Katharina Spath

The origin and impact of embryonic aneuploidy

Despite the clinical importance of aneuploidy, surprisingly little is known concerning its impact during the earliest stages of human development. This study aimed to shed light on the genesis, progression, and survival of different types of chromosome anomaly from the fertilized oocyte through the final stage of preimplantation development (blastocyst). 2,204 oocytes and embryos were examined using comprehensive cytogenetic methodology. A diverse array of chromosome abnormalities was detected, including many forms never recorded later in development. Advancing female age was associated with dramatic increase in aneuploidy rate and complex chromosomal abnormalities. Anaphase lag and congression failure were found to be important malsegregation causing mechanisms in oogenesis and during the first few mitotic divisions. All abnormalities appeared to be tolerated until activation of the embryonic genome, after which some forms started to decline in frequency. However, many aneuploidies continued to have little impact, with affected embryos successfully reaching the blastocyst stage. Results from the direct analyses of female meiotic divisions and early embryonic stages suggest that chromosome errors present during preimplantation development have origins that are more varied than those seen in later pregnancy, raising the intriguing possibility that the source of aneuploidy might modulate impact on embryo viability. The results of this study also narrow the window of time for selection against aneuploid embryos, indicating that most survive until the blastocyst stage and, since they are not detected in clinical pregnancies, must be lost around the time of implantation or shortly thereafter.

Altered Levels of Mitochondrial DNA Are Associated with Female Age, Aneuploidy, and Provide an Independent Measure of Embryonic Implantation Potential

Mitochondria play a vital role in embryo development. They are the principal site of energy production and have various other critical cellular functions. Despite the importance of this organelle, little is known about the extent of variation in mitochondrial DNA (mtDNA) between individual human embryos prior to implantation. This study investigated the biological and clinical relevance of the quantity of mtDNA in 379 embryos. These were examined via a combination of microarray comparative genomic hybridisation (aCGH), quantitative PCR and next generation sequencing (NGS), providing information on chromosomal status, amount of mtDNA, and presence of mutations in the mitochondrial genome. The quantity of mtDNA was significantly higher in embryos from older women (P=0.003). Additionally, mtDNA levels were elevated in aneuploid embryos, independent of age (P=0.025). Assessment of clinical outcomes after transfer of euploid embryos to the uterus revealed that blastocysts that successfully implanted tended to contain lower mtDNA quantities than those failing to implant (P=0.007). Importantly, an mtDNA quantity threshold was established, above which implantation was never observed. Subsequently, the predictive value of this threshold was confirmed in an independent blinded prospective study, indicating that abnormal mtDNA levels are present in 30% of non-implanting euploid embryos, but are not seen in embryos forming a viable pregnancy. NGS did not reveal any increase in mutation in blastocysts with elevated mtDNA levels. The results of this study suggest that increased mtDNA may be related to elevated metabolism and are associated with reduced viability, a possibility consistent with the ‘quiet embryo’ hypothesis. Importantly, the findings suggest a potential role for mitochondria in female reproductive aging and the genesis of aneuploidy. Of clinical significance, we propose that mtDNA content represents a novel biomarker with potential value for in vitro fertilisation (IVF) treatment, revealing chromosomally normal blastocysts incapable of producing a viable pregnancy.

Morphological and cytogenetic assessment of cleavage and blastocyst stage embryos

Morphological assessments are the main way in which fertility clinics select in vitro generated embryo(s) for transfer to the uterus. However, it is widely acknowledged that the microscopic appearance of an embryo is only weakly correlated with its viability. Furthermore, the extent to which morphology is affected by aneuploidy, a genetic defect common in human preimplantation embryos, remains unclear. Aneuploidy is of great relevance to embryo selection as it represents one of the most important causes of implantation failure and miscarriage. The current study aimed to examine whether morphological appearance can assist in identifying embryos at risk of aneuploidy. Additionally, the data produced sheds light on how chromosomal anomalies impact development from the cleavage to the blastocyst stage. A total of 1213 embryos were examined. Comprehensive chromosome analysis was combined with well-established criteria for the assessment of embryo morphology. At the cleavage stage, chromosome abnormalities were common even amongst embryos assigned the best morphological scores, indicating that aneuploidy has little effect on microscopic appearance at fixed time points up until Day 3 of development. However, at the blastocyst stage aneuploidies were found to be significantly less common among embryos of optimal morphological quality, while such abnormalities were overrepresented amongst embryos considered to be of poor morphology. Despite the link between aneuploidy and blastocyst appearance, many chromosomally abnormal embryos were able to achieve the highest morphological scores. In particular, blastocysts affected by forms of aneuploidy with the greatest capacity to produce clinical pregnancies (e.g. trisomy 21) were indistinguishable from euploid embryos. The sex ratio was seen to be equal throughout preimplantation development. Interestingly, however, females were overrepresented amongst the fastest growing cleavage-stage embryos, whereas a sex-related skew in the opposite direction was noted for the most rapidly developing blastocysts. In summary, this study confirms that, at the cleavage stage, chromosome abnormalities have little if any effect on morphological scores assigned using traditional criteria. At the blastocyst stage some forms of aneuploidy begin to affect microscopic appearance, but in most instances the impact is subtle. In the case of the most clinically relevant aneuploidies (those capable of forming a pregnancy) there was no detectable effect on morphology at any preimplantation stage.

Characterization and quantification of proteins secreted by single human embryos prior to implantation

The use of in vitro fertilization (IVF) has revolutionized the treatment of infertility and is now responsible for 1–5% of all births in industrialized countries. During IVF, it is typical for patients to generate multiple embryos. However, only a small proportion of them possess the genetic and metabolic requirements needed in order to produce a healthy pregnancy. The identification of the embryo with the greatest developmental capacity represents a major challenge for fertility clinics. Current methods for the assessment of embryo competence are proven inefficient, and the inadvertent transfer of non‐viable embryos is the principal reason why most IVF treatments (approximately two‐thirds) end in failure. In this study, we investigate how the application of proteomic measurements could improve success rates in clinical embryology. We describe a procedure that allows the identification and quantification of proteins of embryonic origin, present in attomole concentrations in the blastocoel, the enclosed fluid‐filled cavity that forms within 5‐day‐old human embryos. By using targeted proteomics, we demonstrate the feasibility of quantifying multiple proteins in samples derived from single blastocoels and that such measurements correlate with aspects of embryo viability, such as chromosomal (ploidy) status. This study illustrates the potential of high‐sensitivity proteomics to measure clinically relevant biomarkers in minute samples and, more specifically, suggests that key aspects of embryo competence could be measured using a proteomic‐based strategy, with negligible risk of harm to the living embryo. Our work paves the way for the development of “next‐generation” embryo competence assessment strategies, based on functional proteomics.

Clinical application of a protocol based on universal next-generation sequencing for the diagnosis of beta-thalassaemia and sickle cell anaemia in preimplantation embryos

RESEARCH QUESTION Mutations of the beta-globin gene (HBB) cause beta-thalassaemia and sickle cell anaemia. These are the most common cause of severe inherited disease in humans. Traditional preimplantation genetic testing protocols for detecting HBB mutations frequently involve labour intensive, patient-specific test designs owing to the wide diversity of disease-associated HBB mutations. We, therefore, asked the question whether a universally applicable preimplantation genetic testing method can be developed to test for HBB gene mutations. DESIGN A multiplex polymerase chain reaction protocol was designed, allowing simultaneous amplification of multiple overlapping DNA fragments encompassing the entire HBB gene sequence in addition to 17 characterized, closely linked single nucleotide polymorphisms (SNP). Amplicons were then analysed using a next-generation sequencing method, revealing mutations and SNP genotypes. The protocol was extensively validated, optimized and eventually clinically applied on whole-genome amplified DNA derived from embryos of three couples carrying different combinations of beta-thalassaemia mutations. RESULTS The HBB mutation status and associated SNP haplotypes were successfully determined in all 21 embryos. Analysis of 141 heterozygous sites showed no instances of allele dropout and the test displayed 100% concordance compared with the results obtained from karyomapping. This suggests that the combination of trophectoderm biopsy and highly sensitive next-generation sequencing may provide superior accuracy than typically achieved using traditional preimplantation genetic testing methods. Importantly, no patient-specific test design or optimization was needed. CONCLUSIONS It is hoped that protocols that deliver almost universally applicable low-cost tests, without compromising diagnostic accuracy, will improve patient access to preimplantation genetic testing, especially in less affluent parts of the world.

Deep impact: sequencing embryo biopsy specimens at increasing depth.

Despite major advances in assisted reproductive technologies (ART), it remains the case that more than half of all IVF treatments do not ultimately result in the birth of a child. It is clear that the majority of embryos created through IVF are not capable of producing a viable pregnancy, with most failing to implant in the uterus or miscarrying soon afterwards (Fragouli et al., 2013). In order to increase a patient’s chance of a live birth, embryos are usually assessed according to morphological criteria and those considered to display optimal developmental features are prioritised for transfer. However, morphology and viability are only weakly correlated and consequently the selection of embryos on this basis has only a limited capacity to improve the outcome of IVF treatment (Alfarawati et al., 2011; Fragouli et al., 2014). Importantly, aneuploidy (an incorrect number of chromosomes) cannot be reliably identified via morphological evaluation. Aneuploidy is extremely common in preimplantation embryos and is believed to be the leading cause of implantation failure as well as being responsible for at least two-thirds of all miscarriages (Morales et al., 2008). To aid selection of viable embryos, the analysis of chromosome copy number in cells biopsied during preimplantation development (often referred to as preimplantation genetic screening, PGS) has been proposed. The efficiency of PGS has been a matter of debate ever since its introduction into clinical practice in the mid-1990s. Although, in theory, the transfer of euploid embryos should increase live birth rates per transfer, initial PGS approaches based on chromosomal analysis using fluorescent in situ hybridisation (FISH) failed to enhance overall IVF outcomes in randomised clinical trials (Mastenbroek et al., 2011). The success of PGS is highly dependent on the accuracy of the aneuploidy detection method used. Methods utilising FISH have the obvious limitation that only a handful of chromosomes can be tested in each embryo. Additionally, published clinical trials were exclusively carried out at the cleavage stage, a time of genetic instability during which mitotic errors lead to a high rate of mosaicism. Incomplete cytogenetic evaluation, combined with the possibility that the biopsied cell might not be fully representative of the embryo due to mosaisicm, create a significant risk of diagnostic error and thus it is not surprising that early randomised PGS studies failed to yield clear benefits. During the past two decades, PGS strategies have evolved and changed dramatically, overcoming former limitations. In an effort to offer more accurate screening of embryos, a variety of comprehensive aneuploidy detection techniques have been validated. These include microarray-based methods such as comparative genomic hybridisation (aCGH) or singlenucleotide polymorphism analysis (SNP array) (Gutierrez-Mateo et al., 2011; Treff et al., 2010) as well as quantitative realtime polymerase chain reaction (qPCR) (Treff et al., 2012). Furthermore, advances in embryology, specifically extended embryo culture and trophectoderm biopsy, have enabled a shift away from analysis at the cleavage stage towards testing of blastocysts. Trophectoderm biopsy is thought to be associated with a lower risk of damage to the embryo and, as a greater number of cells are collected, to provide a more representative and reliable indication of the chromosomal content of the embryo, reducing the risk of misdiagnosis due to mosaicism. Several randomized controlled clinical trials have now been performed utilising comprehensive aneuploidy screening at the blastocyst stage (Forman et al., 2013; Schoolcraft et al., 2012; Scott et al., 2013; Yang et al., 2012). The cumulative data obtained from all of these trials provides strong evidence in support of the hypothesis that PGS-based embryo selection can significantly improve IVF outcomes. Importantly, beneficial effects were not only observed in patients at high risk of generating aneuploid embryos, but also in those considered to be of ‘good prognosis’ and ‘low risk’. Given the continuous development of innovative, accurate and cost-effective screening technologies, coupled with a growing weight of clinical evidence, it can be expected that PGS will be increasingly utilised as an embryo selection tool. Indeed, it seems likely that the proportion of IVF cycles in the United States that employ PGS will reach 20% during 2015. In particular, next generation sequencing (NGS) is emerging as a powerful technology to assess chromosome copy number