Influence of fibroids on cell-free DNA screening accuracy.

OBJECTIVE: Cell-free DNA (cfDNA) screening assesses both maternal and placental cfDNA. Fibroids are common and release cfDNA into maternal serum. Genetic abnormality is seen in 50% of fibroids. We aimed to assess the impact of fibroids on the accuracy of genome-wide cfDNA screening. METHODS: This was a prospective cohort study of singleton pregnancies examined at one of two centers in Melbourne and Sydney, Australia, between 1 November 2019 and 31 December 2020. All cases underwent pretest ultrasound examination to confirm an ongoing pregnancy of at least 10 weeks' gestation, and, at this stage, the number and volume of any uterine fibroid were documented. Genome-wide cfDNA screening was performed to detect all copy-number variants (CNV) > 7 megabases. The incidence of a false-positive result was compared between cases with and those without fibroids. RESULTS: Over the 14-month study period, 13 184 patients underwent cfDNA screening, of whom 1017 (7.7%) had fibroids. Fibroids were not identified in any of the 17 participants who had a false-positive result for chromosomes 13, 18, 21, X or Y. Ninety-five (0.7%) cases were screen-positive for subchromosomal aberration (SA), rare autosomal trisomy (RAT) or multiple abnormalities (MA), with 10 of these cases having a fetal genetic abnormality. The incidence of a false-positive RAT, MA or SA result was significantly higher in participants with fibroids (20/1017 (2.0%)) than in those without fibroids (64/12 167 (0.5%)). Women with fibroids were approximately six times as likely to have a false-positive result for SA, and this was associated positively with both fibroid number and volume. CONCLUSIONS: Most women with fibroids do not have an abnormal result on genome-wide cfDNA screening. However, CNVs due to fibroids are associated with false-positive SA findings, although fibroids do not appear to influence cfDNA screening accuracy for the common autosomal trisomies or sex-chromosomal abnormalities. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Defining the limits of detection for chromosome rearrangements in the preimplantation embryo using next generation sequencing.

STUDY QUESTION: Is next generation sequencing (NGS) capable of detecting smaller sub-chromosomal rearrangements in human embryos than the manufacturer's quoted resolution suggests? SUMMARY ANSWER: NGS was able to detect unbalanced chromosome segments smaller than the manufacturer's resolution. WHAT IS KNOWN ALREADY: Array Comparative Genomic Hybridization (array-CGH) has been the gold standard platform used for PGD of chromosome rearrangements. NGS is a viable alternative to array-CGH for PGD of chromosome arrangements given that the manufacturer's guidelines quote a resolution of ≥20 Mb. However, as many patients carry a chromosome rearrangement <20 Mb, the detection limits of NGS warrant further investigation. STUDY DESIGN, SIZE, DURATION: This study involved a retrospective assessment of stored DNA samples from embryos that had previously been diagnosed as unbalanced by array-CGH as part of routine PGD in two separate IVF clinics between November 2013 and April 2017. SurePlex whole genome amplification (WGA) products derived from DNA extracted from an embryo biopsy sample known to carry an unbalanced form of a chromosome rearrangement were subjected to a specific NGS workflow (VeriSeq PGS). The results from the two technologies were compared for each sample. PARTICIPANTS/MATERIALS, SETTING, METHODS: WGA products from 200 embryos known to carry unbalanced rearrangements were sequenced and analysed. These embryos had been created by 75 patients known to carry a chromosome rearrangement (68 reciprocal translocations, 3 pericentric inversions, 1 paracentric inversion, 2 insertions and 1 dual reciprocal and inversion). Each sample was assessed for the size of the segmental gain/loss (Mb), copy number for each segment and chromosome, segregation pattern, the number of bins in the analysis software used and concordance with array-CGH results. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 294 unbalanced chromosome segments were assessed. NGS was capable of detecting 285/294 (97%) unbalanced segments previously identified using array-CGH. The final PGD diagnosis was concordant for 200/200 (100%) embryos. In total, 44/75 (59%) patients contained an unbalanced chromosome segment below the quoted 20 Mb manufacturer's stated resolution. Of these, 35/44 (80%) patients had segments that were able to be detected using NGS, whilst maintaining clinical outcome concordance. LIMITATIONS, REASONS FOR CAUTION: Our study subset did not include any rearrangements involving the Y chromosome. NGS has less available bins per chromosome compared to the array-CGH platform used, thus it remains possible that chromosome rearrangements predicted to be small but still detectable by array-CGH may not be feasible for testing using NGS. This should be considered when undertaking a theoretical feasibility assessment for detecting the chromosome rearrangement in question. Only one specific workflow for WGA and NGS was investigated in this study. WIDER IMPLICATIONS OF THE FINDINGS: This study has shown that NGS is available for the detection of unbalanced chromosome rearrangements ≥10 Mb. STUDY FUNDING/COMPETING INTEREST(S): Part sponsorship of the VeriSeq PGS kits used was provided by Illumina. The remainder of the kits were provided by two commercial IVF clinics. None of the authors has any conflicting interests to declare. TRIAL REGISTRATION NUMBER: N/A.

The current state of reproductive biology research in Australia and New Zealand: core themes from the Society for Reproductive Biology Annual Meeting, 2016.

Because reproduction is essential for all life, it is central to our understanding of all aspects of biology. The Society for Reproductive Biology (SRB) 2016 conference held on the Gold Coast (Qld, Australia) displayed the current breadth of reproductive research in Australia and New Zealand, with additional insights from world leaders in the field. This conference review provides a focused summary of the key questions, emerging ideas and novel technologies that were presented in the symposia. Presented research demonstrated key advances in how stem cell biology may allow us to better understand pluripotency, as well as how environmental and lifestyle factors, such as circadian disruption, smoking, alcohol and diet, affect gametogenesis, embryo implantation, placental function and reproductive capacity. Sessions also highlighted the role of reproductive biology in providing insight into the mechanisms and processes governing a wide range of biological science disciplines, including cancer research and therapies, oncofertility, conservation of native species and chronic non-communicable diseases. Recurring themes included the importance of male and female gamete quality for reproductive potential and the critical and varied roles of the placenta in the maintenance of a healthy pregnancy. Dysregulation of reproductive processes can contribute to a variety of pathological states that affect future health, fertility and fecundity. Research being conducted by the SRB has the potential to shape not only the fertility of the current generation, but also the health and reproductive viability of future generations.

When two obese parents are worse than one! Impacts on embryo and fetal development.

The prevalence of overweight and obesity in reproductive-age adults is increasing worldwide. While the effects of either paternal or maternal obesity on gamete health and subsequent fertility and pregnancy have been reported independently, the combination of having both parents overweight/obese on fecundity and offspring health has received minimal attention. Using a 2 × 2 study design in rodents we established the relative contributions of paternal and maternal obesity on fetal and embryo development and whether combined paternal and maternal obesity had an additive effect. Here, we show that parental obesity reduces fetal and placental weights without altering pregnancy establishment and is not dependent on an in utero exposure to a high-fat diet. Interestingly combined parental obesity seemed to accumulate both the negative influences of paternal and maternal obesity had alone on embryo and fetal health rather than an amplification, manifested as reduced embryo developmental competency, reduced blastocyst cell numbers, impaired mitochondrial function, and alterations to active and repressive embryonic chromatin marks, resulting in aberrant placental gene expression and reduced fetal liver mtDNA copy numbers. Further understanding both the maternal cytoplasmic and paternal genetic interactions during this early developmental time frame will be vital for understanding how developmental programming is regulated and for the proposition of interventions to mitigate their effects.

Diet-induced paternal obesity in the absence of diabetes diminishes the reproductive health of two subsequent generations of mice.

BACKGROUND: Obesity and related conditions, notably subfertility, are increasingly prevalent. Paternal influences are known to influence offspring health outcome, but the impact of paternal obesity and subfertility on the reproductive health of subsequent generations has been overlooked. METHODS: A high-fat diet (HFD) was used to induce obesity but not diabetes in male C57Bl6 mice, which were subsequently mated to normal-weight females. First-generation offspring were raised on a control diet and their gametes were investigated for signs of subfertility. Second-generation offspring were generated from both first generation sexes and their gametes were similarly assessed. RESULTS: We demonstrate a HFD-induced paternal initiation of subfertility in both male and female offspring of two generations of mice. Furthermore, we have shown that diminished reproductive and gamete functions are transmitted through the first generation paternal line to both sexes of the second generation and via the first generation maternal line to second-generation males. Our previous findings that founder male obesity alters the epigenome of sperm, could provide a basis for the developmental programming of subfertility in subsequent generations. CONCLUSIONS: This is the first observation of paternal transmission of diminished reproductive health to future generations and could have significant implications for the transgenerational amplification of subfertility observed worldwide in humans.

Screening and cell-based assessment of mutations in the Aristaless-related homeobox (ARX) gene.

ARX mutations cause a diverse spectrum of human disorders, ranging from severe brain and genital malformations to non-syndromic intellectual disability (ID). ARX is a transcription factor with multiple domains that include four polyalanine (pA) tracts, the first two of which are frequently expanded by mutations. We progressively screened DNA samples from 613 individuals with ID initially for the most frequent ARX mutations (c.304ins(GCG)(7)'expansion' of pA1 and c.429_452dup 'dup24bp' of pA2). Five hundred samples without pA1 or pA2 mutations had the entire ARX ORF screened by single stranded polymorphism conformation (SSCP) and/or denaturing high pressure liquid chromatography (dHPLC) analysis. Overall, eight families with six mutations in ARX were identified (1.31%): five duplication mutations in pA2 (0.82%) with three new clinical reports of families with the dup24bp and two duplications larger than the dup24bp mutation discovered (dup27bp, dup33bp); and three point mutations (0.6%), including one novel mutation in the homeodomain (c.1074G>T). Four ultraconserved regions distal to ARX (uc466-469) were also screened in a subset of 94 patients, with three unique nucleotide changes identified in two (uc466, uc467). The subcellular localization of full length ARX proteins was assessed for 11 variants. Protein mislocalization increased as a function of pA2 tract length and phenotypic severity, as has been previously suggested for pA1. Similarly, protein mislocalization of the homeodomain mutations also correlated with clinical severity, suggesting an emerging genotype vs cellular phenotype correlation.

A review of fundamental principles for animal models of DOHaD research: an Australian perspective.

Epidemiology formed the basis of 'the Barker hypothesis', the concept of 'developmental programming' and today's discipline of the Developmental Origins of Health and Disease (DOHaD). Animal experimentation provided proof of the underlying concepts, and continues to generate knowledge of underlying mechanisms. Interventions in humans, based on DOHaD principles, will be informed by experiments in animals. As knowledge in this discipline has accumulated, from studies of humans and other animals, the complexity of interactions between genome, environment and epigenetics, has been revealed. The vast nature of programming stimuli and breadth of effects is becoming known. As a result of our accumulating knowledge we now appreciate the impact of many variables that contribute to programmed outcomes. To guide further animal research in this field, the Australia and New Zealand DOHaD society (ANZ DOHaD) Animals Models of DOHaD Research Working Group convened at the 2nd Annual ANZ DOHaD Congress in Melbourne, Australia in April 2015. This review summarizes the contributions of animal research to the understanding of DOHaD, and makes recommendations for the design and conduct of animal experiments to maximize relevance, reproducibility and translation of knowledge into improving health and well-being.