists annotated MTL cortex subregions on digitized (20X resolution) slices with 5 mm spacing. Parcellations, terminology, and border placement were compared among neuroanatomists. Cytoarchitectonic features of each subregion are described in detail. Qualitative analysis of the annotations showed higher agreement in the definitions of the entorhinal cortex and BA35, while definitions of BA36 and the parahippocampal cortex exhibited less overlap among neuroanatomists. The degree of overlap of cytoarchitectonic definitions was partially reflected in the neuroanatomists' agreement on the respective delineations. Lower agreement in annotations was observed in transitional zones between structures where seminal cytoarchitectonic features are expressed more gradually. The results highlight that definitions and parcellations of the MTL cortex differ among neuroanatomical schools and thereby increase understanding of why these differences may arise. This work sets a crucial foundation to further advance anatomically-informed human neuroimaging research on the MTL cortex