Alan R. Thornhill

Karyomapping: a universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes

The use of genome wide single nucleotide polymorphism (SNP) arrays for high resolution molecular cytogenetic analysis using a combination of quantitative and genotype analysis is well established. This study demonstrates that by Mendelian analysis of the SNP genotypes of the parents and a sibling or other appropriate family member to establish phase, it is possible to identify informative loci for each of the four parental haplotypes across each chromosome and map the inheritance of these haplotypes and the position of any crossovers in the proband. The resulting ‘karyomap’, unlike a karyotype, identifies the parental and grandparental origin of each chromosome and chromosome segment and is unique for every individual being defined by the independent segregation of parental chromosomes and the pattern of non-recombinant and recombinant chromosomes. Karyomapping, therefore, enables both genome wide linkage based analysis of inheritance and detection of chromosome imbalance where either both haplotypes from one parent are present (trisomy) or neither are present (monosomy/deletion). The study also demonstrates that karyomapping is possible at the single cell level following whole genome amplification and, without any prior patient or disease specific test development, provides a universal linkage based methodology for preimplantation genetic diagnosis readily available worldwide.

The causes of misdiagnosis and adverse outcomes in PGD

The European Society of Human Reproduction and Embryology PGD Consortium has collected data on PGD cycles and deliveries since 1997. From 15,158 cycles, 24 misdiagnoses and adverse outcomes have been reported; 12/2538 cycles after polymerase chain reaction and 12/12,620 cycles after fluorescence in situ hybridization. The causes of misdiagnosis include confusion of embryo and cell number, transfer of the wrong embryo, maternal or paternal contamination, allele dropout, use of incorrect and inappropriate probes or primers, probe or primer failure and chromosomal mosaicism. Unprotected sex has been mentioned as a cause of adverse outcome not related to technical and human errors. The majority of these causes can be prevented by using robust diagnostic methods within laboratories working to appropriate quality standards. However, diagnosis from a single cell remains a technically challenging procedure, and the risk of misdiagnosis cannot be eliminated.

The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting

This paper reports the proceedings of an international consensus meeting on oocyte and embryo morphology assessment. Following background presentations about current practice, the expert panel developed a set of consensus points to define the minimum criteria for oocyte and embryo morphology assessment. It is expected that the definition of common terminology and standardization of laboratory practice related to embryo morphology assessment will result in more effective comparisons of treatment outcomes. This document is intended to be referenced as a global consensus to allow standardized reporting of the minimum dataset required for the accurate description of embryo development. This paper reports the proceedings and outcomes of an international consensus meeting on human oocyte and embryo morphology assessment. An expert panel developed a series of consensus points to define the minimum criteria for such assessments. The definition of common terminology, and standardization of laboratory practices related to these morphological assessments, will permit more effective comparisons of treatment outcomes around the world. This report is intended to be referenced as a global consensus to allow standardized reporting of the minimum descriptive criteria required for routine clinical evaluations of human embryo development in vitro.

What next for preimplantation genetic screening

Preimplantation genetic diagnosis for aneuploidy screening (preimplantation genetic screening-PGS) has been used to detect chromosomally normal embryos from subfertile patients. The main indications are advanced maternal age (AMA), repeated implantation failure, repeated miscarriages and severe male factor infertility. Many non-randomized PGS studies have been published and report an increase in implantation rate, and/or a decrease in miscarriage rate. Recently, two randomized controlled trials have been conducted on patients with AMA as the only indication. Neither study showed a benefit in performing PGS using live birth rate as the measure of success. The debate on the usefulness of PGS is ongoing; the only effective way to resolve the debate is to perform more well-designed and well-executed randomized clinical trials.

Genome-wide maps of recombination and chromosome segregation in human oocytes and embryos show selection for maternal recombination rates

Crossover recombination reshuffles genes and prevents errors in segregation that lead to extra or missing chromosomes (aneuploidy) in human eggs, a major cause of pregnancy failure and congenital disorders. Here we generate genome-wide maps of crossovers and chromosome segregation patterns by recovering all three products of single female meioses. Genotyping >4 million informative SNPs from 23 complete meioses allowed us to map 2,032 maternal and 1,342 paternal crossovers and to infer the segregation patterns of 529 chromosome pairs. We uncover a new reverse chromosome segregation pattern in which both homologs separate their sister chromatids at meiosis I; detect selection for higher recombination rates in the female germ line by the elimination of aneuploid embryos; and report chromosomal drive against non-recombinant chromatids at meiosis II. Collectively, our findings show that recombination not only affects homolog segregation at meiosis I but also the fate of sister chromatids at meiosis II.

Body mass index and uterine receptivity in the oocyte donation model

OBJECTIVE To evaluate the relationship of body mass index (BMI) to uterine receptivity under conditions of programmed hormonal support and standardized embryo quality. DESIGN Retrospective cohort study.A tertiary referral center. PATIENTS Ninety-seven consecutive first-cycle recipients of anonymous oocyte donation. After programmed hormone replacement, recipients had transfer of embryos derived from oocyte donation. Anonymous oocyte donors received ovarian stimulation and underwent transvaginal ultrasound-guided oocyte retrieval. SETTING A receiver operator characteristic (ROC) curve of implantation versus BMI. Area under the ROC curve was 0.51, 95% confidence interval (CI) 0.41-0.62, suggesting no relationship between BMI and implantation. There was no difference in implantation rates between obese (BMI >or=30) and nonobese (BMI <30) recipients, odds ratio 1.1, 95% CI 0.5-2.4. CONCLUSION(S) Uterine receptivity was unimpaired in women with increased BMI when hormonal support and embryo quality were standardized.

The effect of cryopreservation on the genome of gametes and embryos: principles of cryobiology and critical appraisal of the evidence

BACKGROUND Cryopreservation has been extensively used in assisted reproductive technology, agriculture and conservation programmes for endangered species. The literature reports largely positive results regarding the survival of frozen-thawed cells and clinical outcomes. Nonetheless, it is unclear whether or not cryopreservation of sperm, oocytes and embryos causes any disruption in their genetic integrity. Drawing on the available published evidence, this review paper describes in detail the physical and biochemical factors of cryopreservation that could potentially affect genomic integrity. METHODS A critical review of the published literature using PubMed with particular emphasis on studies which include assessment of genetic stability after cryopreservation of oocyte, sperm and embryos. The search was performed in 2014 and covered the period from the beginning of electronic records until July 2014. No language restrictions were applied. RESULTS Cryopreservation is associated with extensive damage to cell membranes, and results in alteration of the functional and metabolic status of the cells and mitochondria. Some evidence suggests an increase in DNA single-strand breaks, and degree of DNA condensation or fragmentation in sperm after cryopreservation. The extent of these changes may vary between different individuals and different techniques. The addition of antioxidants to the cryopreservation media and the use of well-controlled cooling regimes could potentially improve such outcomes. Limited numbers of studies on oocytes provide controversial results regarding the effect on DNA fragmentation, sister chromatid exchange (SCE) and aneuploidy. The only study on human embryos suggested that vitrification affects DNA integrity to a much lesser extent than slow freezing. Animal studies show increases in mitochondrial DNA mutations in embryos after cryopreservation. The limited numbers of long-term follow-up studies in humans provide reassurance that derives mostly from retrospective studies with some methodological weaknesses. CONCLUSIONS This review provides an overview of studies performed to date on the effect of cryopreservation on the oocyte, sperm and embryos. Controversy of the reported data has highlighted the gaps in our knowledge not only for clinical studies, but also for basic research in human embryos. New perspectives for future research are proposed.

Array comparative genomic hybridisation on first polar bodies suggests that non-disjunction is not the predominant mechanism leading to aneuploidy in humans

Introduction Aneuploidy (the presence of extra or missing chromosomes) arises primarily through chromosome segregation errors in the oocyte at meiosis I but the details of mechanism by which such errors occur in humans are the subject of some debate. It is generally believed that aneuploidy arises primarily as a result of segregation of a whole chromosome to the same pole as its homologue (non-disjunction). Nonetheless, classical cytogenetic studies suggest that this model does not fully account for the patterns observed in human oocytes. An alternative model (precocious separation of sister chromatids) has thus been proposed, but recurring criticism of this model purports that technical issues may have led to interpretation errors. Materials and methods Array comparative genomic hybridisation (aCGH) was used on 164 human first polar bodies to distinguish between whole chromosome (non-disjunction) and chromatid (precocious separation) errors. Results Single chromatid errors were over 11 times more common than whole chromosome errors, consistent with prior classical cytogenetic and fluorescence in situ hybridisation (FISH) studies. Discussion The received wisdom that non-disjunction is the primary mechanism leading to human aneuploidy should be reconsidered.

Molecular Diagnostics in Preimplantation Genetic Diagnosis

Preimplantation genetic diagnosis (PGD) is a procedure that allows embryos to be tested for genetic disorders before they enter the uterus and before pregnancy has begun. Embryos obtained by in vitro fertilization undergo a biopsy procedure in which one or two cells are removed and tested for a specific disorder. If the cell is unaffected, the embryo from which it was taken is judged to be free of the disorder. The embryo can then be transferred to the uterus to initiate pregnancy. Couples whose children are at increased risk for a specific genetic disorder can benefit from PGD. Some of these couples may have affected family members or family ancestry that puts them at high risk for transmitting a particular disorder to their offspring. PGD is an alternative to prenatal tests such as amniocentesis or chorionic villus sampling and since it is performed before a pregnancy has begun, it may be more acceptable to couples who have either had an affected child, previous termination of pregnancy, or who have objections to termination of pregnancy. PGD tests have largely focused on two methodologies: fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). This review will focus on the use of PCR-based methodologies to diagnose single gene disorders in single cells; specifically describing the characteristics and limitations of single cell PCR and mutation detection strategies which have been developed for use in clinical PGD. The hundreds of cycles of preimplantation diagnosis performed to date have resulted in the birth of several hundred healthy children. 1 As shown in Table 1​1 , the genetic conditions for which PGD has been applied are numerous and the various methods used for diagnosis reflect the heterogeneity of causative mutations. Table 1. Strategies for PCR-Based Tests Used for Clinical Preimplantation Genetic Diagnosis The first clinical application of PGD used a generic PCR protocol for gender determination to avoid the transfer of male embryos which have a 50% probability of being affected by an X-linked recessive disorder. Gender was determined in a single blastomere by a single round of PCR using primers for Y-chromosome specific repetitive DNA sequences. The presence of Y-specific PCR amplification products was indicative of a male embryo and the absence of products was scored as female. 2 Although this approach had some success, a misdiagnosis, presumably due to amplification failure, did occur and emphasized the challenges inherent in single cell analysis and, more specifically, the danger in relying on the absence of amplification to diagnose genotype. 3 Subsequently, PCR protocols for preimplantation gender determination were refined to include primer sets which simultaneously amplify sequences common to both sex chromosomes (for example single copy genes such as ZFX/ZFY, 4 AMELX/AMELY, 5 ) and repetitive sequences such as DXZ1 and DYZ1. 6, 7 Sequences common to the sex chromosomes are identical at the site of primer annealing but differ internally in terms of size or include minor polymorphisms. Despite these technical improvements, the majority of embryo sexing is now accomplished using fluorescent in situ hybridization (FISH) which is less prone to contamination and can also provide the copy number for each chromosome tested thereby potentially avoiding the transfer of common chromosome abnormalities such as triploidy or X-monosomy. 8, 9 Although FISH has largely superseded PCR for sex determination, the specific diagnosis of single-gene defects remains dependent on DNA amplification with PCR. In the case of X-linked disorders, testing of the specific gene has the added advantage of ensuring that all embryos free of the mutant gene can be selected for transfer, irrespective of gender. 10, 11, 12 The list of disorders and the particular mutation detection strategies used for PCR-based clinical PGD application are given in Table 1​1 .

Preimplantation Diagnosis of Genetic and Chromosomal Disorders

The mean success rate per cycle is about 10%; it decreases with the number of insemination cycles, from 10.3% during the first 6 months to 2.3% after 24 cycles of treatment. This indicates that more fertile women conceive more readily. This success rate is dependent on the womens ages, the AID indication, the semen quality, and especially, the postthaw motility. The cumulative success rate (dropout excluded) for all women is 48% of pregnancies at 6 cycles, 66% at 12 cycles, and 80% at 24 cycles. The mean success rate per cycle is 15% with intrauterine insemination and 23% with IVFD (Table III).