Methods for comprehensive chromosome screening of oocytes and embryos: capabilities, limitations, and evidence of validity

Preimplantation aneuploidy screening of cleavage stage embryos using fluorescence in situ hybridization (FISH) may no longer be considered the standard of care in reproductive medicine. Over the last few years, there has been considerable development of novel technologies for comprehensive chromosome screening (CCS) of the human genome. Among the notable methodologies that have been incorporated are whole genome amplification, metaphase and array based comparative genomic hybridization, single nucleotide polymorphism microarrays, and quantitative real-time PCR. As these methods become more integral to treating patients with infertility, it is critical that clinicians and scientists obtain a better understanding of their capabilities and limitations. This article will focus on reviewing these technologies and the evidence of their validity

Chromosomal Preimplantation Genetic Diagnosis: 25 Years and Counting.

Preimplantation genetic diagnosis (PGD), first successfully carried out in humans in the early 1990s, initially involved the PCR sexing of embryos by Y- (and later also X-) chromosome specific detection. Because of the problems relating to misdiagnosis and contamination of this technology however the PCR based test was superseded by a FISH-based approach involving X and Y specific probes. Sexing by FISH heralded translocation screening, which was shortly followed by preimplantation genetic screening (PGS) for Aneuploidy. Aneuploidy is widely accepted to be the leading cause of implantation failure in assisted reproductive technology (ART) and a major contributor to miscarriage, especially in women of advanced maternal age. PGS (AKA PGD for aneuploidy PGD-A) has had a chequered history, with conflicting lines of evidence for and against its use. The current practice of trophectoderm biopsy followed by array CGH or next generation sequencing is gaining in popularity however as evidence for its efficacy grows. PGS has the potential to identify viable embryos that can be transferred thereby reducing the chances of traumatic failed IVF cycles, miscarriage or congenital abnormalities and facilitating the quickest time to live birth of chromosomally normal offspring. In parallel to chromosomal diagnoses, technology for PGD has allowed for improvements in accuracy and efficiency of the genetic screening of embryos for monogenic disorders. The number of genetic conditions available for screening has increased since the early days of PGD, with the human fertilization and embryology authority currently licensing 419 conditions in the UK [1]. A novel technique known as karyomapping that involves SNP chip screening and tracing inherited chromosomal haploblocks is now licensed for the PGD detection of monogenic disorders. Its potential for the universal detection of chromosomal and monogenic disorders simultaneously however, has yet to be realized

Chromosomal Preimplantation Genetic Diagnosis: 25 Years and Counting.

Preimplantation genetic diagnosis (PGD), first successfully carried out in humans in the early 1990s, initially involved the PCR sexing of embryos by Y- (and later also X-) chromosome specific detection. Because of the problems relating to misdiagnosis and contamination of this technology however the PCR based test was superseded by a FISH-based approach involving X and Y specific probes. Sexing by FISH heralded translocation screening, which was shortly followed by preimplantation genetic screening (PGS) for Aneuploidy. Aneuploidy is widely accepted to be the leading cause of implantation failure in assisted reproductive technology (ART) and a major contributor to miscarriage, especially in women of advanced maternal age. PGS (AKA PGD for aneuploidy PGD-A) has had a chequered history, with conflicting lines of evidence for and against its use. The current practice of trophectoderm biopsy followed by array CGH or next generation sequencing is gaining in popularity however as evidence for its efficacy grows. PGS has the potential to identify viable embryos that can be transferred thereby reducing the chances of traumatic failed IVF cycles, miscarriage or congenital abnormalities and facilitating the quickest time to live birth of chromosomally normal offspring. In parallel to chromosomal diagnoses, technology for PGD has allowed for improvements in accuracy and efficiency of the genetic screening of embryos for monogenic disorders. The number of genetic conditions available for screening has increased since the early days of PGD, with the human fertilization and embryology authority currently licensing 419 conditions in the UK [1]. A novel technique known as karyomapping that involves SNP chip screening and tracing inherited chromosomal haploblocks is now licensed for the PGD detection of monogenic disorders. Its potential for the universal detection of chromosomal and monogenic disorders simultaneously however, has yet to be realized

Current issues in medically assisted reproduction and genetics in Europe: research, clinical practice, ethics, legal issues and policy. European Society of Human Genetics and European Society of Human Reproduction and Embryology.

In March 2005, a group of experts from the European Society of Human Genetics and European Society of Human Reproduction and Embryology met to discuss the interface between genetics and assisted reproductive technology (ART), and published an extended background paper, recommendations and two Editorials. Seven years later, in March 2012, a follow-up interdisciplinary workshop was held, involving representatives of both professional societies, including experts from the European Union Eurogentest2 Coordination Action Project. The main goal of this meeting was to discuss developments at the interface between clinical genetics and ARTs. As more genetic causes of reproductive failure are now recognised and an increasing number of patients undergo testing of their genome before conception, either in regular health care or in the context of direct-to-consumer testing, the need for genetic counselling and preimplantation genetic diagnosis (PGD) may increase. Preimplantation genetic screening (PGS) thus far does not have evidence from randomised clinical trials to substantiate that the technique is both effective and efficient. Whole-genome sequencing may create greater challenges both in the technological and interpretational domains, and requires further reflection about the ethics of genetic testing in ART and PGD/PGS. Diagnostic laboratories should be reporting their results according to internationally accepted accreditation standards (International Standards Organisation - ISO 15189). Further studies are needed in order to address issues related to the impact of ART on epigenetic reprogramming of the early embryo. The legal landscape regarding assisted reproduction is evolving but still remains very heterogeneous and often contradictory. The lack of legal harmonisation and uneven access to infertility treatment and PGD/PGS fosters considerable cross-border reproductive care in Europe and beyond. The aim of this paper is to complement previous publications and provide an update of selected topics that have evolved since 2005

Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts

Preimplantation genetic testing for aneuploidy (PGT-A) is widely used in IVF and aims to improve outcomes by avoiding aneuploid embryo transfers. Chromosomal mosaicism is extremely common in early development and could affect the efficacy of PGT-A by causing incorrect embryo classification. Recent innovations have allowed accurate mosaicism detection in trophectoderm samples taken from blastocysts. However, there is little data concerning the impact of mosaicism on viability, and the optimal clinical pathway for such embryos is unclear. This study provides new information concerning the extent to which mosaic preimplantation embryos are capable of producing pregnancies and births. Archived trophectoderm biopsy specimens from transferred blastocysts were analyzed using next generation sequencing (NGS). Unlike other PGT-A methods, NGS accurately detects mosaicism in embryo biopsies. 44 mosaic blastocysts were identified. Their clinical outcomes were compared to 51 euploid blastocysts, derived from a well-matched, contemporary control group. Mosaic embryos were associated with outcomes that were significantly poorer than those of the control group: implantation 30.1 versus 55.8% (P = 0.038); miscarriage rate 55.6 versus 17.2% (P = 0.036); and ongoing pregnancy 15.4 versus 46.2% (P = 0.003). 61% of the mosaic errors affected whole chromosomes and 39% were segmental aneuploidies. Embryo viability is compromised by the presence of aneuploid cells. However, a minority of affected embryos can produce successful pregnancies. Hence, such embryos should not necessarily be excluded, but given a lower priority for transfer than those that are fully euploid. It is recommended that pregnancies established after mosaic embryo transfers be subjected to prenatal testing, with appropriate patient counselling

Cytogenetic analysis of human blastocysts with the use of FISH, CGH and aCGH: scientific data and technical evaluation.

BACKGROUND: Recent studies have suggested that biopsy of several trophectoderm (TE) cells from blastocysts followed by comparative genomic hybridization (CGH) analysis might represent an optimal strategy for aneuploidy detection, but few data on accuracy are available. The main question concerns the rate of mosaicism at the blastocyst stage, and to what extent this might cause misdiagnoses. We assessed blastocyst aneuploidy and mosaicism rates and evaluated the accuracy and efficiency of CGH and microarray-CGH (aCGH) for TE analysis. METHODS: A total of 52 blastocysts, from 20 couples, were biopsied and their chromosomes examined by CGH. The remaining cells were spread and tested by fluorescent in situ hybridization (FISH). Of the 52 blastocysts, 20 underwent a second TE biopsy and were tested using aCGH. RESULTS: CGH and aCGH produced results for 98% of TE samples. 42.3% of blastocysts were uniformly euploid, 30% were uniformly aneuploid and 32.4% were mosaic. Of the mosaic embryos, 15.4% were found to be composed of a mixture of different aneuploid cell lines, while 17% contained both normal and aneuploid cells. Mosaic diploid-aneuploid blastocysts with >30% normal cells accounted for <6% of analysed embryos. CONCLUSIONS: Comprehensive chromosome screening and follow-up assessment of large numbers of cells provided a unique insight into the cytogenetics of human blastocysts. Meiotic and post-zygotic errors leading to mosaicism were common. However, most mosaic blastocysts contained no normal cells. Hence, CGH or aCGH TE analysis is an accurate aneuploidy detection tool and may assist in identifying viable euploid embryos with higher implantation potential

Investigation of sperm telomere length as a potential marker of paternal genome integrity and semen quality

Recent studies have reported shorter sperm telomere length (STL) in men with idiopathic infertility. The aim of this study was to measure STL in semen samples from men to evaluate whether STL variation is associated with chromosomal abnormality, DNA fragmentation, traditional semen parameters, IVF outcome, or all four factors. A significant correlation between telomere length and diploidy was observed (P = 0.037). Additionally, STL was found to be positively associated with sperm count (P = 0.006); oligospermic samples had particularly short telomeres (0.9 ± 0.1 versus 1.4 ± 0.1; P = 0.0019). The results confirmed a link between sperm DNA fragmentation and aneuploidy, previously proposed (P = 0.009). A negative relationship was demonstrated between sperm concentration and aneuploidy and Sperm DNA framentation (P = 0.03, P < 0.0001, respectively). For a subset of 51 of the 73 sperm samples used for fertilization, IVF outcomes were known. A total of 17.6% of these samples had atypical STLs. None of these samples produced an ongoing pregnancy. In contrast, the pregnancy rate for samples that had STLs in the normal range was 35.7% (P = 0.044). In conclusion, STL has potential as a fast and inexpensive form of sperm quality assessment

Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts

Preimplantation genetic testing for aneuploidy (PGT-A) is widely used in IVF and aims to improve outcomes by avoiding aneuploid embryo transfers. Chromosomal mosaicism is extremely common in early development and could affect the efficacy of PGT-A by causing incorrect embryo classification. Recent innovations have allowed accurate mosaicism detection in trophectoderm samples taken from blastocysts. However, there is little data concerning the impact of mosaicism on viability, and the optimal clinical pathway for such embryos is unclear. This study provides new information concerning the extent to which mosaic preimplantation embryos are capable of producing pregnancies and births. Archived trophectoderm biopsy specimens from transferred blastocysts were analyzed using next generation sequencing (NGS). Unlike other PGT-A methods, NGS accurately detects mosaicism in embryo biopsies. 44 mosaic blastocysts were identified. Their clinical outcomes were compared to 51 euploid blastocysts, derived from a well-matched, contemporary control group. Mosaic embryos were associated with outcomes that were significantly poorer than those of the control group: implantation 30.1 versus 55.8% (P = 0.038); miscarriage rate 55.6 versus 17.2% (P = 0.036); and ongoing pregnancy 15.4 versus 46.2% (P = 0.003). 61% of the mosaic errors affected whole chromosomes and 39% were segmental aneuploidies. Embryo viability is compromised by the presence of aneuploid cells. However, a minority of affected embryos can produce successful pregnancies. Hence, such embryos should not necessarily be excluded, but given a lower priority for transfer than those that are fully euploid. It is recommended that pregnancies established after mosaic embryo transfers be subjected to prenatal testing, with appropriate patient counselling

Comparative genomic hybridization of oocytes and first polar bodies from young donors.

Chromosome abnormalities are common in oocytes derived from patients undergoing IVF treatment. The proportion of oocytes displaying aneuploidy is closely related to maternal age and may exceed 60% in patients over 40 years old. However, little information currently exists concerning the incidence of such anomalies in oocytes derived from young fertile women. A total of 121 metaphase II oocytes and their corresponding first polar bodies (PB) were analysed with the use of a comprehensive cytogenetic method, comparative genomic hybridization (CGH). The oocytes were donated from 13 young women (average age 22 years) without any known fertility problems. All oocytes were mature at the time of retrieval and were unexposed to spermatozoa. A low aneuploidy rate (3%) was detected. These results clearly indicate that meiosis I segregation errors are not frequent in oocytes of young fertile women. The higher aneuploidy rates reported in embryos derived from donor oocytes could be due to aggressive hormonal stimulation, in combination with male factors. However a definite contributing factor remains to be elucidated. The data obtained during this study also illustrate that CGH accurately and efficiently detects aneuploidy, confirming that it is suitable for application in a clinical setting for the assessment of oocytes, via PB analysis