Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos

Next-generation sequencing: challenges in reproductive genetics

Deep impact: sequencing embryo biopsy specimens at increasing depth

Preimplantation genetic diagnosis (PGD) is gradually widely used in prevention of gene diseases and chromosomal abnormalities. Much improvement has been achieved in biopsy technique and molecular diagnosis. Blastocyst biopsy can increase diagnostic accuracy and reduce allele dropout. It is cost-effective and currently plays an important role. Whole genome amplification permits subsequent individual detection of multiple gene loci and screening all 23 pairs of chromosomes. For PGD of chromosomal translocation, fluorescence in-situ hybridization (FISH) is traditionally used, but with technical difficulty. Array comparative genomic hybridization (CGH) can detect translocation and 23 pairs of chromosomes that may replace FISH. Single nucleotide polymorphisms array with haplotyping can further distinguish between normal chromosomes and balanced translocation. PGD may shorten time to conceive and reduce miscarriage for patients with chromosomal translocation. PGD has a potential value for mitochondrial diseases. Preimplantation genetic haplotyping has been applied for unknown mutation sites of single gene disease. Preimplantation genetic screening (PGS) using limited FISH probes in the cleavage-stage embryo did not increase live birth rates for patients with advanced maternal age, unexplained recurrent abortions, and repeated implantation failure. Polar body and blastocyst biopsy may circumvent the problem of mosaicism. PGS using blastocyst biopsy and array CGH is encouraging and merit further studies. Cryopreservation of biopsied blastocysts instead of fresh transfer permits sufficient time for transportation and genetic analysis. Cryopreservation of embryos may avoid ovarian hyperstimulation syndrome and possible suboptimal endometrium.

Thawing cryopreserved oocytes: genetic screening of thawed oocytes and ongoing pregnancy success rate

Preimplantation genetic screening by massively parallel sequencing on a MiSeq using transposase-based library preparation

Preimplantation genetic screening (PGS) for aneuploidy in embryos after in vitro fertilization (IVF) does not improve reproductive outcome in women over 35: a prospective controlled randomized study

In the human, mosaicism may occur before implantation; but, to determine when it first occurs, it is necessary to study the chromosomal complement of all blastomeres. Full karyotypes of blastomeres from 2- to 8-cell human embryos by conventional karyotyping of metaphase spreads are difficult to obtain. The aim of this study was to assess the stage at which mosaicism occurred in preimplantation human embryos through use of fluorescence in situ hybridization (FISH) with multiple probes. All or most blastomeres from 2- to 12-cell human embryos were analyzed by FISH using probes for gonosomes and chromosome 18. FISH was performed on blastomeres from 117 morphologically normal monospermic embryos that were not transferred after preimplantation diagnosis because of their risk of carrying X-linked disease; 20 (17.1%) of these embryos were mosaic. Another group of 163 arrested or morphologically abnormal monospermic embryos were also analyzed by FISH; 47 (28.8%) of these embryos were mosaic. In addition, 37 dispermic embryos were analyzed, and 28 (75.7%) of these were found to be mosaic. Mosaicism first occurred at the second cleavage division when the monospermic embryo was mostly diploid and at the first cleavage division when the embryo was mostly haploid, polyploid, or dispermic.

The widespread use of in vitro fertilization (IVF) throughout the world provides the opportunity to study human development at the very earliest stages before implantation. Nonetheless, the study of human embryos poses a series of unique ethical and moral implications. The unique totipotent nature of a human embryo and its potential to develop into a child necessitates a level of restriction and regulatory control that is not present when studying other cell types. Although some governments outlaw any experimental procedure on human embryonic material, others allow it under appropriate control. In the latter case (e.g., in the UK), experimentation can be justified on the basis of development of a diagnostic test and/or the goal of improving patient care. A further challenge to effective study is the paucity of material available. Much of the work reported in this chapter arises from the study of only single nuclei. For these reasons, research on interphase cytogenetics in human preimplantation embryos is less advanced than in other cell types. Despite this, a fundamental insight into chromosome copy number and nuclear organization can be gleaned from this material through collaboration with an appropriate clinical program. As attested by other chapters in this book, fluorescent in situ hybridization (FISH) was first adopted for research, but clinical applications rapidly followed. Prenatal and cancer diagnostics are the best examples of this,, but the increasing use of assisted reproductive technologies, namely IVF, precipitated the use of FISH in the field of preimplantation genetic diagnosis (PGD). PGD is defined as the diagnosis of genetic disorders in human preimplantation embryos. The purpose is selective implantation of unaffected embryos in the hope of establishing genetically normal ongoing pregnancies. PGD by interphase cytogenetics was first applied for sexing (to screen for sex-linked disorders), then for chromosome translocations, and later for chromosome copy number. In the latter case, termed preimplantation genetic screening (PGS), families at risk of adverse obstetrical outcomes (referral categories include advanced maternal age and recurrent miscarriage) are targeted, rather than families at risk of transmitting inherited disorders in a classical Mendelian fashion. Clinical application of interphase cytogenetics in the IVF world has allowed the subsequent study of chromosome copy number and nuclear organization. This chapter provides an overview of interphase cytogenetics in human embryos, highlighting the progress and the sometimes contentious pitfalls that it has encountered.

Gender does not impact the developmental rate of in vitro fertilization-derived human embryos as confirmed by day 3 blastomere biopsy and preimplantation genetic diagnosis (PGD)

Study question Does the selection of blastocysts for single embryo transfer, through the diagnosis of aneuploidy, improves the live birth rate in patients undergoing in vitro fertilization? Summary answer There seems to be no statistical difference in live birth rates between embryos with preimplantational genetic diagnosis (PGD) and those without. What is known already: Initial reports indicate that reproductive results improve after the selection of embryos to be transfer after performing a biopsy of the blastomeres, or trophectoderm cells, with the subsequent comprehensive analysis of the chromosomes. However, these results are now questioned. Reports in the literature are contrasting, so the real utility of selecting all embryos through comprehensive chromosome analysis calls for a more careful analysis that compares the risks, costs, and benefits of these techniques and their actual utility in reproductive results of patients treated with in vitro fertilization. Specifically results related to live birth rate. Study design, size, duration A systematic review of prospective studies evaluating live birth rate after embryo transfer of embryos selected by blastocyst biopsy for aneuploidy analysis compared with reproductive outcomes in embryo transfers of embryos selected morphologically, without biopsy nor screening for aneuploidies. Participants/materials, setting, methods A literature search was performed in PubMed, EmBase, and the Cochrane library (from January 2000 to december 2019). A cumulative meta-analysis and evaluation of heterogeneity was performed for the clinical pregnancy rate. The quality of the included studies was assessed using Cochrane’s Risk of Bias tool and ROBINS I for observational studies Main results and the role of chance Seven studies were included, three were randomized controlled trials and four were non-randomized studies of intervention (NRSI). The included studies were published between 2013 and 2019. For the preimplantational genetic diagnosis, three studies used array comparative genomic hybridization, three studies used next generation sequencing and only one study used qPCR. A total of 1638 patients were included, only two studies excluded patients with advanced maternal age (>35 years), two studies studied patients with recurrent implantation failure and three studies patients with recurrent pregnancy loss. Regarding the assisted reproduction techniques (ART), only studies where embryos where biopsied after day five for the genetic diagnosis where considered, most used ICSI and performed frozen-thawed transfer of up to two embryos, only one study allowed patients to be transferred with more than two embryos per cycle. Reproductive outcomes (live birth rate, miscarriage rate, clinical pregnancy) were extracted considering the events per embryo transfer and calculating the pooled odds ratios (OR) with 95% confidence intervals (95%CI) as our main outcome, sensitivity analyses will be performed using the events per cycles to assess the robustness of the effect estimate. Preliminary meta-analyses resulted in a pooled OR of 1.45 (95%CI 0.24–8.78) for NRSI and 1.34 (95%CI 0.85–2.11) for RCT. Limitations, reasons for caution The main limitation was the quantity of studies with acceptable methodology. This generated heterogeneity, hindering the evaluation of the true impact of PGD in ART outcomes. The use of events per embryo transfer as a main outcome could bias the results favoring PGD as less embryos are usually transferred. Wider implications of the findings: Our results show that there are too few studies with adequate methodology to generate a conclusion about the true benefit of PGD. However, a slight tendency favoring the reproductive outcomes of PGD was found. Trial registration number PROSPERO CRD42020198866

Preimplantation genetic screening using fluorescence in situ hybridization in patients with repetitive implantation failure and advanced maternal age: two randomized trials

Non-invasive preimplantationsexingusingRT-PCRon the spent embryo culture media

Comparison of two embryo biopsy methods in the preimplantation genetic diagnosis

Three-parent in vitro fertilization: gene replacement for the prevention of inherited mitochondrial diseases

Selection of high potential embryos using time-lapse imaging: the era of morphokinetics