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Background: Preimplantation genetic testing (PGT) encompasses methods that allow embryos to be tested for severe inherited conditions or for chromosome abnormalities, relevant to embryo health and viability. In order to obtain embryonic genetic material for analysis, a biopsy is required, involving the removal of one or more cells. This invasive procedure greatly increases the costs of PGT and there have been concerns that embryo viability could be compromised in some cases. The recent discovery of DNA within the blastocoele fluid (BF) of blastocysts and in spent embryo culture media (SCM) has led to interest in the development of non-invasive methods of PGT (niPGT)., Objective and Rationale: This review evaluates the current scientific evidence regarding non-invasive genetic assessment of preimplantation embryos. The success of different PGT methodologies in collecting and analysing extra-embryonic DNA is evaluated, and consideration is given to the potential biological and technical hindrances to obtaining a reliable clinical diagnosis., Search Methods: Original research and review papers concerning niPGT were sourced by searching PubMed and Google Scholar databases until July 2019. Searches comprised the keywords: 'non-invasive'; 'cell-free DNA'; 'blastocentesis'; 'blastocoel fluid'; 'spent culture media'; 'embryo culture medium'; 'preimplantation genetic testing'; 'preimplantation genetic diagnosis'; 'preimplantation genetic screening'; and 'aneuploidy'., Outcomes: Embryonic DNA is frequently detectable in BF and SCM of embryos produced during IVF treatment. Initial studies have achieved some success when performing cytogenetic and molecular genetic analysis. However, in many cases, the efficiency has been restricted by technical complications associated with the low quantity and quality of the DNA. Reported levels of ploidy agreement between SCM/BF samples and biopsied embryonic cells vary widely. In some cases, a discrepancy with respect to cytogenetic data obtained after trophectoderm biopsy may be attributable to embryonic mosaicism or DNA contamination (usually of maternal origin). Some research indicates that aneuploid cells are preferentially eliminated from the embryo, suggesting that their DNA might be over-represented in SCM and BF samples; this hypothesis requires further investigation., Wider Implications: Available data suggest that BF and SCM samples frequently provide DNA templates suitable for genetic analyses, offering a potential means of PGT that is less expensive than traditional methods, requires less micromanipulation skill and poses a lower risk to embryos. Critically, DNA isolation and amplification protocols must be optimised to reproducibly obtain an accurate clinical diagnosis, whilst minimising the impact of confounding factors such as contamination. Further investigations are required to understand the mechanisms underlying the release of embryonic DNA and to determine the extent to which this material reflects the true genetic status of the corresponding embryo. Currently, the clinic al potential of niPGT remains unknown., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: To evaluate the benefit of next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) for embryo selection in frozen-thawed embryo transfer., Design: Randomized controlled trial., Setting: Not applicable., Patient(s): Women aged 25-40 years undergoing IVF with at least two blastocysts that could be biopsied., Intervention(s): Randomization for single frozen-thawed embryo transfer with embryo selection based on PGT-A euploid status versus morphology., Main Outcome Measure(s): Ongoing pregnancy rate (OPR) at 20 weeks' gestation per embryo transfer., Result(s): A total of 661 women (average age 33.7 ± 3.6 years) were randomized to PGT-A (n = 330) or morphology alone (n = 331). The OPR was equivalent between the two arms, with no significant difference per embryo transfer (50% [137/274] vs. 46% [143/313]) or per intention to treat (ITT) at randomization (41.8% [138/330] vs. 43.5% [144/331]). Post hoc analysis of women aged 35-40 years showed a significant increase in OPR per embryo transfer (51% [62/122] vs. 37% [54/145]) but not per ITT., Conclusion(s): PGT-A did not improve overall pregnancy outcomes in all women, as analyzed per embryo transfer or per ITT. There was a significant increase in OPR per embryo transfer with the use of PGT-A in the subgroup of women aged 35-40 years who had two or more embryos that could be biopsied, but this was not significant when analyzed by ITT., Clinical Trial Registration Number: NCT02268786., (Copyright © 2019 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

This study aimed to determine whether karyomapping can be applied to couples requiring preimplantation genetic diagnosis (PGD) for single gene disorder (SGD) and/or chromosomal rearrangement. 75/82 (91.5%) and 6/82 (7.3%) couples were referred for autosomal SGD and X-linked disease, respectively. One couple (1.2%) was referred for SGD and chromosomal rearrangement. Of 608 embryos, 146 (24%, 95% CI 21-28) day-3 and 462 (76%, 95% CI 72-79) blastocyst biopsies were performed. A total of 81 embryo transfers were performed; 16/81 (20%) were following day-3 embryo biopsy, 65/81 (80%) were following blastocyst biopsy and cryopreserved embryo transfer. Of 81 embryo transfers with known pregnancy outcome, 51 (63%, 95% CI 52-73) were on-going pregnancies, 6/81 (7%, 95% CI 3-15) resulted in first trimester miscarriages and 24/81 (30%, 95% CI 21-40) were failed implantations. Of the 51 on-going pregnancies, 15 (29%, 95% CI 19-43) couples had a singleton live birth at the time of write up. There have been no reports of abnormal prenatal, genetic testing or diagnosis of phenotype at birth. Karyomapping is reliable, efficient and accurate for couples requiring PGD for SGD and/or chromosomal rearrangement. Additionally, it provides aneuploidy screening, minimising risks of miscarriage and implantation failure., (Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Objective: To determine whether undetected aneuploidy contributes to pregnancy loss after transfer of euploid embryos that have undergone array comparative genomic hybridization (aCGH)., Design: Case-control study., Setting: University-based fertility center., Patient(s): Cases included 38 patients who underwent frozen euploid ET as determined by aCGH, resulting in miscarriage. Controls included 38 patients who underwent frozen euploid ET as determined by aCGH, resulting in a live birth., Intervention(s): Next-generation sequencing (NGS) protocols were internally validated. Saved amplified DNA samples from the blastocyst trophectoderm biopsies previously diagnosed as euploid by aCGH were reanalyzed using NGS. Cytogenetic reports of the products of conception for 20 of the pregnancies resulting in miscarriage were available for comparison., Main Outcome Measure(s): The incidence of aneuploidy and mosaicism using NGS within embryos resulting in miscarriage and live birth., Result(s): Of euploid embryos analyzed by aCGH resulting in miscarriage, 31.6% were mosaic and 5.2% were polyploid by NGS. The rate of chromosomal abnormalities was significantly higher in embryos resulting in miscarriage (36.8%) than in those resulting in live births (15.8%). The rate of mosaicism was twice as high among embryos resulting in miscarriage than those resulting in live birth, but this was not statistically significant. Next-generation sequencing detected more cases of mosaicism than cytogenetic analysis of products of conception., Conclusion(s): Undetected aneuploidy may increase the risk of first trimester pregnancy loss. Next-generation sequencing may detect mosaicism and triploidy more frequently than aCGH, which could help to identify embryos at high risk of miscarriage. Mosaic embryos, however, should not be discarded as some can result in live births., (Published by Elsevier Inc.)

Study Question: We wanted to probe the opinions and current practices on preimplantation genetic screening (PGS), and more specifically on PGS in its newest form: PGS 2.0?, Study Finding: Consensus is lacking on which patient groups, if any at all, can benefit from PGS 2.0 and, a fortiori, whether all IVF patients should be offered PGS., What Is Known Already: It is clear from all experts that PGS 2.0 can be defined as biopsy at the blastocyst stage followed by comprehensive chromosome screening and possibly combined with vitrification. Most agree that mosaicism is less of an issue at the blastocyst stage than at the cleavage stage but whether mosaicism is no issue at all at the blastocyst stage is currently called into question., Study Design, Samples/materials, Methods: A questionnaire was developed on the three major aspects of PGS 2.0: the Why, with general questions such as PGS 2.0 indications; the How, specifically on genetic analysis methods; the When, on the ideal method and timing of embryo biopsy. Thirty-five colleagues have been selected to address these questions on the basis of their experience with PGS, and demonstrated by peer-reviewed publications, presentations at meetings and participation in the discussion. The first group of experts who were asked about 'The Why' comprised fertility experts, the second group of molecular biologists were asked about 'The How' and the third group of embryologists were asked about 'The When'. Furthermore, the geographical distribution of the experts has been taken into account. Thirty have filled in the questionnaire as well as actively participated in the redaction of the current paper., Main Results and the Role of Chance: The 30 participants were from Europe (Belgium, Germany, Greece, Italy, Netherlands, Spain, UK) and the USA. Array comparative genome hybridization is the most widely used method amongst the participants, but it is slowly being replaced by massive parallel sequencing. Most participants offering PGS 2.0 to their patients prefer blastocyst biopsy. The high efficiency of vitrification of blastocysts has added a layer of complexity to the discussion, and it is not clear whether PGS in combination with vitrification, PGS alone, or vitrification alone, followed by serial thawing and eSET will be the favoured approach. The opinions range from in favour of the introduction of PGS 2.0 for all IVF patients, over the proposal to use PGS as a tool to rank embryos according to their implantation potential, to scepticism towards PGS pending a positive outcome of robust, reliable and large-scale RCTs in distinct patient groups., Limitations, Reasons for Caution: Care was taken to obtain a wide spectrum of views from carefully chosen experts. However, not all invited experts agreed to participate, which explains a lack of geographical coverage in some areas, for example China. This paper is a collation of current practices and opinions, and it was outside the scope of this study to bring a scientific, once-and-for-all solution to the ongoing debate., Wider Implications of the Findings: This paper is unique in that it brings together opinions on PGS 2.0 from all different perspectives and gives an overview of currently applied technologies as well as potential future developments. It will be a useful reference for fertility specialists with an expertise outside reproductive genetics., Large Scale Data: none., Study Funding and Competing Interests: No specific funding was obtained to conduct this questionnaire., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Massively parallel genome sequencing, also known as next-generation sequencing (NGS), is the latest approach for preimplantation genetic diagnosis. The purpose of this study was to determine whether NGS can accurately detect aneuploidy in human embryos. Low coverage genome sequencing was applied to trophectoderm biopsies of embryos at the blastocyst stage of development. Sensitivity and specificity of NGS was determined by comparison of results with a previously validated platform, array-comparative genomic hybridization (aCGH). In total, 156 samples (116 were blindly assessed) were tested: 40 samples were re-biopsies of blastocysts where the original biopsy specimen was previously tested for aCGH; four samples were re-biopsies of single blastomeres from embryos previously biopsied at the cleavage stage and tested using aCGH; 18 samples were single cells derived from well-characterized cell lines; 94 samples were whole-genome amplification products from embryo biopsies taken from previous preimplantation genetic screening cycles analysed using aCGH. Per embryo, NGS sensitivity was 100% (no false negatives), and 100% specificity (no false positives). Per chromosome, NGS concordance was 99.20%. With more improvement, NGS will allow the simultaneous diagnosis of single gene disorders and aneuploidy, and may have the potential to provide more detailed insight into other aspects of embryo viability., (Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Objective: To determine the usefulness of semiconductor-based next-generation sequencing (NGS) for cleavage-stage preimplantation genetic diagnosis (PGD) of aneuploidy., Design: Prospective case-control study., Setting: A private center for reproductive medicine., Patient(s): A total of 45 patients underwent day-3 embryo biopsy with PGD and fresh cycle transfer. Additionally, 53 patients, matched according to age, anti-Müllerian hormone levels, antral follicles count, and infertility duration were selected as controls., Intervention(s): Choice of embryos for transfer was based on the PGD NGS results., Main Outcome Measure(s): Clinical pregnancy rate (PR) per embryo transfer (ET) was the primary outcome. Secondary outcomes were implantation and miscarriage rates., Result(s): The PR per transfer was higher in the NGS group (84.4% vs. 41.5%). The implantation rate (61.5% vs. 34.8%) was higher in the NGS group. The miscarriage rate was similar in the 2 groups (2.8% vs. 4.6%)., Conclusion(s): We demonstrate the technical feasibility of NGS-based PGD involving cleavage-stage biopsy and fresh ETs. Encouraging data were obtained from a prospective trial using this approach, arguing that cleavage-stage NGS may represent a valuable addition to current aneuploidy screening methods. These findings require further validation in a well-designed randomized controlled trial., Clinical Trial Registration Number: ACTRN12614001035617., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: The majority of human embryos created using in vitro fertilisation (IVF) techniques are aneuploid. Comprehensive chromosome screening methods, applicable to single cells biopsied from preimplantation embryos, allow reliable identification and transfer of euploid embryos. Recently, randomised trials using such methods have indicated that aneuploidy screening improves IVF success rates. However, the high cost of testing has restricted the availability of this potentially beneficial strategy. This study aimed to harness next-generation sequencing (NGS) technology, with the intention of lowering the costs of preimplantation aneuploidy screening., Methods: Embryo biopsy, whole genome amplification and semiconductor sequencing., Results: A rapid (<15 h) NGS protocol was developed, with consumable cost only two-thirds that of the most widely used method for embryo aneuploidy detection. Validation involved blinded analysis of 54 cells from cell lines or biopsies from human embryos. Sensitivity and specificity were 100%. The method was applied clinically, assisting in the selection of euploid embryos in two IVF cycles, producing healthy children in both cases. The NGS approach was also able to reveal specified mutations in the nuclear or mitochondrial genomes in parallel with chromosome assessment. Interestingly, elevated mitochondrial DNA content was associated with aneuploidy (p<0.05), a finding suggestive of a link between mitochondria and chromosomal malsegregation., Conclusions: This study demonstrates that NGS provides highly accurate, low-cost diagnosis of aneuploidy in cells from human preimplantation embryos and is rapid enough to allow testing without embryo cryopreservation. The method described also has the potential to shed light on other aspects of embryo genetics of relevance to health and viability., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Chromosome abnormalities are extremely common in human oocytes and embryos and are associated with a variety of negative outcomes for both natural cycles and those using assisted conception techniques. Embryos containing the wrong number of chromosomes (aneuploidy) may fail to implant in the uterus, miscarry, or lead to children with serious medical problems (e.g., Down syndrome). Preimplantation genetic screening (PGS) is a method that seeks to improve the outcomes of assisted reproductive treatments, such as in vitro fertilization (IVF), by ensuring that the embryos chosen for transfer to the uterus are chromosomally normal. Here we summarize published and novel data concerning the frequency and variety of chromosomal abnormalities seen in oocytes and embryos at the cleavage and blastocyst stages of development. Clinical outcomes of studies using PGS are presented, and the controversy over the use of chromosome screening as a tool for embryo selection is discussed. We describe validation and preliminary clinical data from the new generation of methods being used for PGS, including comparative genomic hybridization (CGH), microarrays (aCGH and single nucleotide polymorphism arrays), and quantitative polymerase chain reaction. These methodologies allow comprehensive chromosomal analysis, provide high accuracy, and have yielded encouraging preliminary clinical data. The combination of advances in genetics and embryology seems poised to usher in a new era in the treatment of infertility., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Objective: To evaluate a new strategy for comprehensive chromosome screening at the blastocyst stage., Design: Clinical research study., Setting: An IVF clinic and a specialist preimplantation genetic diagnosis laboratory., Patient(s): Forty-five infertile couples participated in the study. The mean maternal age was 37.7 years, and most couples had at least one previous unsuccessful IVF treatment cycle (mean 2.4)., Intervention(s): This study used a novel chromosome screening approach, combining biopsy of several trophectoderm cells on day 5 after fertilization and detailed analysis of all 24 types of chromosome using comparative genomic hybridization., Main Outcome Measure(s): Proportion of embryos yielding a diagnostic result, aneuploidy rate, implantation rate, and pregnancy rate., Result(s): A diagnosis was obtained from 93.7% of embryos tested. The aneuploidy rate was 51.3%. The probability of an individual transferred embryo forming a pregnancy reaching the third trimester/birth was 68.9%, an implantation rate 50% higher than contemporary cycles from the same clinic. The pregnancy rate was 82.2%., Conclusion(s): The comprehensive chromosome screening method described overcomes many of the problems that limited earlier aneuploidy screening techniques and may finally allow preimplantation genetic screening to achieve the benefits predicted by theory. The high embryo implantation rate achieved is particularly encouraging and, if confirmed in subsequent studies, will be of great significance for IVF clinics attempting to reduce the number of embryos transferred or to implement single embryo transfer., (Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Chromosome abnormalities are common among human oocytes and are usually lethal to any embryos they produce. It therefore seems logical that a reliable technique for distinguishing between normal and aneuploid embryos would be a useful tool for physicians and embryologists, assisting the choice of which embryo(s) to prioritize for uterine transfer. This concept has led to the development of a variety of methods for the detection of chromosome abnormalities in oocytes and embryos, most often referred to as preimplantation genetic screening (PGS). However, several well-controlled studies have been unable to show an advantage of chromosome screening in terms of pregnancy and birth rates. Some investigators have suggested that damage to embryos, sustained during cleavage-stage biopsy, might explain why PGS has not always provided the anticipated benefits. This paper asks whether there is evidence that a non-invasive, morphological analysis could allow chromosomally normal embryos to be accurately identified and reviews data from the most recent publication concerning IVF outcome following PGS., (Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

One of the most important factors influencing embryo viability is chromosome imbalance (aneuploidy). Embryos derived from aneuploid gametes have little potential for forming a viable pregnancy, but cannot be distinguished from normal embryos using standard morphological evaluation. For more than a decade, preimplantation genetic screening (PGS) has been used to assist in the identification of aneuploid embryos. However, current strategies, based upon cell biopsy followed by fluorescent in situhybridization, allow less than half of the chromosomes to be screened. In this review, we discuss methods that overcome the limitations of earlier PGS strategies and provide screening of the entire chromosome complement in oocytes and embryos. In recent months, there has been a rapid growth in the number of PGS cycles utilizing one such method, comparative genomic hybridization (CGH). Data from IVF cycles utilizing CGH must be considered preliminary, but appear to indicate a dramatic increase in embryo implantation following comprehensive chromosomal screening. It is expected that methods based upon microarrays will yield similar clinical results and may be sufficiently rapid to permit comprehensive screening without the need for embryo cryopreservation. Some microarray platforms also offer the advantage of embryo fingerprinting and the potential for combined aneuploidy and single gene disorder diagnosis. However, more data concerning accuracy and further reductions in the price of tests will be necessary before microarrays can be widely applied.

Chromosome testing strategies, such as preimplantation genetic testing for aneuploidy (PGT-A), improve initial IVF outcomes by avoiding unwitting transfer of aneuploid embryos in morphology-based selection practices. Newer technologies have revealed that some embryos may appear to have intermediate whole chromosome (or parts of a chromosome termed segmental) copy number results suggesting trophectoderm mosaicism. An embryo with a trophectoderm mosaic-range result may be the only option for transfer for some patients. Recent data suggest that such mosaic embryos can be transferred without added risk of abnormal birth outcomes but may be associated with increased implantation failure and miscarriage rates, with higher values of mosaicism appearing to be less favourable for producing good outcomes. In this Position Statement, we provide guidance to laboratories, clinics, clinicians and counsellors to assist in discussions on the utility and transfer of mosaic embryos. [ABSTRACT FROM AUTHOR]

Irregular cleavage divisions are expected to produce chromosomally deviant embryos. We investigated whether embryos from irregular cleavages could develop into euploid blastocysts, and, if so, whether any evidence existed of a self-correction mechanism of the embryo. We also investigated the role of different dynamic aspects of morula compaction in this process. A total of 791 embryos from 141 patients undergoing pre-implantation genetic screening were retrospectively analysed using a time-lapse imaging system, and multiple cell divisions were evaluated. A total of 276 embryos developed into blastocysts suitable for biopsy and chromosome screening through array-comparative genomic hybridization. As well as testing trophectoderm biopsy specimens for aneuploidy, excluded cells of 18 blastocysts, which developed from partially compacted morulas, were also analysed. Unique data on the developmental fate of embryos with cleavage abnormalities are presented, and a potential mechanism of ‘aneuploidy rescue’ is postulated through which mosaic embryos may form partially compacted morulas to exclude aneuploid cells. In addition, this process seems to be less efficient in older women. The data obtained also provide further evidence that excluded cells should not be used to infer the cytogenetic status of the embryo. [ABSTRACT FROM AUTHOR]