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Recent advances in preimplantation genetic testing for aneuploidy (PGT-A) have significantly enhanced its application in ART, providing critical insights into embryo viability, and potentially reducing both the time spent in fertility treatments and the risk of pregnancy loss. With the integration of next-generation sequencing, PGT-A now offers greater diagnostic precision, although challenges related to segmental aneuploidies and mosaicism remain. The emergence of non-invasive PGT-A (niPGT-A), which analyzes DNA in spent embryo culture media, promises a simpler aneuploidy screening method. This mini review assesses the methodological criteria for test validation, the current landscape of PGT-A, and the potential of niPGT-A, while evaluating its advantages and potential pitfalls. It underscores the importance of a robust three-phase validation process to ensure the clinical reliability of PGT-A. Despite initial encouraging data, niPGT-A not only confronts issues of DNA amplification failure and diagnostic inaccuracies but also has yet to meet the three-prong criteria required for appropriate test validation, necessitating further research for its clinical adoption. The review underscores that niPGT-A, like traditional PGT-A, must attain the high standards of precision and reliability expected of any genetic testing platform used in clinical settings before it can be adopted into routine ART protocols. [ABSTRACT FROM AUTHOR]

The New England Journal of Medicine recently published a large study addressing the efficacy of preimplantation genetic testing for aneuploidy (PGT-A). The 14-centre randomized control non-inferiority trial used cumulative live birth rate (CLBR) as a clinical endpoint to examine the value of PGT-A and concluded that conventional IVF was not inferior to IVF with PGT-A. Unfortunately, the experimental design was highly flawed; and in fact, the data generated in the study do not support the major conclusions presented in the publication. The embryos in each patient's three-embryo pool, which were available for transfer, were selected solely by morphology. The investigators then randomized patients to either the PGT-A group or the control group. It is important to note that PGT-A screening in the study group was done only after the embryos were selected. PGT-A was not really used in a meaningful way, which would have been for the PGT-A results to help in selecting which embryos would be in the three-embryo group. Thus, the outcomes were wholly determined prior to the study intervention. The ultimate delivery rate for each group of three embryos was determined when they were selected by morphology. The randomization, which occurred after embryo selection, would assure equal distribution of those cohorts destined to deliver and those destined to fail to the two study groups, the PGT-A and control groups. Thus, there was no potential for PGT-A to enhance selection and thus no possible way to improve the cumulative outcomes. Since there was no possible way for the control group to be inferior, the experimental design precluded any chance of evaluating the primary endpoint of the study. The primary question of the study was never evaluated. Another serious flaw was that the study was initiated prior to knowing how to interpret the data provided in the PGT-A analytical result. Specifically, the design excluded mosaic embryos from transfer despite the literature demonstrating the significant reproductive potential for these embryos. When accounting for the lost deliveries induced by this non-evidence-based decision, the expected delivery rates in the two groups become virtually identical. That is an important issue because the data from the study actually demonstrate the safety of PGT-A without diminution in outcomes from the impact of trophectoderm biopsy or the discarding of competent embryos which had wrongfully been considered aneuploid. A final serious flaw in the experimental design and interpretation of the data surrounding the issue of the miscarriage rate. The investigators noted that the miscarriage rate was lower in the PGT-A group but stated that its impact was insufficient to alter the CLBR. Of course, by design, the CLBRs were limited to being equivalent. There was no potential for enhanced outcomes in the PGT-A group and thus no possibility that the lower risk of miscarriage in the PGT-A group would raise the CLBR. The benefit of a lower miscarriage rate is real and significant. Its relevance should not be diminished based on the lack of a change in the CLBR since that was never possible in this study. The investigators of the study concluded that the CLBR with conventional ART is equivalent to that with PGT-A, but a simple review of the experiment reassigns their genuine findings to those of a safety study. Significantly, the data in the study demonstrate that the intervention of PGT-A is safe. This study neither supports nor refutes the efficacy of clinical PGT-A. [ABSTRACT FROM AUTHOR]

The hypothesis of preimplantation genetic testing for aneuploidy (PGT-A) was first proposed 20 years ago, suggesting that during IVF elimination of aneuploid embryos prior to transfer will improve implantation rates of remaining embryos and, therefore, increase pregnancy and live birth rates, while also reducing miscarriages. Subsequently, unvalidated and increasingly unrestricted clinical utilization of PGT-A called for at least one properly randomized controlled trial (RCT) to assess cumulative live birth rates following a single oocyte retrieval, utilizing all fresh and frozen embryos of an IVF cycle. Only recently two such RCTs were published, however both, when properly analysed, not only failed to demonstrate significant advantages from utilization of PGT-A, but actually demonstrated outcome deficits in comparison to non-use of PGT-A, when patient selection biases in favour of PGT-A were reversed. Moreover, because of high embryo mosaicism at the blastocyst stage and, therefore, high false-positive rates from trophectoderm biopsies, large numbers of chromosomal-normal embryos with normal pregnancy potential are unnecessarily left unused or discarded, indisputably causing harm to affected couples. We, therefore, strongly call for restricting PGT-A to only research protocols and, as of this point in time, encourage professional societies in the field to follow suit with appropriate practice guidelines. [ABSTRACT FROM AUTHOR]

Study Question: Can the embryo tracking system (ETS) increase safety, efficacy and scalability of massively parallel sequencing-based preimplantation genetic testing (PGT)?Summary Answer: Applying ETS-PGT, the chance of sample switching is decreased, while scalability and efficacy could easily be increased substantially.What Is Known Already: Although state-of-the-art sequencing-based PGT methods made a paradigm shift in PGT, they still require labor intensive library preparation steps that makes PGT cost prohibitive and poses risks of human errors. To increase the quality assurance, efficiency, robustness and throughput of the sequencing-based assays, barcoded DNA fragments have been used in several aspects of next-generation sequencing (NGS) approach.Study Design, Size, Duration: We developed an ETS that substantially alleviates the complexity of the current sequencing-based PGT. With (n = 693) and without (n = 192) ETS, the downstream PGT procedure was performed on both bulk DNA samples (n = 563) and whole-genome amplified (WGAed) few-cell DNA samples (n = 322). Subsequently, we compared full genome haplotype landscapes of both WGAed and bulk DNA samples containing ETS or no ETS.Participants/materials, Setting, Methods: We have devised an ETS to track embryos right after whole-genome amplification (WGA) to full genome haplotype profiles. In this study, we recruited 322 WGAed DNA samples derived from IVF embryos as well as 563 bulk DNA isolated from peripheral blood of prospective parents. To determine possible interference of the ETS in the NGS-based PGT workflow, barcoded DNA fragments were added to DNA samples prior to library preparation and compared to samples without ETS. Coverages and variants were determined.Main Results and the Role Of Chance: Current PGT protocols are quality sensitive and prone to sample switching. To avoid sample switching and increase throughput of PGT by sequencing-based haplotyping, six control steps should be carried out manually and checked by a second person in a clinical setting. Here, we developed an ETS approach in which one step only in the entire PGT procedure needs the four-eyes principal. We demonstrate that ETS not only precludes error-prone manual checks but also has no effect on the genomic landscape of preimplantation embryos. Importantly, our approach increases efficacy and throughput of the state-of-the-art PGT methods.Limitations, Reasons For Caution: Even though the ETS simplified sequencing-based PGT by avoiding potential errors in six steps in the protocol, if the initial assignment is not performed correctly, it could lead to cross-contamination. However, this can be detected in silico following downstream ETS analysis. Although we demonstrated an approach to evaluate purity of the ETS fragment, it is recommended to perform a pre-PGT quality control assay of the ETS amplicons with non-human DNA, such that the purity of each ETS molecule can be determined prior to ETS-PGT.Wider Implications Of the Findings: The ETS-PGT approach notably increases efficacy and scalability of PGT. ETS-PGT has broad applicative value, as it can be tailored to any single- and few-cell sequencing approach where the starting specimen is scarce, as opposed to other methods that require a large number of cells as the input. Moreover, ETS-PGT could easily be adapted to any sequencing-based diagnostic method, including PGT for structural rearrangements and aneuploidies by low-pass sequencing as well as non-invasive prenatal testing.Study Funding/competing Interest(s): M.Z.E. is supported by the EVA (Erfelijkheid Voortplanting & Aanleg) specialty program (grant no. KP111513) of Maastricht University Medical Centre (MUMC+), and the Horizon 2020 innovation (ERIN) (grant no. EU952516) of the European Commission.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Is there an efficient and cost-effective detection platform for different genetic conditions about embryos?Summary Answer: A multifunctional haplotyping-based preimplantation genetic testing platform was provided for detecting different genetic conditions.What Is Known Already: Genetic disease and chromosomal rearrangement have been known to significantly impact fertility and development. Therefore, preimplantation genetic testing for aneuploidy (PGT-A), monogenic disorders (PGT-M) and structural rearrangements (PGT-SR), a part of ART, has been presented together to minimize the fetal genetic risk and increase pregnancy rate. For patients or their families who are suffering from chromosome abnormality, monogenic disease, unexplained repeated spontaneous abortion or implantation failure, after accepting genetic counseling, they may be suggested to accept detection from more than one PGT platforms about the embryos to avoid some genetic diseases. However, PGT platforms work through different workflows. The high costliness, lack of material and long-time operation of combined PGT platforms limit their application.Study Design, Size, Duration: All 188 embryonic samples from 43 families were tested with HaploPGT platform, and most of their genetic abnormalities had been determined by different conventional PGT methods beforehand. Among them, there were 12 families only carrying structural rearrangements (115 embryos) in which 9 families accepted implantation and 5 families had normal labor ART outcomes, 7 families only carrying monogenic diseases (26 embryos) and 3 families carrying both structural rearrangements and monogenic diseases (26 embryos). Twelve monopronucleated zygotes (1PN) samples and 9 suspected triploid samples were collected from 21 families.Participants/materials, Settings, Methods: Here, we raised a comprehensive PGT method called HaploPGT, combining reduced representation genome sequencing, read-count analysis, B allele frequency and haplotyping analysis, to simultaneously detect different genetic disorders in one single test.Main Results and the Role Of Chance: With 80 million reads (80M) genomic data, the proportion of windows (1 million base pairs (Mb)) containing two or more informative single nucleotide polymorphism (SNP) sites was 97.81%, meanwhile the genotyping error rate stabilized at a low level (2.19%). Furthermore, the informative SNPs were equally distributed across the genome, and whole-genomic haplotyping was established. Therefore, 80M was chosen to balance the cost and accuracy in HaploPGT. HaploPGT was able to identify abnormal embryos with triploid, global and partial loss of heterozygosity, and even to distinguish parental origin of copy number variation in mosaic and non-mosaic embryos. Besides, by retrospectively analyzing 188 embryonic samples from 43 families, HaploPGT revealed 100% concordance with the available results obtained from reference methods, including PGT-A, PGT-M, PGT-SR and PGT-HLA.Limitations, Reason For Caution: Despite the numerous benefits HaploPGT could bring, it still required additional family members to deduce the parental haplotype for identifying balanced translocation and monogenic mutation in tested embryos. In terms of PGT-SR, the additional family member could be a reference embryo with unbalanced translocation. For PGT-M, a proband was normally required. In both cases, genomic information from grandparents or parental siblings might help for haplotyping theoretically. Another restriction was that haploid, and diploid resulting from the duplication of a haploid, could not be told apart by HaploPGT, but it was able to recognize partial loss of heterozygosity in the embryonic genome. In addition, it should be noted that the location of rearrangement breakpoints and the situation of mutation sites were complicated, which meant that partial genetic disorders might not be completely detected.Wider Implications Of the Findings: HaploPGT is an efficient and cost-effective detection platform with high clinical value for detecting genetic status. This platform could promote the application of PGT in ART, to increase pregnancy rate and decrease the birth of children with genetic diseases.Study Funding/competing Interest(s): This study was supported by grants from the National Natural Science Foundation of China (81873478, to L.H.), National Key R&D Program of China (2018YFC1003100, to L.H.), the Natural Science Foundation of Hunan Province (Grant 2022JJ30414, to P.X.), Hunan Provincial Grant for Innovative Province Construction (2019SK4012) and the Scientific Research Foundation of Reproductive and Genetic Hospital of China International Trust & Investment Corporation (CITIC)-Xiangya (YNXM-201910). Haplotyping analysis has been licensed to Basecare Co., Ltd. L.K., Y.M., K.K., D.Z., N.L., J.Z. and R.D. are Basecare Co., Ltd employees. The other authors declare no competing interests.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Can an artificial intelligence (AI) model predict human embryo ploidy status using static images captured by optical light microscopy?Summary Answer: Results demonstrated predictive accuracy for embryo euploidy and showed a significant correlation between AI score and euploidy rate, based on assessment of images of blastocysts at Day 5 after IVF.What Is Known Already: Euploid embryos displaying the normal human chromosomal complement of 46 chromosomes are preferentially selected for transfer over aneuploid embryos (abnormal complement), as they are associated with improved clinical outcomes. Currently, evaluation of embryo genetic status is most commonly performed by preimplantation genetic testing for aneuploidy (PGT-A), which involves embryo biopsy and genetic testing. The potential for embryo damage during biopsy, and the non-uniform nature of aneuploid cells in mosaic embryos, has prompted investigation of additional, non-invasive, whole embryo methods for evaluation of embryo genetic status.Study Design, Size, Duration: A total of 15 192 blastocyst-stage embryo images with associated clinical outcomes were provided by 10 different IVF clinics in the USA, India, Spain and Malaysia. The majority of data were retrospective, with two additional prospectively collected blind datasets provided by IVF clinics using the genetics AI model in clinical practice. Of these images, a total of 5050 images of embryos on Day 5 of in vitro culture were used for the development of the AI model. These Day 5 images were provided for 2438 consecutively treated women who had undergone IVF procedures in the USA between 2011 and 2020. The remaining images were used for evaluation of performance in different settings, or otherwise excluded for not matching the inclusion criteria.Participants/materials, Setting, Methods: The genetics AI model was trained using static 2-dimensional optical light microscope images of Day 5 blastocysts with linked genetic metadata obtained from PGT-A. The endpoint was ploidy status (euploid or aneuploid) based on PGT-A results. Predictive accuracy was determined by evaluating sensitivity (correct prediction of euploid), specificity (correct prediction of aneuploid) and overall accuracy. The Matthew correlation coefficient and receiver-operating characteristic curves and precision-recall curves (including AUC values), were also determined. Performance was also evaluated using correlation analyses and simulated cohort studies to evaluate ranking ability for euploid enrichment.Main Results and the Role Of Chance: Overall accuracy for the prediction of euploidy on a blind test dataset was 65.3%, with a sensitivity of 74.6%. When the blind test dataset was cleansed of poor quality and mislabeled images, overall accuracy increased to 77.4%. This performance may be relevant to clinical situations where confounding factors, such as variability in PGT-A testing, have been accounted for. There was a significant positive correlation between AI score and the proportion of euploid embryos, with very high scoring embryos (9.0-10.0) twice as likely to be euploid than the lowest-scoring embryos (0.0-2.4). When using the genetics AI model to rank embryos in a cohort, the probability of the top-ranked embryo being euploid was 82.4%, which was 26.4% more effective than using random ranking, and ∼13-19% more effective than using the Gardner score. The probability increased to 97.0% when considering the likelihood of one of the top two ranked embryos being euploid, and the probability of both top two ranked embryos being euploid was 66.4%. Additional analyses showed that the AI model generalized well to different patient demographics and could also be used for the evaluation of Day 6 embryos and for images taken using multiple time-lapse systems. Results suggested that the AI model could potentially be used to differentiate mosaic embryos based on the level of mosaicism.Limitations, Reasons For Caution: While the current investigation was performed using both retrospectively and prospectively collected data, it will be important to continue to evaluate real-world use of the genetics AI model. The endpoint described was euploidy based on the clinical outcome of PGT-A results only, so predictive accuracy for genetic status in utero or at birth was not evaluated. Rebiopsy studies of embryos using a range of PGT-A methods indicated a degree of variability in PGT-A results, which must be considered when interpreting the performance of the AI model.Wider Implications Of the Findings: These findings collectively support the use of this genetics AI model for the evaluation of embryo ploidy status in a clinical setting. Results can be used to aid in prioritizing and enriching for embryos that are likely to be euploid for multiple clinical purposes, including selection for transfer in the absence of alternative genetic testing methods, selection for cryopreservation for future use or selection for further confirmatory PGT-A testing, as required.Study Funding/competing Interest(s): Life Whisperer Diagnostics is a wholly owned subsidiary of the parent company, Presagen Holdings Pty Ltd. Funding for the study was provided by Presagen with grant funding received from the South Australian Government: Research, Commercialisation, and Startup Fund (RCSF). 'In kind' support and embryology expertise to guide algorithm development were provided by Ovation Fertility. 'In kind' support in terms of computational resources provided through the Amazon Web Services (AWS) Activate Program. J.M.M.H., D.P. and M.P. are co-owners of Life Whisperer and Presagen. S.M.D., M.A.D. and T.V.N. are employees or former employees of Life Whisperer. S.M.D, J.M.M.H, M.A.D, T.V.N., D.P. and M.P. are listed as inventors of patents relating to this work, and also have stock options in the parent company Presagen. M.V. sits on the advisory board for the global distributor of the technology described in this study and also received support for attending meetings.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Is it possible to develop a comprehensive pipeline for all-in-one preimplantation genetic testing (PGT), also suitable for parents-only haplotyping and, for the first time, third-party reproduction?Summary Answer: Optimized reduced representation sequencing (RRS) by GENType, along with a novel analysis platform (Hopla), enables cheap, accurate and comprehensive PGT of blastocysts, even without the inclusion of additional family members or both biological parents for genome-wide embryo haplotyping.What Is Known Already: Several haplotyping strategies have proven to be effective for comprehensive PGT. However, these methods often rely on microarray technology, whole-genome sequencing (WGS) or a combination of strategies, hindering sample throughput and cost-efficiency. Moreover, existing tools (including other RRS-based strategies) require both prospective biological parents for embryo haplotyping, impeding application in a third-party reproduction setting.Study Design, Size, Duration: This study included a total of 257 samples. Preliminary technical validation was performed on 81 samples handpicked from commercially available cell lines. Subsequently, a clinical validation was performed on a total of 72 trophectoderm biopsies from 24 blastocysts, tested for a monogenic disorder (PGT-M) (n = 15) and/or (sub)chromosomal aneuploidy (PGT-SR/PGT-A) (n = 9). Once validated, our pipeline was implemented in a diagnostic setting on 104 blastocysts for comprehensive PGT.Participants/materials, Setting, Methods: Samples were whole-genome amplified (WGA) and processed by GENType. Quality metrics, genome-wide haplotypes, b-allele frequencies (BAFs) and copy number profiles were generated by Hopla. PGT-M results were deduced from relative haplotypes, while PGT-SR/PGT-A results were inferred from read-count analysis and BAF profiles. Parents-only haplotyping was assessed by excluding additional family members from analysis and using an independently diagnosed embryo as phasing reference. Suitability for third-party reproduction through single-parent haplotyping was evaluated by excluding one biological parent from analysis. Results were validated against reference PGT methods.Main Results and the Role Of Chance: Genome-wide haplotypes of single cells were highly accurate (mean > 99%) compared to bulk DNA. Unbalanced chromosomal abnormalities (>5 Mb) were detected by GENType. For both PGT-M as well as PGT-SR/PGT-A, our technology demonstrated 100% concordance with reference PGT methods for diverse WGA methods. Equally, for parents-only haplotyping and single-parent haplotyping (of autosomal dominant disorders and X-linked disorders), PGT-M results were fully concordant. Furthermore, the origin of trisomies in PGT-M embryos was correctly deciphered by Hopla.Limitations, Reasons For Caution: Intrinsic to linkage-analysis strategies, de novo single-nucleotide variants remain elusive. Moreover, parents-only haplotyping is not a stand-alone approach and requires prior diagnosis of at least one reference embryo by an independent technology (i.e. direct mutation analysis) for haplotype phasing. Using a haplotyping approach, the presence of a homologous recombination site across the chromosome is biologically required to distinguish meiotic II errors from mitotic errors during trisomy origin investigation.Wider Implications Of the Findings: We offer a generic, fully automatable and accurate pipeline for PGT-M, PGT-A and PGT-SR as well as trisomy origin investigation without the need for personalized assays, microarray technology or WGS. The unique ability to perform single-parent assisted haplotyping of embryos paves the way for cost-effective PGT in a third-party reproduction setting.Study Funding/competing Interest(s): L.D.W. is supported by the Research Foundation Flanders (FWO; 1S74619N). L.R. and B.M. are funded by Ghent University and M.B., S.S., K.T., F.V.M. and A.D. are supported by Ghent University Hospital. Research in the N.C. lab was funded by Ghent University, VIB and Kom op Tegen Kanker. A.D.K and N.C. are co-inventors of patent WO2017162754A1. The other authors have no conflicts of interest.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: What are the outcomes for patients who choose to move embryos diagnosed as abnormal by preimplantation genetic testing for aneuploidy (PGT-A) to a new institution for transfer after the diagnosing institution refused to transfer them?Summary Answer: Many patients seek to have selected embryos with PGT-A abnormal trophectoderm biopsies transferred recognizing that these embryos can still offer a chance of pregnancy and live birth.What Is Known Already: : PGT-A is a widely practiced method of selecting embryos for transfer based on biopsy of a few cells. Many clinical practices refuse to transfer PGT-A abnormal embryos even when there are no other 'normal' embryos available.Study Design, Size, Duration: This is a prospective cohort of 69 couples who, since 2014, moved a total of 444 PGT-A abnormal embryos previously refused transfer at their parent institutions to our practice. Among these, 50 patients have, thus far, undergone 57 transfer cycles of 141 embryos.Participants/materials, Setting, Methods: Embryos diagnosed at other institutions by PGT-A as abnormal (mostly using next generation sequencing) were moved to our academically affiliated private fertility and research center in New York City. Female age at retrieval was 41.35 ± 3.98 years, 74% were Caucasian, 12% Asian and 10% were of African descent. All embryos identified as PGT-A abnormal among prospectively identified couples were recorded in our center's registry.Main Results and the Role Of Chance: Among the 144 embryos transferred 102 (72.3%) had only 1 or 2 chromosomal abnormalities, 30 (21.3%) had 3 or more and 9 (6.4%) were 'undiagnosed' because of degraded DNA, yet still had been refused transfer. Transfer of PGT-A abnormal embryos resulted in 8 live births, 11 miscarriages and no voluntary terminations. One child was born with a segmental duplication and required repair of coarctation of the aorta as a newborn. Many couples with only PGT-A abnormal embryos are willing to have their PGT-A abnormal embryos transferred and such transfers can result in the establishment of ongoing euploid pregnancies and live births.Limitations, Reasons For Caution: Findings in this case series represent couples who chose to have their embryos transferred after having been refused transfer elsewhere and may not be representative of the wider population of couples undergoing IVF with PGT-A in general. Not all abnormal phenotypes present in the immediate postnatal period so it will be important to continue to follow the development of these children.Wider Implications Of the Findings: PGT-A can result in a clinics refusal to transfer embryos with abnormal PGT-A biopsies, even those with mosaic findings, consequently large numbers of infertile women are prematurely advised that their only chance of motherhood is through third-party egg-donation.Study Funding/competing Interest(s): This work was supported by intramural funds from the Center for Human Reproduction and the not-for-profit research Foundation for Reproductive Medicine, both in New York, NY, USA. N.G. and D.H.B. are listed as co-inventors on several U.S. patents. One of these patents (US Patent# 7,615,544) relates to pre-supplementation of hypo-androgenic infertile women with androgens, such as DHEA and testosterone and, therefore, at least peripherally related to the subject of this manuscript. N.G. and D.F.A. also received travel funds and speaker honoraria from several pharmaceutical and medical device companies, though none related to the here presented subject and manuscript. N.G. is a shareholder in Fertility Nutraceuticals and he and D.H.B. receive royalty payments from Fertility Nutraceuticals LLC.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Can non-invasive imaging with fluorescence lifetime imaging microscopy (FLIM) detect metabolic differences in euploid versus aneuploid human blastocysts?Summary Answer: FLIM has identified significant metabolic differences between euploid and aneuploid blastocysts.What Is Known Already: Prior studies have demonstrated that FLIM can detect metabolic differences in mouse oocytes and embryos and in discarded human blastocysts.Study Design, Size, Duration: This was a prospective observational study from August 2019 to February 2020. Embryo metabolic state was assessed using FLIM to measure the autofluorescence metabolic factors nicotinamide adenine dinucleotide dehydrogenase together with nicotinamide adenine phosphate dinucleotide dehydrogenase (NAD(P)H) and flavin adenine dinucleotide (FAD). Eight metabolic FLIM parameters were obtained from each blastocyst (four for NAD(P)H and four for FAD): short (T1) and long (T2) fluorescence lifetime, fluorescence intensity (I) and fraction of the molecules engaged with enzymes (F). The redox ratio (NAD(P)H-I)/(FAD-I) was also calculated for each image.Participants/materials, Setting, Methods: This study was performed at a single academically affiliated centre where there were 156 discarded frozen blastocysts (n = 17 euploids; 139 aneuploids) included. Ploidy status was determined by pre-implantation genetic testing for aneuploidy (PGT-A). Discarded human blastocysts were compared using single FLIM parameters. Additionally, inner cell mass (ICM) and trophectoderm (TE) were also evaluated. Multilevel models were used for analysis. A post-hoc correction used Benjamini-Hochberg's false discovery rate, at a q-value of 0.05.Main Results and the Role Of Chance: Comparing euploid (n = 17) versus aneuploid (n = 139) embryos, a significant difference was seen in NAD(P)H-F (P < 0.04), FAD-I (P < 0.04) and redox ratio (P < 0.05). Euploid ICM (n = 15) versus aneuploid ICM (n = 119) also demonstrated significantly different signatures in NAD(P)H-F (P < 0.009), FAD-I (P < 0.03) and redox ratio (P < 0.03). Similarly, euploid TE (n = 15) versus aneuploid TE (n = 119) had significant differences in NAD(P)H-F (P < 0.0001) and FAD-I (P < 0.04).Limitations, Reasons For Caution: This study utilized discarded human blastocysts, and these embryos may differ metabolically from non-discarded human embryos. The blastocysts analysed were vitrified after PGT-A biopsy and it is unclear how the vitrification process may affect the metabolic profile of blastocysts. Our study was also limited by the small number of rare donated euploid embryos available for analysis. Euploid embryos are very rarely discarded due to their value to patients trying to conceive, which limits their use for research purposes. However, we controlled for the imbalance with the bootstrap resampling analysis.Wider Implications Of the Findings: These findings provide preliminary evidence that FLIM may be a useful non-invasive clinical tool to assist in identifying the ploidy status of embryos.Study Funding/competing Interest(s): The study was supported by the Blavatnik Biomedical Accelerator Grant at Harvard University. Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. is an inventor on patent US20170039415A1. There are no other conflicts of interest to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Can label-free, non-invasive optical imaging by hyperspectral autofluorescence microscopy discern between euploid and aneuploid cells within the inner cell mass (ICM) of the mouse preimplantation embryo?Summary Answer: Hyperspectral autofluorescence microscopy enables discrimination between euploid and aneuploid ICM in mouse embryos.What Is Known Already: Euploid/aneuploid mosaicism affects up to 17.3% of human blastocyst embryos with trophectoderm biopsy or spent media currently utilized to diagnose aneuploidy and mosaicism in clinical in vitro fertilization. Based on their design, these approaches will fail to diagnose the presence or proportion of aneuploid cells within the foetal lineage ICM of some blastocyst embryos.Study Design, Size, Duration: The impact of aneuploidy on cellular autofluorescence and metabolism of primary human fibroblast cells and mouse embryos was assessed using a fluorescence microscope adapted for imaging with multiple spectral channels (hyperspectral imaging). Primary human fibroblast cells with known ploidy were subjected to hyperspectral imaging to record native cell fluorescence (4-6 independent replicates, euploid n = 467; aneuploid n = 969). For mouse embryos, blastomeres from the eight-cell stage (five independent replicates: control n = 39; reversine n = 44) and chimeric blastocysts (eight independent replicates: control n = 34; reversine n = 34; 1:1 (control:reversine) n = 30 and 1:3 (control:reversine) n = 37) were utilized for hyperspectral imaging. The ICM from control and reversine-treated embryos were mechanically dissected and their karyotype confirmed by whole genome sequencing (n = 13 euploid and n = 9 aneuploid).Participants/materials, Setting, Methods: Two models were employed: (i) primary human fibroblasts with known karyotype and (ii) a mouse model of embryo aneuploidy where mouse embryos were treated with reversine, a reversible spindle assembly checkpoint inhibitor, during the four- to eight-cell division. Individual blastomeres were dissociated from control and reversine-treated eight-cell embryos and either imaged directly or used to generate chimeric blastocysts with differing ratios of control:reversine-treated cells. Individual blastomeres and embryos were interrogated by hyperspectral imaging. Changes in cellular metabolism were determined by quantification of metabolic co-factors (inferred from their autofluorescence signature): NAD(P)H and flavins with the subsequent calculation of the optical redox ratio (ORR: flavins/[NAD(P)H + flavins]). Autofluorescence signals obtained from hyperspectral imaging were examined mathematically to extract features from each cell/blastomere/ICM. This was used to discriminate between different cell populations.Main Results and the Role Of Chance: An increase in the relative abundance of NAD(P)H and decrease in flavins led to a significant reduction in the ORR for aneuploid cells in primary human fibroblasts and reversine-treated mouse blastomeres (P < 0.05). Mathematical analysis of endogenous cell autofluorescence achieved separation between (i) euploid and aneuploid primary human fibroblast cells, (ii) control and reversine-treated mouse blastomeres cells, (iii) control and reversine-treated chimeric blastocysts, (iv) 1:1 and 1:3 chimeric blastocysts and (v) confirmed euploid and aneuploid ICM from mouse blastocysts. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve of 0.97, 0.99, 0.87, 0.88 and 0.93, respectively. We believe that the role of chance is low as mathematical features separated euploid from aneuploid in both human fibroblasts and ICM of mouse blastocysts.Large Scale Data: N/A.Limitations, Reasons For Caution: Although we were able to discriminate between euploid and aneuploid ICM in mouse blastocysts, confirmation of this approach in human embryos is required. While we show this approach is safe in mouse, further validation is required in large animal species prior to implementation in a clinical setting.Wider Implications Of the Findings: We have developed an original, accurate and non-invasive optical approach to assess aneuploidy within the ICM of mouse embryos in the absence of fluorescent tags. Hyperspectral autofluorescence imaging was able to discriminate between euploid and aneuploid human fibroblast and mouse blastocysts (ICM). This approach may potentially lead to a new diagnostic for embryo analysis.Study Funding/competing Interest(s): K.R.D. is supported by a Mid-Career Fellowship from the Hospital Research Foundation (C-MCF-58-2019). This study was funded by the Australian Research Council Centre of Excellence for Nanoscale Biophotonics (CE140100003) and the National Health and Medical Research Council (APP2003786). The authors declare that there is no conflict of interest. [ABSTRACT FROM AUTHOR]

Study Question: What is the clinical validity and utility of preconception Expanded Carrier Screening (ECS) application on the management of prospective parents?Summary Answer: The high detection rate of at-risk couples (ARCs) and the high proportion opting for IVF/preimplantation genetic testing (PGT) treatment demonstrate the clinical utility of ECS in the preconception space in IVF and general population.What Is Known Already: About 2-4% of couples are at risk of conceiving a child with an autosomal recessive or X-linked genetic disorder. In recent years, the increasing cost-effectiveness of genetic diagnostic techniques has allowed the creation of ECS panels for the simultaneous detection of multiple recessive disorders. Comprehensive preconception genetic screening holds the potential to significantly improve couple's genetic risk assessment and reproductive planning to avoid detectable inheritable genetic offspring.Study Design, Size, Duration: A total of 3877 individuals without a family history of genetic conditions were analyzed between January 2017 and January 2020. Of the enrolled individuals, 1212 were gamete donors and 2665 were patients planning on conceiving from both the IVF and the natural conception group. From the non-donor cohort, 1133 were analyzed as individual patients, while the remaining ones were analyzed as couples, for a total of 766 couples.Participants/materials, Setting, Methods: A focused ECS panel was developed following American College of Obstetrics and Gynecology ACOG-recommended criteria (prevalence, carrier rate, severity), including highly penetrant severe childhood conditions. Couples were defined at-risk when both partners carried an autosomal recessive pathogenic/likely pathogenic variant (PLP) on the same gene or when the woman was a carrier of an X-linked PLP variant. ARC detection rate defined the clinical validity of the ECS approach. Clinical utility was evaluated by monitoring ARCs reproductive decision making.Main Results and the Role Of Chance: A total of 402 individuals (10.4%) showed PLP for at least one of the genes tested. Among the 766 couples tested, 173 showed one carrier partner (22.6%), whereas 20 couples (2.6%) were found to be at increased risk. Interestingly, one ARC was identified as a result of cascade testing in the extended family of an individual carrying a pathogenic variant on the Survival Of Motor Neuron 1SMN1 gene. Of the identified ARCs, 5 (0.7%) were at risk for cystic fibrosis, 5 (0.7%) for fragile X syndrome, 4 (0.5%) for spinal muscular atrophy, 4 (0.5%) for Beta-Thalassemia/Sickle Cell Anemia, 1 (0.1%) for Smith-Lemli-Opitz Syndrome and 1 (0.1%) for Duchenne/Becker Dystrophy. Fifteen ARCs were successfully followed up from both the IVF and the natural conception groups. All of these (15/15) modified their reproductive planning by undergoing ART with Preimplantation Genetic Testing for Monogenic disease and Aneuploidies (PGT-M and PGT-A). To date, 6/15 (40%) couples completed their PGT cycle with euploid/unaffected embryos achieving a pregnancy after embryo transfer and three of them have already had an unaffected baby.Limitations, Reasons For Caution: The use of a limited panel of core gene-disease pairs represents a limitation on the research perspective as it can underestimate the rate of detectable carriers and ARCs in this cohort of prospective parents. Expanding the scope of ECS to a larger panel of conditions is becoming increasingly feasible, thanks to a persistent technological evolution and progressive cataloging of gene-disease associations.Wider Implications Of the Findings: These results highlight the potential clinical validity and utility of ECS in reducing the risk of a pregnancy affected by a detectable inheritable genetic condition. The steady reduction in the costs of genetic analyses enables the expansion of monogenic testing/screening applications at the preimplantation stage, thus, providing valid decisional support and reproductive autonomy to patients, particularly in the context of IVF.Study Funding/competing Interest(s): No external funding was used for this study. A.C., M.F., S.C., M.P., L.G., and C.P. are employees of Igenomix Italy. C.S. is the head of the scientific board of Igenomix.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: What is the potential of applying non-invasive preimplantation genetic testing (niPGT) for chromosome abnormalities in blastocysts reported with a mosaic trophectoderm (TE) biopsy?Summary Answer: niPGT of cell-free DNA in blastocyst culture medium exhibited a good diagnostic performance in putative mosaic blastocysts.What Is Known Already: Advances in niPGT have demonstrated the potential reliability of cell-free DNA as a resource for genetic assessment, but information on mosaic embryos is scarce because the mosaicism may interfere with niPGT. In addition, the high incidence of mosaicism reported in the context of PGT and the viability of mosaic blastocysts raise questions about whether mosaicism really exists.Study Design, Size, Duration: The study was performed between May 2020 and July 2020. First, clinical data collected by a single-center over a 6-year period on PGT for chromosome aneuploidies (PGT-A) or chromosomal structural rearrangements (PGT-SR) were analyzed. After confirming the reliability of niPGT, 41 blastocysts classified as mosaics by trophectoderm (TE) biopsy were re-cultured. The chromosomal copy number of the blastocyst embryo (BE, the gold standard), TE re-biopsy, and corresponding cell-free DNA in the culture medium was assessed.Participants/materials, Setting, Methods: Data on patients enrolled for PGT at a single center from 2014 to 2019 were collected and the cycles with available putative mosaic blastocysts were evaluated. To verify the diagnostic validity of niPGT, eight aneuploid blastocysts were thawed and re-cultured for 14-18 h. The concordance of the niPGT diagnosis results and the whole blastocyst testing results was analyzed. Forty-one blastocysts reported as mosaics from 22 patients were included and re-cultured for 14-18 h. The genetic material of the BE, TE re-biopsy, and corresponding cell-free DNA in the culture medium was amplified using multiple annealing and looping-based amplification cycles. The karyotype data from niPGT and TE re-biopsy were compared with that from the whole blastocyst, and the efficiency of niPGT was assessed.Main Results and the Role Of Chance: Data on 3738 blastocysts from 785 PGT-A or PGT-SR cycles of 677 patients were collected. According to the TE biopsy report, of the 3662 (98%) successfully amplified samples, 24 (0.6%) yielded no results, 849 (23.2%) were euploid, 2245 (61.3%) were aneuploid, and 544 (14.9%) were mosaic. Sixty patients without euploid blastocysts opted for a single mosaic blastocyst transfer, and 30 (50%) of them obtained a clinical pregnancy. With the BE chromosome copy number as the gold standard, niPGT and TE re-biopsy showed reliable detection ability and diagnostic efficiency in eight putative aneuploid blastocysts. Of the 41 putative mosaic blastocysts re-cultured and re-tested, 35 (85.4%) showed euploid BE results. All but two of the blastocysts previously diagnosed with segmental chromosomal mosaic were actually euploid. In addition, all blastocysts previously classified as low degree (20-50%) mosaics were identified as euploid by BE PGT, whereas four of the six putative high degree (50-80%) mosaic blastocysts showed chromosomal abnormalities. The raw concordance rates of spent culture medium (SCM) and TE re-biopsies compared with BE were 74.4% and 82%, respectively, in terms of overall ploidy and 96.2% and 97.6%, respectively, per single chromosome when considering all degree mosaic results as true positives. However, when we set a mosaicism identification threshold of 50%, the concordance rates of SCM and TE re-biopsies compared with BE were 87.2% and 85% at the overall ploidy level and 98.8% and 98.3% at the chromosomal level, respectively. At the full ploidy level, the sensitivity and false negative rates for niPGT were 100% and 0, respectively. After adjustment of the threshold for mosaicism, the specificity of niPGT increased from 69.7% to 84.8% in terms of overall ploidy and from 96.1% to 98.9% at the chromosomal level.Limitations, Reasons For Caution: The primary limitation of this study is the small sample size, which decreases the strength of our conclusions. If possible, identifying the clinical outcome of niPGT on reassessed mosaic blastocysts would be further progress in this field.Wider Implications Of the Findings: This study is the first to explore the practicability of niPGT in diagnostic reassessment of putative mosaicism. The present study provides a novel opportunity for patients with only mosaic blastocysts and no euploid blastocysts, regardless of the technical or biological basis of mosaicism. Employing niPGT after 14-18 h of re-culturing might be a superior option for the best use of blastocysts because of its minimally invasive nature.Study Funding/competing Interest(s): This work was supported by grants from National Key Technology Research and Development Program of China (No. 2017YFC1002004), the Central Guiding the Science and Technology Development of the Local (2018080802D0081) and College Natural Science Project of Anhui Province (KJ2019A0287). There are no competing interests to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: What is the true incidence of chromosomal mosaicism in embryos analyzed by preimplantation genetic testing (PGT).Summary Answer: The true incidence of chromosomal mosaicism is much lower than we usually surmise.What Is Known Already: In recent years, contemporary methods for chromosome analysis, along with the biopsy of more than one cell, have given rise to an increased rate of chromosomal mosaicism detection after preimplantation genetic testing for aneuploidy. However, the exorbitant incidence of mosaicism represents a dilemma and imposes restrictions on the application of PGT treatment. Concern has been raised about the possibility that the incidence of chromosomal mosaicism is overestimated and quite a few of the results are false-positive errors. However, studies verifying the diagnosis of chromosomal mosaicism and assessing the true incidence of chromosomal mosaicism are limited.Study Design, Size, Duration: A total of 1719 blastocysts from 380 patients who underwent PGT treatment were retrospectively analyzed to evaluate the typical incidence of mosaicism. Then 101 embryos donated by 70 couples were re-biopsied and dissected into three portions if available: trophectoderm (TE), inner cell mass (ICM), and the remaining portions. All the portions were tested using next-generation sequencing (NGS), and the results were compared to the original diagnosis.Participants/materials, Setting, Methods: The setting for this study was a university-affiliated center with an in-house PGT laboratory. All samples were amplified with multiple annealing and looping-based amplification cycles (MALBACs) and the NGS was carried out on a Life Technologies Ion Proton platform.Main Results and the Role Of Chance: A clinical TE biopsy revealed an incidence of 11.9% for diploid-aneuploid mosaicism (DAM), 17.3% for aneuploid mosaicism (AM) and 29.1% in total. After rebiopsy, 94.1% whole-chromosome aneuploidies and 82.8% segmental-chromosome aneuploidies were confirmed in the embryos. As for the mosaic errors, only 32 (31.7%) out of 101 embryos presented with uniform chromosomal aberrations in agreement with the original biopsy results, 15 (14.8%) embryos presented with de novo chromosomal aberrations, and 54 (53.5%) embryos showed a euploid profile in all portions. Among the 32 uniform embryos, the true mosaicism was confirmed in only 4 cases, where a reciprocal chromosomal aberration was identified; 14 embryos presented with identical mosaicism, providing the moderate evidence for true mosaicism; and 14 embryos displayed uniform full aneuploidies in all portions of embryo, revealing a high-grade mosaicism or a false-negative diagnosis. Logistical regression analysis revealed that the concordance rate with ICM was associated with the type and level of mosaicism. The concordance rate of segmental-chromosome mosaicism was significantly lower than whole-chromosome mosaicism (adjusted Odds Ratio (aOR): 5.137 (1.061, 24.876), P = 0.042) and compared to DAM, the concordance rate of AM was significantly higher (aOR: 6.546 (1.354, 31.655), P = 0.019). The concordance rate also increased with increasing levels of mosaicism (P < 0.001).Limitations, Reasons For Caution: This study was limited by a small sample size and the use of a single whole-genome amplification (WGA) method and NGS platform. These findings are only applicable to samples subjected to MALBAC amplification and Ion Proton platform, and studies involving larger sample sizes and multiple WGA methods and NGS platforms are required to prove our findings.Wider Implications Of the Findings: TE biopsy is reliable to detect whole-chromosome aneuploidies, but the ability to diagnose mosaicism is doubtful. More attention should be paid to false-positive and false-negative errors in NGS-based PGT, especially for laboratories using less stringent criteria for mosaicism classification (i.e. 20-80%), which might be subject to a much higher false-positive mosaicism rate in the practice.Study Funding/competing Interest(s): This study was supported by grants from the National Key R&D Program of China (No. 2016YFC1000206-5) and the National Natural Science Foundation of China (No. 81701509).Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Do embryos with different developmental competence exhibit different DNA methylation profiles at the blastocyst stage?Summary Answer: We established genome-wide DNA methylome analysis for embryo trophectoderm (TE) biopsy samples and our findings demonstrated correlation of methylation profile of trophectoderm with euploidy status and with maternal age, indicating that genome-wide methylation level might be negatively correlated with embryo quality.What Is Known Already: DNA methylation is a fundamental epigenetic regulatory mechanism that affects differentiation of cells into their future lineages during pre-implantation embryo development. Currently there is no established approach available to assess the epigenetic status of the human preimplantation embryo during routine IVF treatment.Study Design, Size, Duration: In total, we collected trophectoderm biopsy samples from 30 randomly selected human blastocysts and conducted whole-genome bisulfite sequencing (WGBS) to evaluate their DNA methylation profile. Nested linear models were used to assess association between DNA methylation level and ploidy status (aneuploidy [n = 20] vs. euploidy [n = 10]), maternal age (29.4-42.5 years old), and time of blastulation (day 5 [n = 16] vs. day 6 [n = 14]), using embryo identity as a covariate.Participants/materials, Setting, Methods: TE biopsy samples were obtained and submitted to bisulfite conversion. For WGBS, whole-genome sequencing libraries were then generated from the converted genome. An average of 75 million reads were obtained for each sample, and about 63% of the reads aligned to human reference. An average of 40 million reads used for the final analysis after the unconverted reads were filtered out.Main Results and the Role Of Chance: We revealed an increase of genome-wide DNA methylation level in aneuploid embryo TE biopsies compared to euploid embryos (25.4% ± 3.2% vs. 24.7% ± 3.2%, P < 0.005). We also found genome-wide DNA methylation level to be increased with the maternal age (P < 0.005). On a chromosomal scale, we found monosomic embryos have lower methylation levels on the involved chromosome while no drastic change was observed for the involved chromosome in trisomies. Additionally, we revealed that WGBS data precisely revealed the chromosome copy number variance.Limitations, Reasons For Caution: Though our results demonstrated a negative correlation of genome-wide methylation level and embryo quality, further WGBS analysis on a greater number of embryos and specific investigation of its correlation with implantation and live birth are needed before any practical use of this approach for evaluation of embryo competence.Wider Implications Of the Findings: This study revealed a change in genome-wide DNA methylation profile among embryos with different developmental potentials, reinforcing the critical role of DNA methylation in early development.Study Funding/competing Interest(s): No external funding was received for this study. Intramural funding was provided by the Foundation for Embryonic Competence (FEC). E.S. is a consultant for and receives research funding from the Foundation for Embryonic Competence; he is also co-founder and a shareholder of ACIS LLC and coholds patent US2019/055906 issued for utilizing electrical resistance measurement for assessing cell viability and cell membrane piercing.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Human embryos utilise an array of processes to eliminate the very high prevalence of aneuploid cells in early embryo stages. Human embryo self-correction was recently demonstrated by their ability to eliminate/expel abnormal blastomeres as cell debris/fragments. A whole genome amplification study has demonstrated that 63.6% of blastocysts expelled cell debris with abnormal chromosomal rearrangements. Moreover, 55.5% of euploid blastocysts expel aneuploid debris, strongly suggesting that the primary source of cell free DNA in culture media is expelled aneuploid blastomeres and/or their fragments. Such a substantial ability to self-correct downstream from the blastocyststage, therefore, renders any chromosomal diagnosis at the blastocyststage potentially useless, and this, unfortunately, also must particularly include non-invasive PGT-A based on cell-free DNA in spent medium. High rates of false-positive diagnoses of human embryos often lead to non-use and/or disposal of embryos with entirely normal pregnancy potential. Before adopting yet another round of unvalidated PGT-A as a routine adjunct to IVF, we here present facts that deserve to be considered. [ABSTRACT FROM AUTHOR]

Study Question: Can whole genome sequencing (WGS) offer a relatively cost-effective approach for embryonic genome-wide haplotyping and preimplantation genetic testing (PGT) for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR)?Summary Answer: Reliable genome-wide haplotyping, PGT-M, PGT-A and PGT-SR could be performed by WGS with 10× depth of parental and 4× depth of embryonic sequencing data.What Is Known Already: Reduced representation genome sequencing with a genome-wide next-generation sequencing haplarithmisis-based solution has been verified as a generic approach for automated haplotyping and comprehensive PGT. Several low-depth massively parallel sequencing (MPS)-based methods for haplotyping and comprehensive PGT have been developed. However, an additional family member, such as a sibling, or a proband, is required for PGT-M haplotyping using low-depth MPS methods.Study Design, Size, Duration: In this study, 10 families that had undergone traditional IVF-PGT and 53 embryos, including 13 embryos from two PGT-SR families and 40 embryos from eight PGT-M families, were included to evaluate a WGS-based method. There were 24 blastomeres and 29 blastocysts in total. All embryos were used for PGT-A. Karyomapping validated the WGS results. Clinical outcomes of the 10 families were evaluated.Participants/materials, Setting, Methods: A blastomere or a few trophectoderm cells from the blastocyst were biopsied, and multiple displacement amplification (MDA) was performed. MDA DNA and bulk DNA of family members were used for library construction. Libraries were sequenced, and data analysis, including haplotype inheritance deduction for PGT-M and PGT-SR and read-count analysis for PGT-A, was performed using an in-house pipeline. Haplotyping with a proband and parent-only haplotyping without additional family members were performed to assess the WGS methodology. Concordance analysis between the WGS results and traditional PGT methods was performed.Main Results and the Role Of Chance: For the 40 PGT-M and 53 PGT-A embryos, 100% concordance between the WGS and single-nucleotide polymorphism (SNP)-array results was observed, regardless of whether additional family members or a proband was included for PGT-M haplotyping. For the 13 embryos from the two PGT-SR families, the embryonic balanced translocation was detected and 100% concordance between WGS and MicroSeq with PCR-seq was demonstrated.Limitations, Reasons For Caution: The number of samples in this study was limited. In some cases, the reference embryo for PGT-M or PGT-SR parent-only haplotyping was not available owing to failed direct genotyping.Wider Implications Of the Findings: WGS-based PGT-A, PGT-M and PGT-SR offered a comprehensive PGT approach for haplotyping without the requirement for additional family members. It provided an improved complementary method to PGT methodologies, such as low-depth MPS- and SNP array-based methods.Study Funding/competing Interest(s): This research was supported by the research grant from the National Key R&D Program of China (2018YFC0910201 and 2018YFC1004900), the Guangdong province science and technology project of China (2019B020226001), the Shenzhen Birth Defect Screening Project Lab (JZF No. [2016] 750) and the Shenzhen Municipal Government of China (JCYJ20170412152854656). This work was also supported by the National Natural Science Foundation of China (81771638, 81901495 and 81971344), the National Key R&D Program of China (2018YFC1004901 and 2016YFC0905103), the Shanghai Sailing Program (18YF1424800), the Shanghai Municipal Commission of Science and Technology Program (15411964000) and the Shanghai 'Rising Stars of Medical Talent' Youth Development Program Clinical Laboratory Practitioners Program (201972). The authors declare no competing interests.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Questions continue to be raised regarding the benefit of genetic assessment of embryos prior to transfer in IVF, specifically with regards to preimplantation genetic testing for aneuploidy (PGT-A). To evaluate and quantify these concerns, we appraised the most recent (2012-2019) randomized controlled trials on the topic. Only two of these six studies listed cumulative live birth rates per started cycle, with both eliciting a statistically non-significant result. This article describes the concern that a focus on results from the first embryo transfer compared to cumulative outcomes falsely construes PGT-A as having superior outcomes, whilst its true benefit is not confirmed, and it cannot actually improve the true pregnancy outcome of an embryo pool. [ABSTRACT FROM AUTHOR]

Study Question: Does preimplantation genetic testing for aneuploidy (PGT-A) increase the likelihood of live birth among women undergoing autologous IVF who have fertilized embryos?Summary Answer: PGT-A is associated with a greater probability of live birth among women 35 years old and older who are undergoing IVF.What Is Known Already: Previous studies evaluating the association between PGT-A and the incidence of live birth may be prone to confounding by indication, as women whose embryos undergo PGT-A may have a lower probability of live birth due to other factors associated with their increased risk of aneuploidy (e.g. advancing age, history of miscarriage). Propensity score matching can reduce bias where strong confounding by indication is expected.Study Design, Size, Duration: We conducted a retrospective cohort study utilizing data from women who underwent autologous IVF treatment, had their first oocyte retrieval at our institution from 1 January 2011 through 31 October 2017 and had fertilized embryos from this retrieval. If a woman elected to use PGT-A, all good quality embryos (defined as an embryo between Stages 3 and 6 with Grade A or B inner or outer cell mass) were tested. We only evaluated cycles associated with the first oocyte retrieval in this analysis.Participants/materials, Setting, Methods: Our analytic cohort included 8227 women. We used multivariable logistic regression to calculate a propensity score for PGT-A based on relevant demographic and clinical factors available to the IVF provider at the time of PGT-A or embryo transfer. We used the propensity score to match women who did and did not utilize PGT-A in a 1:1 ratio. We then used log-binomial regression to compare the cumulative incidence of embryo transfer, clinical pregnancy, miscarriage and live birth between women who did and did not utilize PGT-A. Because the risk of aneuploidy increases with age, we repeated these analyses among women <35, 35-37 and ≥38 years old based on the Society for Assisted Reproductive Technology's standards.Main Results and the Role Of Chance: Overall, women with fertilized embryos who used PGT-A were significantly less likely to have an embryo transfer (risk ratios (RR): 0.78; 95% CI: 0.73, 0.82) but were more likely to have a cycle that resulted in a clinical pregnancy (RR: 1.15; 95% CI: 1.04, 1.28) and live birth (RR: 1.21; 95% CI: 1.08, 1.35) than women who did not use PGT-A. Among women aged ≥38 years, those who used PGT-A were 67% (RR: 1.67; 95% CI: 1.31, 2.13) more likely to have a live birth than women who did not use PGT-A. Among women aged 35-37 years, those who used PGT-A were also more likely to have a live birth (RR: 1.27; 95% CI: 1.05, 1.54) than women who did not use PGT-A. In contrast, women <35 years old who used PGT-A were as likely to have a live birth (RR: 0.91; 95% CI: 0.78, 1.06) as women <35 years old who did not use PGT-A.Limitations, Reasons For Caution: We were unable to abstract several potential confounding variables from patients' records (e.g. anti-Mullerian hormone levels and prior IVF treatment), which may have resulted in residual confounding. Additionally, by restricting our analyses to cycles associated with the first oocyte retrieval, we were unable to estimate the cumulative incidence of live birth over multiple oocyte retrieval cycles.Wider Implications Of the Findings: Women aged 35 years or older are likely to benefit from PGT-A. Larger studies might identify additional subgroups of women who might benefit from PGT-A.Study Funding/competing Interest(s): No funding was received for this study. D.S. reports that he is a member of the Cooper Surgical Advisory Board. The other authors report no conflicts of interest.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Is there a difference in level of decision regret following IVF treatment between those who choose to complete or not complete preimplantation genetic testing for aneuploidy [PGT-A]?Summary Answer: Approximately one-third of the participants expressed moderate to severe regret (MSR) following their decision to either complete or not complete PGT-A; notably, decision regret was higher in those who chose not to complete PGT-A, primarily driven by significantly higher regret scores in those that experienced a miscarriage after not testing.What Is Known Already: Previous research has found that 39% of participants who completed PGT-A expressed some degree of decision regret and that negative clinical outcomes, such as lack of euploid embryos, negative pregnancy test or miscarriage, were associated with a higher level of decision regret. To date, there are no published studies assessing the possible disparity in decision regret surrounding PGT-A in a population of IVF patients that either chose to pursue PGT-A or not.Study Design, Size, Duration: An anonymous online survey was distributed to 1583 patients who underwent IVF with or without PGT-A at a single university institution between January 2016 and December 2017. In total, 335 women accessed the survey, 220 met eligibility criteria and 130 completed the full study survey. Six participants were excluded due to refusal of medical record review, and nine participants were excluded after record review due to not meeting eligibility based on cycle start date or completing only embryo banking without attempting transfer. One hundred and fifteen participants were included in the final analysis.Participants/materials, Setting, Methods: Of the 115 participants included, 55 (48%) completed PGT-A and 60 (52%) did not complete PGT-A. The online survey included four sections: Demographics; Perceptions about PGT-A risks and benefits [scale from 0 (absolutely not true) to 100 (absolutely true)]; Decision-making factors [scale from 0 (not important) to 100 (very important)]; and Brehaut Decision Regret Scale [DRS] [range 0-100, with >25 indicating MSR]. A retrospective chart review was conducted to confirm study eligibility and collect cumulative clinical outcomes of consenting participants who completed the survey.Main Results and the Role Of Chance: Demographics of the PGT-A and no PGT-A cohorts were similar, with the majority of respondents being Caucasian or Asian, unaffiliated with any religion and with a graduate or professional degree. The two groups differed significantly in mean age, with the PGT-A group being slightly older (mean ± SD: 37 ± 3.7 versus 36 ± 3.4; P = 0.048), and in rate of miscarriages, with fewer participants in the PGT-A cohort experiencing a miscarriage (5% versus 22%; P = 0.012). The majority of participants in both PGT-A and no PGT-A cohorts strongly believed in the purported benefits of PGT-A, including that it decreases the risk of birth defects (median 82 versus 77; P = 0.046), improves the chances of having a healthy baby (median 89 versus 74; P = 0.002) and selects the best embryo for transfer (median 85 versus 80; P = 0.049). When asked to report their motivating factors for decision-making, both groups cited physician counseling as important (median 70 versus 71; P = 0.671); however, the PGT-A cohort was more strongly motivated by a desire to not transfer abnormal embryos (median 84 versus 53; P = 0.0001). Comparison of DRS score between those who did or did not undergo PGT-A showed significantly higher median DRS score after not completing PGT-A (median 15 versus 0; P = 0.013). There was a significantly higher proportion of participants who did not complete PGT-A that expressed mild (36% versus 16%) and MSR (32% versus 24%) compared to those who completed PGT-A (χ2 = 9.03, df = 2; P = 0.011). Sub-group analyses of DRS scores by outcomes of clinical pregnancy, miscarriage and live birth revealed that the higher DRS score in those not completing PGT-A was driven by a large increase in regret noted by those with history of a miscarriage (median 45 versus 0; P = 0.018). Multivariate logistic regression modeling found no evidence that any specific demographic factor, clinical outcome or perception/motivation surrounding PGT-A was independently predictive of increased risk for MSR.Limitations, Reasons For Caution: The retrospective nature of data collection incurs the possibility of sampling and recall bias. As only 59% of eligible respondents completed the full survey, it is possible that mainly those with very positive or negative sentiments following treatment felt compelled to complete their response. This bias, however, would apply to the whole of the population, and not simply to those who did or did not complete PGT-A.Wider Implications Of the Findings: The proportion of participants expressing any degree of decision regret in this PGT-A cohort was 40%, which is comparable to that shown in prior research. This study adds to prior data by also assessing decision regret experienced by those who went through IVF without PGT-A, and showed that 68% expressed some level of regret with their decision-making. These results should not be interpreted to mean that all patients should opt for PGT-A to pre-emptively mitigate their risk of regret. Instead, it suggests that drivers of decision regret are likely multifactorial and unique to the experience of one's personal expectations regarding PGT-A, motivations for pursuing or not pursuing it and resultant clinical outcome. Highlighting the complex nature of regret, these data should encourage physicians to more carefully consider individual patient values toward risk-taking or risk-averse behavior, as well as their own positions regarding PGT-A. Until there are clear recommendations regarding utilization of PGT-A, a strong collaboration between physicians and genetic counselors is recommended to educate patients on the risks and potential benefits of PGT-A in a balanced and individualized manner.Study Funding/competing Interest(s): No funding was utilized for study completion and the authors have no competing interests.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Is de novo segmental aneuploidy (SA) a biological event or an artifact that is erroneously interpreted as partial chromosome imbalance?Summary Answer: The detection of de novo SA in sequential biopsies of preimplantation embryos supports the biological nature of SA.What Is Known Already: Although some SAs are detected in oocytes and in blastocysts, the highest incidence is observed in cleavage-stage embryos. Based on these findings, we can postulate that the majority of cells affected by SAs are eliminated by apoptosis or that affected embryos mainly undergo developmental arrest.Study Design, Size, Duration: This retrospective study includes 342 preimplantation genetic testing for aneuploidy (PGT-A) cycles performed between January 2014 and December 2018. Chromosome analysis was performed on 331 oocytes, 886 cleavage-stage embryos and 570 blastocysts (n = 1787). From 268 expanded blastocysts, the blastocoelic fluid (BF) was also analyzed (resulting in 2025 samples in total). In cases of SAs involving loss or gain in excess of 15 Mb, embryos were not considered for transfer and sequential biopsies were performed at following stages. This resulted in 66 sets where the initial diagnosis of SAs (4 made in polar bodies, 25 in blastomeres and 37 in trophectoderm (TE) cells) was followed up.Participants/materials, Setting, Methods: A total of 2082 samples (2025 + 27 whole embryos) were processed by whole genome amplification followed by array comparative genomic hybridization.Main Results and the Role Of Chance: The incidence of SAs was 6.3% in oocytes, increased to 16.6% in cleavage-stage embryos (P < 0.001) and decreased to 11.2% in blastocysts (P < 0.025 versus oocytes; P < 0.01 versus cleavage-stage embryos). The highest incidence of SAs was found in BFs (26.1%, P < 0.001). The analysis of 66 sets of sequential biopsies revealed that the initial finding was confirmed in all following samples from 39 sets (59.1% full concordance). In 12 additional sets, SAs were detected in some samples while in others the interested chromosome had full aneuploidy (18.2%). In three more sets, there was a partial concordance with the initial diagnosis in some samples, but in all TE samples the interested chromosome was clearly euploid (4.5%). In the remaining 12 sets, the initial SA was not confirmed at any stage and the corresponding chromosomes were euploid (18.2% no concordance). The pattern of concordance was not affected by the number of SAs in the original biopsy (single, double or complex) or by the absence or presence of concomitant aneuploidies for full chromosomes.Limitations, Reasons For Caution: Chromosome analyses were performed on biopsies that might not be representative of the true constitution of the embryo itself due to the occurrence of mosaicism.Wider Implications Of the Findings: The permanence of SAs throughout the following stages of embryo development in more than half of the analyzed sets suggests for this dataset a very early origin of this type of chromosome imbalance, either at meiosis or at the first mitotic divisions. Since SAs remained in full concordance with the initial diagnosis until the blastocyst stage, a corrective mechanism seems not to be in place. In the remaining cases, it is likely that, as for full chromosome aneuploidy, mosaicism derived from mitotic errors could have occurred. In following cell divisions, euploid cell lines could prevail preserving the embryo chances of implantation. Due to the scarcity of data available, the transfer of embryos with SAs should be strictly followed up to establish possible clinical consequences related to this condition.Study Funding/competing Interest(s): No specific funding was obtained. There are no conflicts of interest. [ABSTRACT FROM AUTHOR]

Study Question: Has the number of preimplantation genetic testing (PGT) cycles in the UK and USA changed between 2014 and 2016?Summary Answer: From 2014 to 2016, the number of PGT cycles in the UK has remained the same at just under 2% but in the USA has increased from 13% to 27%.What Is Known Already: PGT was introduced as a treatment option for couples at risk of transmitting a known genetic or chromosomal abnormality to their child. This technology has also been applied as an embryo selection tool in the hope of increasing live birth rates per transfer. ART cycles are monitored in the UK by the Human Fertilisation and Embryology Authority (HFEA) and in the USA by the Society for Assisted Reproductive Technology (SART). Globally, data are monitored via the ESHRE PGT Consortium.Study Design, Size, Duration: This cross-sectional study used the HFEA and SART databases to analyse PGT cycle data and make comparisons with IVF data to examine the success of and changes in patient treatment pathways. Both data sets were analysed from 2014 to 2016. The UK data included 3385 PGT cycles and the USA data included 94 935 PGT cycles.Participants/materials, Setting, Methods: Following an extensive review of both databases, filters were applied to analyse the data. An assessment of limitations of each database was also undertaken, taking into account data collection by the ESHRE PGT Consortium. In the UK and USA, the publicly available information from these datasets cannot be separated into different indications.Main Results and the Role Of Chance: The proportion of PGT cycles as a total of ART procedures has remained the same in the UK but increased annually in the USA from 13% to 27%. Between 2014 and 2016 inclusive, 3385 PGT cycles have been performed in the UK, resulting in 1074 PGT babies being born. In the USA 94 935 PGT cycles have been performed, resulting in 26 822 babies being born. This gave a success rate per egg collection for PGT of 32% for the UK and 28% for the USA. Analysis of the data by maternal age shows very different patient populations between the UK and USA. These differences may be related to the way PGT is funded in the UK and USA and the lack of HFEA support for PGT for aneuploidy.Limitations, Reasons For Caution: Data reported by the HFEA and SART have different limitations. As undertaken by the ESHRE PGT Consortium, both data sets should separate PGT data by indication. Although the HFEA collects data from all IVF clinics in the UK, SART data only represent 83% of clinics in the USA.Wider Implications Of the Findings: Worldwide, a consistent reporting scheme is required in which success rates can convey the effectiveness of PGT approaches for all indications.Study Funding/competing Interest(s): No specific funding was obtained and there are no competing interests to declare that are directly related to this project. Joyce Harper is the director of the Embryology and PGD Academy, which offers education in these fields. [ABSTRACT FROM AUTHOR]

Mosaic embryos have the potential to implant and develop into healthy babies. The transfer of mosaic embryos is now considered to be a possible option for women undergoing ART with preimplantation genetic testing for aneuploidies and in the absence of euploid embryos, particularly those with diminished ovarian reserve and/or advanced maternal age. It can aid in avoiding the discard of potentially viable embryos, which might otherwise result in healthy babies. In over 500 studies on mosaicism, there have been no reports of mosaicism in babies born following the transfer of mosaic embryos. Here, we present a case report of a 39-year-old woman with diminished ovarian reserve with only one blastocyst available for trophectoderm biopsy. The transfer of the embryo, which showed 35% mosaicism of monosomy 2, resulted in pregnancy. Amniocentesis revealed a mosaic trisomic mos46,XX(98)/47,XX,+2(2) karyotype. There were no pathological findings in detailed ultrasonography, and the fetus showed a normal fetal growth with no evidence of intrauterine growth retardation. A healthy female baby was born at Week 37. The peripheral blood chromosome analysis validated with fluorescence in situ hybridization showed 2% mosaic monosomy 2 [mos45,XX,-2(2)/46,XX(98)]. This is the first reported case of true fetal mosaicism resulting in a live birth following the transfer of a known mosaic embryo. Worldwide, prenatal diagnosis has shown the depletion of mosaicism in embryos transferred after they have been reported as mosaics. Our case demonstrates the need for close prenatal monitoring and diagnosis by early amniocentesis, preferably at >14 weeks gestation. [ABSTRACT FROM AUTHOR]

Study Question: Is it possible to haplotype parents using parental siblings to leverage preimplantation genetic testing (PGT) for monogenic diseases and aneuploidy (comprehensive PGT) by genome-wide haplotyping?Summary Answer: We imputed identity-by-state (IBS) sharing of parental siblings to phase parental genotypes.What Is Known Already: Genome-wide haplotyping of preimplantation embryos is being implemented as a generic approach for genetic diagnosis of inherited single-gene disorders. To enable the phasing of genotypes into haplotypes, genotyping the direct family members of the prospective parent carrying the mutation is required. Current approaches require genotypes of either (i) both or one of the parents of the affected prospective parent or (ii) an affected or an unaffected child of the couple. However, this approach cannot be used when parents or children are not attainable, prompting an investigation into alternative phasing options.Study Design, Size, Duration: This is a retrospective validation study, which applied IBS-based phasing of parental haplotypes in 56 embryos derived from 12 PGT families. Genome-wide haplotypes and copy number profiles generated for each embryo using the new phasing approach were compared with the reference PGT method to evaluate the diagnostic concordance.Participants/materials, Setting, Methods: This study included 12 couples with a known hereditary genetic disorder, participating in the comprehensive PGT program and with at least one parental sibling available (e.g. brother and/or sister). Genotyping data from both prospective parents and the parental sibling(s) were used to perform IBS-based phasing and to trace the disease-associated alleles. The outcome of the IBS-based PGT was compared with the results of the clinically implemented reference haplotyping-based PGT method.Main Results and the Role Of Chance: IBS-based haplotyping was performed for 12 PGT families. In accordance with the theoretical prediction of allele sharing between sibling pairs, 6 out of 12 (50%) couples or 23 out of 56 embryos could be phased using parental siblings. In families where phasing was possible, haplotype calling in the locus of interest was 100% concordant between the reference PGT method and IBS-based approach using parental siblings.Large Scale Data: N/A.Limitations, Reasons For Caution: Phasing of parental haplotypes will only be possible when the disease locus lies in an informative region (categorized as IBS1). Phasing prospective parents using relatives with reduced genetic relatedness as a reference (e.g. siblings) decreases the size and the occurrence of informative IBS1 regions, necessary for haplotype calling. By including more than one extended family member, the chance of obtaining IBS1 coverage in the interrogated locus can be increased. A pre-PGT work-up can define whether the carrier couple could benefit from this approach.Wider Implications Of the Findings: Phasing by relatives extends the potential of comprehensive PGT, since it allows the inclusion of couples who do not have access to the standard phasing references, such as parents or offspring.Study Funding/competing Interest(s): The study was funded by the KU Leuven grant (C14/18/092), Research Foundation Flanders (FWO; GA09311N), Horizon 2020 innovation programme (WIDENLIFE, 692065) and Agilent Technologies. J.R.V., T.V. and M.Z.E. are co-inventors of a patent ZL910050-PCT/EP2011/060211-WO/2011/157846 'Methods for haplotyping single-cells' and ZL913096-PCT/EP2014/068315-WO/2015/028576 'Haplotyping and copy number typing using polymorphic variant allelic frequencies' licensed to Agilent Technologies. The other authors have no conflict of interest to declare. [ABSTRACT FROM AUTHOR]

The utilization of preimplantation genetic testing for aneuploidy (PGT-A) has understandable intuitive appeal in reassuring the clinician that 'everything possible' has been done to assure the birth of a healthy baby. Whereas the development of the PGT-A technology is still in a relatively early stage, great strides have nevertheless been made in the understanding of the genetics of the preimplantation human embryo. The problem lies not in the progress that has been achieved, but rather, in the reality that PGT-A is being actively marketed as a mature technology. Those that market the technology overstate its benefits and underestimate the losses of potential implantations that are the consequence of the practice of PGT-A. The implication is that the PGT-A technology is accurate, has minimal errors and is ready to be applied to every case of IVF. This approach is not evidence-based. Substantial losses of potential implantations are even evident in the analysis of the numbers presented by marketing materials themselves. In order to provide accurate, evidence-based counseling for patients undergoing IVF, we need to apply an appropriate level of scientific scrutiny to the data that are available and apply PGT-A selectively to those cases in which the benefits clearly outweigh the costs. [ABSTRACT FROM AUTHOR]

Study Question: What is the current status of assisted reproductive technology (ART) service availability, efficacy and safety in mainland China?Summary Answer: In this first national report on ART status in mainland China, data on treatment numbers, outcomes and complications in 2016 are provided and analyzed, respectively.What Is Known Already: National ART Service Provision Surveys are conducted in mainland China regularly. Data were analyzed, and this manuscript was written by team members from the National Center for Women and Children's Health, China CDC and the Department of Women and Children Health, National Health Commission of the People's Republic of China.Study Design, Size and Duration: A cross-sectional nationwide survey was completed in 2018, in which data regarding ART treatments, performed from 1st January to 31st December2016 in 445 ART clinics located in 31 provinces of mainland China, were collected.Participants/materials, Setting and Methods: There were in total 451 licensed ART clinics (including artificial insemination clinics) in mainland China in 2016, of which 445 submitted service data. A total of 906 840 cycles were provided by 323 in vitro fertilization (IVF) clinics, involving 375 770 conventional IVF cycles, 154 948 intracytoplasmic sperm injection (ICSI) cycles, 367 146 frozen embryo transfer (FET) thawing cycles and 8976 preimplantation genetic diagnosis (PGD) treatment cycles. A total of 161 376 artificial (i.e. intrauterine) insemination (AI) cycles were reported by 443 clinics, with 126 872 cycles using the husband's semen (AIH) and 34 504 using donor semen (AID).Main Results and the Role Of Chance: In total, 98.7% of the licensed clinics, contributing to 100% of the ART services (including AID and AIH cycles), were included in this report. (Six clinics provided institutional information only and were excluded.) There were 906 840 in vitro fertilization cycles performed in mainland China with a population of over 1.3 billion inhabitants, with cycles per million inhabitants (C/M) increasing from 360 in 2013 to 657 in 2016, nationwide (range among provinces: 45-3676). After treatment with conventional IVF, the clinical pregnancy rate (PR) per oocyte retrieval cycle was 23.2%, the delivery rate (DR) per oocyte retrieval cycle was 18.7% and the proportion of twin delivery among the total deliveries was 27.9%. For ICSI cycles, the PR, DR and TDR were 20.5%, 16.7% and 27.2%, respectively. For FET per thawing cycles, the PR, DR and TDR were 48.2%, 37.6% and 24.2%. For PGD per diagnosis cycles, the PR, DR and TDR were 38.1%, 29.7% and 4.2%. For AIH cycles, the PR and DR were 13.3% and 10.5%; for AID cycles, the PR and DR were 24.3% and 21.1%, respectively. The total number of live infants born in mainland China in 2016, was 18.46 million, and the number of infants born through ART conducted in 2016 was 311 309, which accounted for 1.69% of the total. The reported rate of birth defects was about 87/10 000. The incidence of moderate to severe ovarian hyper-stimulation syndrome (OHSS) was 11.5 per 1000 oocyte retrieval cycles, and other complications were much more rare.Limitations and Reasons For Caution: This report is based on the summary data of ART services provided. The success rates were not calculated by age stratification. A low rate of birth defects was reported, which might be confounded by variations in birth follow-up methods, statistical timing and record taking.Wider Implications Of the Findings: ART service availability has improved significantly in recent years in mainland China. Because China is a vast country, significant imbalances in ART service provision do exist; however, the main efficacy and safety indicators were close to those of western countries.Trial Registration Number: N/A.Study Funding/competing Interest(s): The study was funded by the National Key R&D Program of China (2016YFC1000307-2). There are no competing interests. [ABSTRACT FROM AUTHOR]

Study Question: Is it cost-effective to use in vitro fertilisation and preimplantation genetic testing of monogenic defects (IVT/PGT-M) to prevent transmission of BRCA1/2 mutations to second-generation new births in comparison with naturally conceived births?Summary Answer: In this cost-effectiveness analysis, we found that IVF/PGT-M is cost-effective for BRCA1 and BRCA2 mutation carriers if using a willingness to pay of $50 000 per quality-adjusted life-year (QALY).What Is Known Already: Carriers of a BRCA1 or BRCA2 mutation have a significantly increased risk of several types of cancer throughout their lifetime. The cost of risk reduction, screening and treatment of cancer in this population is high. In addition, there is a 50% chance of passing on this genetic mutation to each child. One option to avoid transmission of an inherited deleterious gene to one's offspring involves in vitro fertilisation with preimplantation genetic testing.Study Design, Size, Duration: We implemented a state transition model comparing the healthcare impact of a cohort of healthy children born after IVF/PGT-M, who have a population risk of developing cancer, to a cohort of naturally conceived live-births, half of whom are carriers of the BRCA mutation. Transition probabilities are based on published sources, a lifetime horizon and a perspective of a provincial Ministry of Health in Canada.Participants/materials, Setting, Methods: The target population is the second-generation new births who have at least one parent with a known BRCA1 or BRCA2 mutation.Main Results and the Role Of Chance: At a willingness-to-pay threshold of $50 000 per QALY, IVF/PGT-M is a cost-effective intervention for carriers of either BRCA mutation. For BRCA1, the incremental cost-effectiveness ratio (ICER) for IVF/PGT-M is $14 242/QALY. For BRCA2, the ICER of intervention is $12 893/QALY. Probabilistic sensitivity analysis results show that IVF/PGT-M has a 98.4 and 97.3% chance of being cost-effective for BRCA1 and BRCA2 mutation carriers, respectively, at the $50 000/QALY threshold.Limitations, Reasons For Caution: Our model did not include the short-term negative effect of IVF/PGT-M on the woman's quality of life; in addition, our model did not consider any ethical issues related to post-implantation genetic testing.Wider Implications Of the Findings: In countries in which the healthcare of a large segment of the population is covered by a single payer system such as the government, it would be cost-effective for that payer to cover the cost of IVF/PGT-M for couples in which one member has a BRCA mutation, in order to avoid the future costs and disutility of managing offspring with an inherited BRCA mutation.Study Funding/competing Interest(s): Dr Wong's research program was supported by the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council (NSERC), the Canadian Liver Foundation and an Ontario Ministry of Research, Innovation and Science Early Researcher Award. All authors declared no conflict of interests. [ABSTRACT FROM AUTHOR]

Study Question: Can miRNAs be reliably detected in the spent blastocyst media (SBM) after IVF as putative biomarkers of the implantation potential of euploid embryos?Summary Answer: Adjustment of the data for blastocyst quality and the day of full-expansion hinders the predictive power of a fast, inexpensive, reproducible and user-friendly protocol based on the detection of 10 selected miRNAs from SBM.What Is Known Already: Euploidy represents so far the strongest predictor of blastocyst competence. Nevertheless, ~50% of the euploid blastocysts fail to implant. Several studies across the years have suggested that a dialogue exists between the embryo and the endometrium aimed at the establishment of a pregnancy. MicroRNAs have been proposed as mediators of such a dialogue and investigated in this respect. Several expensive, time-consuming and complex protocols have been adopted and promising results have been produced, but conclusive evidence from large clinical studies is missing.Study Design, Size, Duration: This study was conducted in two phases from September 2015 to December 2017. In Phase 1, the human blastocyst miRNome profile was defined from the inner cell mass (ICM) and the corresponding whole-trophectoderm (TE) of six donated blastocysts. Two different protocols were adopted to this end. In parallel, 6 pools of 10 SBM each were run (3 from only implanted euploid blastocysts, IEBs; and 3 from only not-implanted euploid blastocysts, not-IEBs). A fast, inexpensive and user-friendly custom protocol for miRNA SBM profiling was designed. In Phase 2, 239 SBM from IEB and not-IEB were collected at three IVF centres. After 18 SBM from poor-quality blastocysts were excluded from the analysis, data from 107 SBM from not-IEB and 114 from IEB were produced through the previously developed custom protocol and compared. The data were corrected through logistic regressions.Participant/materials, Settings, Methods: Donated blastocysts underwent ICM and whole-TE isolation. SBM were collected during IVF cycles characterized by ICSI, blastocyst culture in a continuous media, TE biopsy without zona pellucida opening in Day 3, quantitative PCR (qPCR)-based aneuploidy testing and vitrified-warmed single euploid embryo transfer. Not-IEB and IEB were clustered following a negative pregnancy test and a live birth, respectively. The Taqman Low Density Array (TLDA) cards and the Exiqon microRNA human panel I+II qPCR analysis protocols were adopted to analyse the ICM and whole-TE. The latter was used also for SBM pools. A custom protocol and plate was then designed based on the Exiqon workflow, validated and finally adopted for SBM analysis in study Phase 2. This custom protocol allows the analysis of 10 miRNAs from 10 SBM in 3 hours from sample collection to data inspection.Main Results and Role Of the Chance: The TLDA cards protocol involved a higher rate of false positive results (5.6% versus 2.8% with Exiqon). There were 44 miRNAs detected in the ICM and TE from both the protocols. One and 24 miRNAs were instead detected solely in the ICM and the TE, respectively. Overall, 29 miRNAs were detected in the pooled SBM: 8 only from not-IEB, 8 only from IEB and 13 from both. Most of them (N = 24/29, 82.7%) were also detected previously in both the ICM and TE with the Exiqon protocol; two miRNAs (N = 2/29, 6.9%) were previously detected only in the TE, and three (N = 3/29, 10.3%) were never detected previously. In study Phase 2, significant differences were shown between not-IEB and IEB in terms of both miRNA detection and relative quantitation. However, when the data were corrected for embryo morphology and day of full development (i.e. SBM collection), no significant association was confirmed.Limitations, Reasons For Caution: This study did not evaluate specifically exosomal miRNAs, thereby reducing the chance of identifying the functional miRNAs. Ex-vivo experiments are required to confirm the role of miRNAs in mediating the dialogue with endometrial cells, and higher throughput technologies need to be further evaluated for miRNA profiling from clinical SBM samples.Wider Implications Of the Findings: Although no clinical predictive power was reported in this study, the absence of invasiveness related with SBM analysis and the evidence that embryonic genetic material can be reliably detected and analysed from SBM make this waste product of IVF an important source for further investigations aimed at improving embryo selection.Study Funding/competing Interest(s): This project has been financially supported by Merck KgaA (Darmstadt, Germany) with a Grant for Fertility Innovation (GFI) 2015. The authors have no conflict of interest to declare related with this study.Trial Registration Number: None. [ABSTRACT FROM AUTHOR]

Study Question: Do monosomy rescue (MR) and trisomy rescue (TR) in preimplantation human embryos affect other developmental processes, such as X-chromosome inactivation (XCI)?Summary Answer: Aneuploid rescue precedes XCI and increases the incidence of XCI skewness by reducing the size of the embryonic progenitor cell pools.What Is Known Already: More than half of preimplantation human embryos harbor aneuploid cells, some of which can be spontaneously corrected through MR or TR. XCI in females is an indispensable process, which is predicted to start at the early-blastocyst phase.Study Design, Size, Duration: We examined the frequency of XCI skewness in young females who carried full uniparental disomy (UPD) resulting from MR or TR/gamete complementation (GC). The results were statistically analyzed using a theoretical model in which XCI involves various numbers of embryonic progenitor cells.Participants/materials, Setting, Methods: We studied 39 children and young adults ascertained by imprinting disorders. XCI ratios were determined by DNA methylation analysis of a polymorphic locus in the androgen receptor gene. We used Bayesian approach to assess the probability of the occurrence of extreme XCI skewness in the MR and TR/GC groups using a theoretical model of 1-12 cell pools.Main Results and the Role Of Chance: A total of 12 of 39 individuals (31%) showed skewed XCI. Extreme skewness was observed in 3 of 15 MR cases (20%) and 1 of 24 TR/GC cases (4.2%). Statistical analysis indicated that XCI in the MR group was likely to have occurred when the blastocyst contained three or four euploid embryonic progenitor cells. The estimated size of the embryonic progenitor cell pools was approximately one-third or one-fourth of the predicted size of normal embryos. The TR/GC group likely had a larger pool size at the onset of XCI, although the results remained inconclusive.Limitations, Reasons For Caution: This is an observational study and needs to be validated by experimental analyses.Wider Implications Of the Findings: This study provides evidence that the onset of XCI is determined by an intrinsic clock, irrespectively of the number of embryonic progenitor cells. Our findings can also be applied to individuals without UPD or imprinting disorders. This study provides a clue to understand chromosomal and cellular dynamics in the first few days of human development, their effects on XCI skewing and the possible implications for the expression of X-linked diseases in females.Study Funding/competing Interest(s): This study was supported by the Grants-in-aid for Scientific Research on Innovative Areas (17H06428) and for Scientific Research (B) (17H03616) from Japan Society for the Promotion of Science (JSPS), and grants from Japan Agency for Medical Research and Development (AMED) (18ek0109266h0002 and 18ek0109278h0002), National Center for Child Health and Development and Takeda Science Foundation. The authors declare no conflict of interest.Trial Registration Number: Not applicable. [ABSTRACT FROM AUTHOR]

Study Question: How reliable are cleavage stage and trophectoderm (TE) biopsies compared to inner cell mass (ICM) biopsies?Summary Answer: The reliability of TE biopsy compared to ICM biopsy is almost perfect, but only substantial between cleavage stage biopsy and ICM biopsy.What Is Known Already: One of the prevailing reasons for implantation failure is presumed to be chromosomal aneuploidy in human preimplantation embryos. Preimplantation genetic testing for aneuploidies (PGT-A) has been introduced into assisted reproduction in an effort to increase pregnancy rates. Increasing evidence indicates that genetic results obtained following blastomere or TEbiopsy may not accurately reflect the true genetic status of the embryo due to the presence of embryonic mosaicism, and therefore the reliability of PGT is highly controversial.Study Design, Size, Duration: This was an observational descriptive study, performed in a private infertility centre from August 2016 to January 2017.Participants/materials, Setting, Methods: The mean female age was 33.9 years, ranging from 24 to 46 years, and the mean number of biopsied embryos per couple was 2.2 (range 1-7 embryos). Blastomere biopsies had been performed at cleavage stage on Day 3 (D3) due to the turnover time of genetic testing and the inability to cryopreserve embryos in accordance with the local law governing ART. To confirm the genetic results in embryos not chosen for transfer, additional biopsies of the TE at blastocyst stage (BLASTO-TE) as well as of the ICM (BLASTO-ICM) were performed on D5. Only surplus blastocysts, which had not been selected for transfer and were not cryopreserved in accordance with the law governing ART, had been included.Main Results and the Role Of Chance: Comparison of all biopsies (D3/BLASTO-ICM/BLASTO-TE) per embryo demonstrated that 50 (59.5%) out of 84 embryos showed concordance in all three results (= full concordance). Thirty-four (40.4%) embryos had at least two discordant results between the three biopsies, regardless of whether the embryo diagnosis (aneuploid/euploid) was discordant or not, or in aneuploid embryos, whether the chromosomal patterns were inconsistent. Nine (= 10.7%) embryos had complete discordance between all three biopsies. False positive results between D3/BLASTO-TE, D3/BLASTO-ICM and BLASTO-TE/BLASTO-ICM were 26.4%/30.2% and 7.5%, respectively, while the Kappa agreement between the different approaches was 0.647, 0.553 and 0.857, respectively. Therefore the reliability of D3/BLASTO-TE, D3/BLASTO-ICM and BLASTO-TE/BLASTO-ICM can be interpreted as substantial, as moderate and as almost perfect.Limitations, Reasons For Caution: The limitation of this study is the possible bias in the concordance/discordance rate because embryos that had been selected for transfer did not undergo biopsy on D5.Wider Implications Of the Findings: The obvious discordance between the three different approaches for PGT-A underlines the limitations of genetic testing and highlights the importance of ongoing research in order to improve the accuracy of PGT-A results. Until then reproductive specialists will continue to make challenging decisions on whether to transfer or discard an embryo in light of current evidence questioning the reliability of genetic results.Study Funding/competing Interest(s): This study was supported by Igenomix. The funder provided support in the form of salary for R.C. The co-author R.C. is an employee of Igenomix. She participated in the blinded analysis of the samples; however the final data collection and statistical analysis of the results, as well as the decision to publish, was taken by B.L, I.E. and H.F. The authors B.L., I.E., A.L., A.B., A.A., N.D. and H.F. have no competing interests. The funder did not have any additional role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. The commercial affiliation of R.C. did not play any role in the study.Trial Registration Number: This study was approved by the Ethics Committee of IVIRMA Middle East Fertility Clinic, Abu Dhabi, UAE (Research Ethics Committee IVI-MEREFA009a/2017). [ABSTRACT FROM AUTHOR]

The accurate genetic screening of pre-implantation embryos currently entails the use of technically challenging and biologically invasive biopsies of the human embryos. Investigating a more conservative sampling approach has emerged as a timely and desired alternative. Circulating cell-free embryonic DNA is present in the blastocoel fluid and spent culture media of blastocysts, and this has lately been sought as an attractive source of genetic information. The genetic analysis of cell-free embryonic DNA has been reported, to be useful in evaluating the genetic constitution of embryos; thus, providing a potential alternative to conventional biopsy-derived pre-implantation genetic testing (PGT). In this review, we have summarized these non-invasive alternative applications of PGT and discussed their current limitations and future clinical implications. [ABSTRACT FROM AUTHOR]

Study Question: Does PGD increase the risk on adverse cognitive and socio-emotional development?Summary Answer: The cognitive and socio-emotional development in children born after PGD seems to be normal when compared to control groups.What Is Known Already: A limited number of studies with small sample sizes indicate that the cognitive and socio-emotional development of (pre)school-aged children born after either PGD or PGS seem to be comparable to those of children born after IVF/ICSI and to naturally conceived (NC) children from the general population.Study Design, Size, Duration: For this study we invited 72 5-year-old PGD children, 128 5-year-old IVF/ICSI children and 108 5-year-old NC children from families with a genetic disorder. All children were invited between January 2014 and July 2016.Participants/materials, Setting, Methods: In total, 51 PGD children, 52 IVF/ICSI children and 35 NC children underwent neuropsychological testing (WPPSI-III-NL and AWMA). The children's parent(s) and teachers filled in questionnaires evaluating children's executive functioning (Behaviour Rating Inventory of Executive Functions; BRIEF) and socio-emotional development (Child Behaviour Checklist; CBCL and Caregiver-Teacher Report Form; C-TRF).Main Results and the Role Of Chance: The mean full-scale intelligence quotient scores (P = 0.426) and performance on the AWMA Listening Span task (P = 0.873) and Spatial Span task (P = 0.458) were comparable between the three groups. Regarding socio-emotional development, the teachers' scores revealed more externalizing (P = 0.011) and total problem (P = 0.019) behaviour in PGD children than for IVF/ICSI children; both groups did not differ significantly from the NC children (P = 0.11). More children (13%) with an affected first-degree family member (mostly parent) were included in the PGD group than in the NC group. Scores in all groups fell within the normal population range and should be considered normal.Limitations, Reasons For Caution: The number of NC children from families with a genetic disorder was relatively small. Furthermore, the fathers' CBCL results were based on small samples.Wider Implications Of the Findings: PGD children show levels of cognitive and socio-emotional development at 5 years that are within the normal range, despite the biopsy involved in PGD and the potential extra psychological burden associated with the presence of a genetic disorder in the family.Study Funding/competing Interest(s): This study was funded by ZonMw (70-71300-98-106). None of the authors have any competing interests to declare.Trial Registration Number: NCT02149485. [ABSTRACT FROM AUTHOR]

Study Question: Can a second round of biopsy, vitrification and chromosomal testing provide a valid diagnosis where the first attempt fails?Summary Answer: The risk of inconclusive chromosomal-assessment after trophectoderm biopsy was 2.5% but a further biopsy and vitrification-warming appeared not to impair the competence of euploid blastocysts.What Is Known Already: The increasing implementation of multicell trophectoderm biopsy has significantly reduced the risk of inconclusive diagnosis after preimplantation-genetic-testing (PGT). Yet, few reports have defined the variables that influence the risk of failure or described the technical and clinical outcomes after re-biopsy.Study Design, Size, Duration: Retrospective multicenter study involving 8990 blastocyst biopsies conducted between April 2013 and September 2017 at six IVF centers but analyzed at a single genetic laboratory. A total of 206 blastocysts were successfully re-biopsied after warming and re-expansion, then re-vitrified. And 49 of these blastocysts were diagnosed euploid and used in single-embryo-transfers (SETs). Logistic regression analyses were conducted.Participants/materials, Setting, Methods: A total of 3244 PGT-for-aneuploidies (PGT-A) cycles with a freeze-all approach, vitrification and qPCR-based analysis were performed by 2687 consenting couples. DNA amplification failure (AF) or non-concurrent data resulted in inconclusive diagnoses. In case of DNA amplification, the cellularity of the biopsy was estimated according to a previously validated method. Euploid SETs were performed. Clinical pregnancy, miscarriage, live birth rates (LBR) and perinatal outcomes were monitored.Main Results and the Role Of Chance: Overall, 2.5% of trophectoderm biopsies resulted in an inconclusive diagnosis (N = 228/8990). Specifically, 2% (N = 176/8990) resulted in AF and 0.5% (N = 52/8990) in non-concurrent results. The only parameters significantly associated with inconclusive diagnoses were the IVF center and the embryo age (days) at biopsy. Among samples with successful amplification, the number of cells in the biopsy and the day of biopsy were critical to limit non-concurrent results. In total, 213 blastocysts with an inconclusive diagnosis were warmed for re-analysis and the survival rate was 96.7% (N = 206/213). The euploidy rate in blastocysts biopsied twice was 51.9% (N = 107/206) and the euploid embryos were re-vitrified. Overall, 49 euploid embryos were warmed for replacement and all survived. The LBR after SET was 38.8% (N = 19/49). No minor/major obstetrical/perinatal complication was reported.Limitations, Reasons For Caution: A single aneuploidy-testing method was adopted in this retrospective analysis. A more powered report of the clinical and obstetrical/perinatal outcomes after re-biopsied and re-vitrified blastocysts euploid SET requires a larger sample size.Wider Implications Of the Findings: It is important to re-biopsy and re-vitrify undiagnosed blastocysts since healthy live births can result from them.Study Funding/competing Interest(s): None.Trial Registration Number: None. [ABSTRACT FROM AUTHOR]

Study Question: Does preimplantation genetic testing for aneuploidy (PGT-A) by comprehensive chromosome screening (CCS) of the first and second polar body to select embryos for transfer increase the likelihood of a live birth within 1 year in advanced maternal age women aged 36-40 years planning an ICSI cycle, compared to ICSI without chromosome analysis?Summary Answer: PGT-A by CCS in the first and second polar body to select euploid embryos for transfer does not substantially increase the live birth rate in women aged 36-40 years.What Is Known Already: PGT-A has been used widely to select embryos for transfer in ICSI treatment, with the aim of improving treatment effectiveness. Whether PGT-A improves ICSI outcomes and is beneficial to the patients has remained controversial.Study Design, Size, Duration: This is a multinational, multicentre, pragmatic, randomized clinical trial with intention-to-treat analysis. Of 396 women enroled between June 2012 and December 2016, 205 were allocated to CCS of the first and second polar body (study group) as part of their ICSI treatment cycle and 191 were allocated to ICSI treatment without chromosome screening (control group). Block randomization was performed stratified for centre and age group. Participants and clinicians were blinded at the time of enrolment until the day after intervention.Participants/materials, Setting, Methods: Infertile couples in which the female partner was 36-40 years old and who were scheduled to undergo ICSI treatment were eligible. In those assigned to PGT-A, array comparative genomic hybridization (aCGH) analysis of the first and second polar bodies of the fertilized oocytes was performed using the 24sure array of Illumina. If in the first treatment cycle all oocytes were aneuploid, a second treatment with PB array CGH was offered. Participants in the control arm were planned for ICSI without PGT-A. Main exclusion criteria were three or more previous unsuccessful IVF or ICSI cycles, three or more clinical miscarriages, poor response or low ovarian reserve. The primary outcome was the cumulative live birth rate after fresh or frozen embryo transfer recorded over 1 year after the start of the intervention.Main Results and the Role Of Chance: Of the 205 participants in the chromosome screening group, 50 (24%) had a live birth with intervention within 1 year, compared to 45 of the 191 in the group without intervention (24%), a difference of 0.83% (95% CI: -7.60 to 9.18%). There were significantly fewer participants in the chromosome screening group with a transfer (relative risk (RR) = 0.81; 95% CI: 0.74-0.89) and fewer with a miscarriage (RR = 0.48; 95% CI: 0.26-0.90).Limitations, Reasons For Caution: The targeted sample size was not reached because of suboptimal recruitment; however, the included sample allowed a 90% power to detect the targeted increase. Cumulative outcome data were limited to 1 year. Only 11 patients out of 32 with exclusively aneuploid results underwent a second treatment cycle in the chromosome screening group.Wider Implications Of the Findings: The observation that the similarity in birth rates was achieved with fewer transfers, less cryopreservation and fewer miscarriages points to a clinical benefit of PGT-A, and this form of embryo selection may, therefore, be considered to minimize the number of interventions while producing comparable outcomes. Whether these benefits outweigh drawbacks such as the cost for the patient, the higher workload for the IVF lab and the potential effect on the children born after prolonged culture and/or cryopreservation remains to be shown.Study Funding/competing Interest(s): This study was funded by the European Society of Human Reproduction and Embryology. Illumina provided microarrays and other consumables necessary for aCGH testing of polar bodies. M.B.'s institution (UZBrussel) has received educational grants from IBSA, Ferring, Organon, Schering-Plough, Merck and Merck Belgium. M.B. has received consultancy and speakers' fees from Organon, Serono Symposia and Merck. G.G. has received personal fees and non-financial support from MSD, Ferring, Merck-Serono, Finox, TEVA, IBSA, Glycotope, Abbott and Gedeon-Richter as well as personal fees from VitroLife, NMC Healthcare, ReprodWissen, BioSilu and ZIVA. W.V., C.S., P.M.B., V.G., G.A., M.D., T.E.G., L.G., G.Ka., G.Ko., J.L., M.C.M., M.P., A.S., M.T., K.V., J.G. and K.S. declare no conflict of interest.Trial Registration Number: NCT01532284.Trial Registration Date: 7 February 2012.Date Of First Patient’s Enrolment: 25 June 2012. [ABSTRACT FROM AUTHOR]

Study Question: Is next generation sequencing (NGS) capable of detecting smaller sub-chromosomal rearrangements in human embryos than the manufacturer's quoted resolution suggests?Summary Answer: NGS was able to detect unbalanced chromosome segments smaller than the manufacturer's resolution.What Is Known Already: Array Comparative Genomic Hybridization (array-CGH) has been the gold standard platform used for PGD of chromosome rearrangements. NGS is a viable alternative to array-CGH for PGD of chromosome arrangements given that the manufacturer's guidelines quote a resolution of ≥20 Mb. However, as many patients carry a chromosome rearrangement <20 Mb, the detection limits of NGS warrant further investigation.Study Design, Size, Duration: This study involved a retrospective assessment of stored DNA samples from embryos that had previously been diagnosed as unbalanced by array-CGH as part of routine PGD in two separate IVF clinics between November 2013 and April 2017. SurePlex whole genome amplification (WGA) products derived from DNA extracted from an embryo biopsy sample known to carry an unbalanced form of a chromosome rearrangement were subjected to a specific NGS workflow (VeriSeq PGS). The results from the two technologies were compared for each sample.Participants/materials, Setting, Methods: WGA products from 200 embryos known to carry unbalanced rearrangements were sequenced and analysed. These embryos had been created by 75 patients known to carry a chromosome rearrangement (68 reciprocal translocations, 3 pericentric inversions, 1 paracentric inversion, 2 insertions and 1 dual reciprocal and inversion). Each sample was assessed for the size of the segmental gain/loss (Mb), copy number for each segment and chromosome, segregation pattern, the number of bins in the analysis software used and concordance with array-CGH results.Main Results and the Role Of Chance: A total of 294 unbalanced chromosome segments were assessed. NGS was capable of detecting 285/294 (97%) unbalanced segments previously identified using array-CGH. The final PGD diagnosis was concordant for 200/200 (100%) embryos. In total, 44/75 (59%) patients contained an unbalanced chromosome segment below the quoted 20 Mb manufacturer's stated resolution. Of these, 35/44 (80%) patients had segments that were able to be detected using NGS, whilst maintaining clinical outcome concordance.Limitations, Reasons For Caution: Our study subset did not include any rearrangements involving the Y chromosome. NGS has less available bins per chromosome compared to the array-CGH platform used, thus it remains possible that chromosome rearrangements predicted to be small but still detectable by array-CGH may not be feasible for testing using NGS. This should be considered when undertaking a theoretical feasibility assessment for detecting the chromosome rearrangement in question. Only one specific workflow for WGA and NGS was investigated in this study.Wider Implications Of the Findings: This study has shown that NGS is available for the detection of unbalanced chromosome rearrangements ≥10 Mb.Study Funding/competing Interest(s): Part sponsorship of the VeriSeq PGS kits used was provided by Illumina. The remainder of the kits were provided by two commercial IVF clinics. None of the authors has any conflicting interests to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Which is the prevalence of a 47,XXY karyotype in human blastocysts biopsied during preimplantation genetic testing for aneuploidies (PGT-A) cycles?Summary Answer: The prevalence of a 47,XXY karyotype amongst male blastocysts without autosomal aneuploides is ~1%.What Is Known Already: The prevalence of Klinefelter syndrome is estimated as 0.1-0.2% in male newborns. However, the KS phenotype is extremely variable and there are men with a 47,XXY karyotype and less evident signs, who may go undetected. No risk factor for the 47,XXY karyotype in products of conception has been yet clearly defined, and no data are available regarding the prevalence of this karyotype among human preimplantation embryos.Study Design, Size, Duration: This multicentre cohort study involved 7549 blastocysts obtained during 2826 PGT-A cycles performed between April 2013 and September 2017 at six IVF clinics in Italy.Participants/materials, Setting, Methods: During 2826 PGT-A cycles, 7549 blastocysts underwent trophectoderm biopsy and quantitative-PCR-based comprehensive chromosomal testing to predict the karyotype of the corresponding embryos. The results were also presented according to ranges of maternal and paternal age at oocyte retrieval as well as sperm factor and blastocyst quality. Univariate and multivariate logistic regression analyses were conducted to investigate the correlation of possible confounding factors with the prevalence of 47,XXY karyotype.Main Results, the Role Of Chance: Overall, 62 blastocysts were 47,XXY or had an XXY karyotype associated with autosomal aneuploidies. After exclusion of the latter, the prevalence of a 47,XXY karyotype among male blastocysts without autosomal aneuploidies was 0.9% (n = 17/1794). A significant correlation was only found for maternal age and blastocyst quality (OR: 1.20, 95% CI: 1.01-1.42; P = 0.04 and OR: 1.6, 95% CI: 1.13-2.45; P = 0.01).Limitations, Reasons For Caution: These retrospective data have been produced based on a population of infertile couples undergoing IVF and PGT-A, and the women were mainly of advanced maternal age. Moreover, the qPCR technique is validated only to detect full-chromosome uniform aneuploidies in trophectoderm biopsies.Wider Implications Of the Findings: The 0.9% prevalence of the 47,XXY karyotype among male blastocysts, when compared with the 0.1-0.2% prevalence reported in the prenatal and postnatal periods, suggests four possible scenarios that require further investigations: (i) the latter prevalence is underestimated; (ii) 47,XXY blastocysts result in a lower implantation rate than euploid embryos (estimated to be ≈50%); (iii) 47,XXY blastocysts result in a higher early miscarriage rate than euploid embryos (estimated to be ≈10%); or (iv) infertile patients of advanced maternal age and referred to IVF/PGT-A produce a higher rate of 47,XXY blastocysts.Study Funding/competing Interest(s): None.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: To what extent does a trophectoderm (TE) biopsy reliably reflect the chromosomal constitution of the inner cell mass (ICM) in human blastocysts?Summary Answer: Concordance between TE and ICM was established in 62.1% of the embryos analysed.What Is Known Already: Next generation sequencing (NGS) platforms have recently been optimised for preimplantation genetic testing for aneuploidies (PGT-A). However, higher sensitivity has led to an increase in reports of chromosomal mosaicism within a single TE biopsy. This has raised substantial controversy surrounding the prevalence of mosaicism in human blastocysts and the clinical implications of heterogeneity between the TE and ICM.Study Design, Size, Duration: To define the distribution and rate of mosaicism in human blastocysts, we assessed chromosomal profiles of the ICM and multiple TE portions obtained from the same embryo. We evaluated donated embryos with an unknown chromosomal profile (n = 34), as well as PGT-A blastocysts, previously diagnosed as abnormal or mosaic (n = 24). Our intra-embryo comparison included a total of 232 samples, obtained from 58 embryos.Participants/materials, Setting, Methods: Four embryo samples, including the ICM and three distinct TE portions, were acquired from good quality blastocysts by micromanipulation. Whole genome amplification (WGA), followed by NGS was performed on all embryo segments. Profiles were compared between samples from the same embryo, while the results from pretested blastocysts were further correlated to the original report. The embryos investigated in our untested group were obtained from good prognosis patients (n = 25), with maternal age ranging from 23 to 39 years. For the pretested embryo group, maternal age ranged from 23 to 40 years (n = 18).Main Results and the Role Of Chance: We uncover chromosomal mosaicism, involving both numerical and structural aberrations, in up to 37.9% of the blastocysts analysed. Within the untested group, the overall concordance between the ICM and all TE portions was 55.9%. A normal ICM was detected in 20.6% of blastocysts for which at least one TE portion showed a chromosomal aberration. Conversely, 17.6% of embryos presented with mosaic or uniform abnormalities within the ICM, while showing normal or mosaic TE profiles. For the pretested blastocysts, the overall concordance between the ICM and all TE samples was 70.8%. However, 50% of embryos previously diagnosed with mosaicism did not confirm the original diagnosis. Notably, 31.3% of embryos with a mosaic aberration reported in the original TE biopsy, revealed a euploid profile in the ICM and all three TE samples. Taken together, concordance between the ICM and all TE portions was established in 62.1% of blastocysts, across both embryo groups. Finally, we could not observe a significant effect of age on embryo mosaicism (P = 0.101 untested group; P = 0.7309 pretested group). Similarly, ICM and TE quality were not found to affect the occurrence of chromosomal mosaicism (P = 0.718 and P = 0.462 untested group; P = 1.000 and P = 0.2885 pretested group).Large Scale Data: All data that support the findings of this study are available online in Vivar (http://cmgg.be/vivar) upon request.Limitations, Reasons For Caution: Evaluating biological variation in some instances remains challenging. The technological limitations of sampling mitotic errors that lead to mosaicism, as well as WGA artefacts, warrant careful interpretation.Wider Implications Of the Findings: Our results highlight the complex nature of genetic (in)stability during early ontogenesis and indicate that blastocysts harbour a higher rate of chromosomal mosaicism than may have been anticipated. Moreover, our findings reveal an overall high diagnostic sensitivity and relatively low specificity in the context of PGT-A. This suggests that a considerable proportion of embryos are potentially being classified as clinically unsuitable. Ultimately, more precise quantification will benefit the clinical management of embryo mosaicism.Study Funding/competing Interest(s): M.P. is supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF01D08114). J.T. and L.D. are supported by the agency for innovation through science (131673, 141441). B.H. and this research are supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF15/GOA/011). The authors declare no competing interests.Trial Registration Number: Not applicable. [ABSTRACT FROM AUTHOR]

Study Question: Is there a benefit to assessing ploidy in delayed embryos reaching the morula stage on Day 6 of development?Summary Answer: Day-6 morulae should be considered for biopsy in women <40 years old undergoing preimplantation genetic testing for aneuploidy (PGT-A) because they are associated with acceptable, albeit reduced, euploidy and implantation rates (IRs).What Is Known Already: Embryo development and morphology have been shown to correlate with aneuploidy and pregnancy rates. During PGT-A cycles, embryos are biopsied if they reach the blastocyst stage by Day 5 or 6, whereas slow-developing embryos are typically deselected and discarded. Determining the viability of slow-developing embryos is particularly relevant for women undergoing PGT-A who have diminished ovarian reserve and a relatively low blastocyst yield.Study Design, Size, Duration: This is a retrospective cohort study that was performed at an academic medical center. Patients who underwent IVF with PGT-A were reviewed for inclusion.Participants/materials, Setting, Methods: A total of 1615 cycles were reviewed. All cycles which involved a biopsy of a cavitating or compacted morula on Day 6 were included (n = 763). PGT-A was performed using array comparative genomic hybridization. The aneuploidy and implantation of morulae were compared to those of blastocysts originating from the same couples.Main Results and the Role Of Chance: The study included 763 cycles in which 1260 morulae and 3014 blastocysts were biopsied. Women were divided into four age groups (<35, 35-37, 38-39 and ≥40 years): the prevalence of aneuploidy was consistently lower among blastocysts (40.3, 50.8, 56 and 78.3%, respectively) than among compacted morulae (68.7, 75.5, 88.9 and 98.1%, respectively) and cavitating morulae (57, 66.4, 81 and 91.6%, respectively) throughout the different age groups (P < 0.001). Of note, the majority of compacted morulae (98.1%) and cavitating morulae (91.6%) were aneuploid in women aged ≥40 years. Compacted and cavitating morulae had significantly higher rates of complex aneuploidy, which involves ≥3 chromosomes, compared with blastocysts (P < 0.001). Furthermore, euploid morulae were associated with a significantly lower IR (28.2 versus 54.6%; P = 0.002) and live birth rate (23.1 versus 55.0%; P = 0.001) compared to euploid blastocysts.Limitations Reasons For Caution: This study confirms that Day-6 morulae should not be discarded in young women undergoing PGT-A. However, a potential drawback of biopsying embryos at the morula stage is the inability to distinguish between inner cell mass and trophectoderm cell origin. The sample size of euploid morula transfer cycles in this study was limited. Thus, a larger cohort would be beneficial to validate the reassuring live birth and spontaneous abortion rates reported here. Furthermore, the reproducibility of our findings should be determined at different centers.Wider Implications Of the Findings: Although Day-6 morulae are associated with higher aneuploidy rates and lower IRs compared to blastocysts, they still yielded successful pregnancies. Therefore, testing Day-6 morulae should be considered, especially for women <40 years old who are undergoing PGT-A with a small cohort of available blastocysts for biopsy.Study Funding/competing Interest(s): The authors have nothing to disclose. They received no specific funding for this work.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Do specific factors affect the segregation patterns of a quadrivalent structure and can the quadrivalent affect genome stability during meiosis?Summary Answer: Meiotic segregation patterns can be affected by the carrier's gender and age, location of breakpoints and chromosome type, and the quadrivalent structure can increase genome instability during meiosis.What Is Known Already: Carriers of reciprocal translocations have an increased genetic reproductive risk owing to the complex segregation patterns of a quadrivalent structure. However, the results of previous studies on the factors that affect segregation patterns seem to be contradictory, and the effect of a quadrivalent on genome stability during meiosis is unknown.Study Design, Size, Duration: We designed a retrospective study to analyze the segregation patterns of 24 chromosomes from reciprocal translocation and non-translocation patients. Data for 356 reciprocal translocation carriers and 53 patients with the risk to transmit monogenic inherited disorders (RTMIDs) undergoing PGD-single nucleotide polymorphism array analysis were collected. The study was performed between March 2014 and July 2017.Participants/materials, Setting, Methods: Segregation patterns of a quadrivalent in 1842 blastocysts from 466 assisted reproduction cycles of reciprocal translocation carriers were analyzed according to the location of chromosome breakpoints, the carrier's gender and age, and chromosome type. In addition, to analyze the effect of quadrivalent structure on genome stability, segregation products of chromosomes which are not involved in the translocation from translocation carriers were compared with those of 23 pairs of chromosomes in 318 blastocysts from 72 assisted reproduction cycles of patients with RTMIDs.Main Results and the Role Of Chance: The percentage of adjacent-2 products with severe asymmetric quadrivalent was significantly higher than those with mild asymmetric quadrivalent (P = 0.020) while, in contrast, the incidence of 4:0/others was lower (P = 0.030). The frequencies of adjacent-1, adjacent-2 and 3:1 products differed between male and female carriers (P < 0.001, P = 0.015 and P = 0.001, respectively), and also for adjacent-1 and 4:0/others products in young versus older carriers (P = 0.04 and P = 0.002, respectively). In addition, adjacent-1 products of a quadrivalent with an acrocentric chromosome were significantly higher than those of a quadrivalent without an acrocentric chromosome (P = 0.001). Moreover, a quadrivalent could significantly increase the frequencies of abnormal chromosomes compared to patients with RTMIDs (P = 0.048, odds ratio (OR) = 1.43, 95% CI = 1.01-2.43), especially for the male carriers (P = 0.018, OR = 1.58, 95% CI = 1.08-2.25). In contrast, for older carriers, no difference was found in both aneuploidy and segmental anomalies compared to patients with RTMIDs.Limitations, Reasons For Caution: The study contained appropriate controls, yet the analysis was limited by a small number of control patients and embryos.Wider Implications Of the Findings: Until now, there had been no definite report about the effect of quadrivalents on genome stability in reciprocal translocation carriers compared with control samples, and in the present study the large sample size ensured a detailed analysis of factors with a possible impact on segregation patterns. These data provide a better insight into the meiotic mechanisms involved in non-disjunction events in gametes from reciprocal translocation carriers. In addition, our results will help to provide each reciprocal translocation carrier couple undergoing PGD with more appropriate genetic counseling and a better understanding of the large numbers of abnormal embryos with chromosome aneuploidy.Study Funding/competing Interest(s): The research was supported by the Research Funding of Shanghai Ji Ai Genetics & IVF Institute and the authors declare a lack of competing interests in this study. [ABSTRACT FROM AUTHOR]

Study Question: Has PGD-HLA been successful relative to diagnostic and clinical efficacy?Summary Answer: The diagnostic efficacy of PGD-HLA protocols was found lower in this study in comparison to published PGD-HLA protocols and to that reported for general PGD by ESHRE (78.5 vs 94.1% and vs 92.6%, respectively), while the clinical efficacy has proven very difficult to assess due to inadequate follow-up of both the ART/PGD and HSCT procedure outcomes.What Is Known Already: The first clinical cases for PGD-HLA were reported in 2001. It is now a well-established procedure, with an increasing number of cycles performed every year. However, PGD-HLA is still offered by relatively few PGD centres, the currently available data is fragmented and most reports on PGD-HLA applications are limited in number and scope. Published systematic details on methodology, diagnostic results, overall ART success and haematopoietic stem cell transplantation (HSCT) outcomes are limited, precluding an evaluation of the true clinical utility of PGD-HLA cycles.Study Design, Size, Duration: This retrospective multi-centre cohort study aimed to investigate the diagnostic and clinical efficacy of the PGD-HLA procedure and the aspects of PGD-HLA cycles influencing positive outcomes: birth of genetically suitable donor-baby (or babies) and HSCT. In April 2014, 32 PGD centres (Consortium members and non-members) with published/known PGD-HLA activity were invited to participate. Between February and September 2015, 14 centres submitted their data, through a custom-designed secure database, with unique login access for each centre. Data parameters covered all aspects of PGD-HLA cycles (ART, embryology and genetic diagnosis), donor-babies born and HSCT.Participants/materials, Setting, Methods: From 716 cycles submitted by 14 centres (performed between August 2001 and September 2015), the quality evaluation excluded 12 cycles, leaving 704, from 364 couples. The online database, based on REDCap, a free, secure, web-based data-capture application, was customized by Centre for Clinical Epidemiology and Outcomes Research (CLEO), Athens. Continuous variables are presented using mean, standard deviation, median and interquartile range, and categorical variables are presented as absolute and relative frequencies.Main Results and the Role Of Chance: The data included 704 HLA-PGD cycles. Mean maternal age was 33.5 years. Most couples (81.3%) requested HLA-typing with concurrent exclusion of a single monogenic disease (58.6% for beta-thalassaemia). In 92.5% couples, both partners were fertile, with an average 1.93 HLA-PGD cycles/couple. Overall, 9751 oocytes were retrieved (13.9/cycle) and 5532 embryos were analysed (7.9/cycle). Most cycles involved fresh oocytes (94.9%) and Day 3 embryo biopsy (85.3%). In 97.5% of cycles, the genotyping method involved PCR only. Of 4343 embryos diagnosed (78.5% of analysed embryos), 677 were genetically suitable (15.4% of those analysed for HLA alone, 11.6% of those analysed for HLA with exclusion of monogenic disease). Of the 364 couples, 56.6% achieved an embryo transfer (ET) and 598 embryos were transferred in 382 cycles, leading to 164 HCG-positive pregnancies (pregnancy rate/ET 41.3%, pregnancy rate/initiated cycle 23.3%) and 136 babies born (live birth rate/ET 34.3%, live birth rate/initiated cycle 19.3%) to 113 couples. Data analysis identified the following limitations to the overall success of the HLA-PGD procedure: the age of the mother undergoing the treatment cycle, the number of oocytes collected per cycle and genetic chance. HSCT was reported for 57 cases, of which 64.9% involved combined umbilical cord-blood and bone marrow transplantation from the HLA-identical sibling donor; 77.3% of transplants reported no complications.Limitations Reasons For Caution: The findings of the study may be limited as not all PGD centres with PGD-HLA experience participated. Reporting bias on completion of the online database may be another potential limitation. Furthermore, the study is based on retrospective data collection from centres with variable practices and strategies for ART, embryology and genetic diagnosis.Wider Implications Of the Findings: This is the first multi-centre study evaluating the clinical utility of PGD-HLA, indicating variations in practice and outcomes throughout 15 years and between centres. The study highlights parameters important for positive outcomes and provides important information for both scientists and couples interested in initiating a cycle. Above all, the study underlines the need for better collaboration between all specialists involved in the ART-PGD/HLA procedure, as well as the need for comprehensive and prospective long-term data collection, and encourages all specialists to aim to properly evaluate and follow-up all procedures, with the ultimate aim to promote best practice and encourage patient informed decision making.Study Funding/competing Interest(s): The study wishes to acknowledge ESHRE for funding the customization of the REDCap database. There are no competing interests.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Does Day-3 cleavage-stage PGS affect neurodevelopment of 9-year-old IVF offspring?Summary Answer: We did not find evidence of adverse consequences of Day-3 cleavage-stage PGS on neurodevelopment of 9-year-old IVF offspring, although children born after IVF with or without PGS often had a non-optimal neurological condition.What Is Known Already: Knowledge on long-term sequelae for development and health of children born following PGS is lacking. This is striking as evidence accumulates that IVF itself is associated with increased risk for impaired health and development in the offspring.Study Design Size, Duration: This prospective, assessor-blinded, multicentre, follow-up study evaluated development and health of 9-year-old IVF children born to women who were randomly assigned to IVF with PGS (PGS group) or without PGS (control group). The follow-up examination at 9 years took place between March 2014 and May 2016.Participants/materials, Setting, Methods: In total, 408 women were included and randomly assigned to IVF with or without Day-3 cleavage-stage PGS. This resulted in 52 ongoing pregnancies in the PGS group and 74 in the control group. In the PGS group, 59 children were born alive; in the control group, 85 children were born alive. At the age of 9 years, 43 children born after PGS and 56 control children participated in the study. Our primary outcome was the neurological optimality score, a sensitive measure of neurological condition assessed with a standardized, age-specific test (Touwen test). Secondary outcomes were adverse neurological condition (neurologically abnormal and the complex form of minor neurological dysfunction), cognitive development (intelligence quotient and specific domains), behaviour (parental and teacher's questionnaires), blood pressure and anthropometrics.Main Results and the Role Of Chance: Neurodevelopmental outcome of PGS children did not differ from that of controls; the neurological optimality scores (mean values [(95% CI]: PGS children 51.5 [49.3; 53.7], control children 53.1 [50.5; 55.7]) were not significantly different. The prevalences of adverse neurological outcome (in all but one child implying the presence of the complex form of minor neurological dysfunction) did not differ between the groups (PGS group 17/43 [40%], control group 19/56 [34%]), although the prevalence of complex minor neurological dysfunction in both groups was rather high. Also intelligence quotient scores of the two groups were not significantly different (PGS group 114 [108; 120]); control group 117 [109; 125]), and the behaviour, blood pressure and anthropometrics of both groups did not differ. Mean blood pressures of both groups were above the 60th percentile.Limitations Reasons For Caution: The power analysis of the study was not based on the number of children needed for the follow-up study, but on the number of women who were needed to detect an increase in ongoing pregnancy rates after PGS. In addition, our study evaluated embryo biopsy in the form of PGS at cleavage stage (Day-3 embryo biopsy), while currently PGS at blastocyst stage (Day-5 embryo biopsy) is recommended and increasingly being used.Wider Implications Of the Findings: Our findings indicate that PGS in cleavage stage embryos is not associated with adverse effects on neurological, cognitive and behavioural development, blood pressure and anthropometrics of offspring at 9 years. This is a reassuring finding as embryo biopsy in the forms of PGS and PGD is increasingly applied. However, both groups of IVF offspring showed high prevalences of the clinically relevant form of minor neurological dysfunction, which is a point of concern for the IVF community. In addition, our study confirms findings of others that IVF offspring may be at risk of an unfavourable cardiovascular outcome. These findings are alarming and highlight the importance of research on the underlying mechanisms of unfavourable neurodevelopmental and cardiovascular outcomes in IVF offspring.Study Funding/competing Interest(s): The randomized controlled trial was financially supported by the Organization for Health Research and Development (ZonMw), The Netherlands (Grant number 945-03-013). The follow-up was financially supported by the University Medical Center Groningen (Grant number: 754510), the Cornelia Foundation, and the graduate schools BCN and Share, Groningen, The Netherlands. The sponsors of the study had no role in study design, data collection, data analysis, data interpretation or writing of the report. There are no conflicts of interest.Trial Registration Number: ISRCTN76355836. [ABSTRACT FROM AUTHOR]

Study Question: What is the optimal timing for blastomere biopsy during the 8-cell stage, at which embryos will have the best implantation potential?Summary Answer: Fast-cleaving embryos that are biopsied during the last quarter (Q4) of the 8-cell stage and are less affected by the biopsy procedure, and their implantation potential is better than that of embryos biopsied earlier during the 8-cell stage (Q1-Q3).What Is Known Already: Blastomer biopsy from cleavage-stage embryos is usually performed on the morning of Day 3 when the embryos are at the 6- to 8-cell stage and is still the preferred biopsy method for preimplantation genetic diagnosis (PGD) for monogentic disorders or chromosomal translocations. Human embryos usually remain at the 8-cell stage for a relatively long 'arrest phase' in which cells grow, duplicate their DNA and synthesize various proteins in preparation for the subsequent division.Study Design, Size, Duration: This is a retrospective cohort study. The study group (195 embryos) included all 8-cell stage embryos that underwent blastomere biopsy for PGD for monogenetic disorders and chromosomal translocations in our unit between 2012-2014 and cultured in the EmbryoScope until transfer. The control group (115 embryos) included all embryos that underwent intracytoplasmic sperm injection without a biopsy during the same period.Participants/materials, Setting, Methods: The 8-cell stage was divided into four quarters: the first 5 h post-t8 (Q1), 5-10 h post-t8 (Q2), 10-15 h post-t8 (Q3) and at 15-20 h post-t8 (Q4). Non-biopsied control embryos were divided into four equivalent quarters. Embryos were evaluated for timing of developmental events following biopsy including timing of first cleavge after biopsy, timing of comapction (tM) and start of blastulation (tSB). Timing of these events were compared between PGD and control embryos, as well as with 56 PGD implanted embryos with Known Implantation Data (PGD-KID-positive embryos).Main Results and the Role Of Chance: Embryos that were biopsied during Q3 (10-15 h from entry into 8-cell stage) were delayed in all three subsequent developmental events, including first cleavage after biopsy, compaction and start of blastulation. In contrast, these events occurred exactly at the same time as in the control group, in embryos that were biopsied during Q1, Q2 or Q4 of the 8-cell stage. The results show also that embryos that were biopsied during Q1, Q2 or Q3 of the 8-cell stage demonstrated a significant delay from the biopsied implanted embryos already in t8 as well as in tM and tSB. However, embryos that were biopsied during Q4 demonstrated dynamics similar to those of the biopsied implanted embryos in t8 and tM, and a delay was noticed only in the last stage of tSB.Limitations, Reasons For Caution: This is a retrospective study that is limited to the timing of biopsy that is routinely performed in the IVF lab. A prospective study in which biopsy will be performed at a desired timing is needed in order to differ between the effect of biopsy itself and the cleavage rate of the embryo.Wider Implications Of the Findings: Our findings showed that blastomere biopsy can be less harmful to further development if it is carried out during a critical period of embryonic growth, i.e during Q4 of the 8-cell stage. They also demonstrated the added value of time-lapse microscopy for determining the optimal timing for blastomere biopsy.Study Funding/competing Interest(s): The study was funded by the routine budget of our IVF unit.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: What is the incidence, origin and clinical significance of segmental aneuploidy in human oocytes and preimplantation embryos?Summary Answer: Segmental aneuploidy occurs at a considerable frequency in preimplantation embryos with a majority being mitotic in origin.What Is Known Already: In recent years, accurate techniques for the detection of aneuploidy in single cells have been developed. Research using such methods has confirmed that aneuploidy is a common feature of human oocytes and preimplantation embryos. However, thus far research has mainly focused on loss or gain of whole chromosomes. We utilized sensitive molecular methods to study another important form of cytogenetic abnormality at the earliest stages of human development, namely segmental aneuploidy.Study Design, Size, Duration: Chromosomal copy number data was obtained from oocytes and embryos of 635 IVF patients, who requested chromosome screening for various reasons, most commonly for advanced maternal age or previously unsuccessful IVF treatments. A total of 3541 samples comprising of 452 human oocytes, 1762 cleavage stage and 1327 blastocyst stage embryos were investigated in the present study.Participants/materials, Setting, Methods: Whole genome amplification (Sureplex, Illumina) was performed on cells biopsied from oocytes and embryos of IVF patients who requested chromosome screening. The samples were subsequently processed and analyzed for their chromosome complement using microarray comparative genomic hybridization (aCGH), (Illumina, Cambridge, UK).Main Results and the Role Of Chance: Segmental abnormalities, involving loss or gain of chromosomal fragments in excess of 15 Mb, were found to occur at a high frequency. The incidence of such abnormalities was 10.4% in oocytes, but this increased dramatically during the first 3 days of embryonic development (24.3%), before starting to decline as embryos reached the final (blastocyst) stage of preimplantation development (15.6%). While some segmental errors were clearly of meiotic origin, most appear to arise during the first few mitoses following fertilization. The reduction in frequency at the blastocyst stage suggests that many cells/embryos affected by segmental abnormalities are eliminated (e.g. via arrest of the affected embryos or apoptosis of abnormal cells). Interestingly, sites of chromosome breakage associated with segmental aneuploidy were not entirely random but tended to occur within distinct chromosomal regions. Some of the identified hotspots correspond to known fragile sites while others may be considered novel and may be specific to gametogenesis and/or embryogenesis.Limitations Reasons For Caution: The cytogenetic analysis was performed on biopsies of embryos, which might not be representative of the true incidence of mosaic segmental aneuploidy of the entire embryo.Wider Implications Of the Findings: The findings of this study are valuable for understanding the origin of subchromosomal duplications and deletions, a clinically important class of abnormalities that are a common cause of congenital abnormalities and miscarriage. Furthermore, the results provide additional evidence that control of the cell cycle is more relaxed during the first few mitotic divisions following fertilization, permitting DNA double-strand breaks to occur and persist through cell division. The data are also of great relevance for preimplantation genetic testing, where the detection of segmental aneuploidy is currently considered problematic for embryo diagnosis and patient counseling.Study Funding/competing Interest(s): This study was supported by institutional funding (Reprogenetics UK). Additionally, DW is supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. DB was supported by the University of Oxford's Clarendon funding. No conflict of interests to declare. [ABSTRACT FROM AUTHOR]

Study Question: Does high gonadotropin dosage affect euploidy and pregnancy rates in PGS cycles with single embryo transfer?Summary Answer: High gonadotropin dosage does NOT affect euploidy and pregnancy rates in PGS cycles with single embryo transfer.What Is Known Already: PGS has been proven to be the most effective and reliable method for embryo selection in IVF cycles. Euploidy and blastulation rates decrease significantly with advancing maternal age. In order to recruit an adequate number of follicles, the average dosage of gonadotropins administered during controlled ovarian stimulation in IVF cycles often increases significantly with advancing maternal age.Study Design, Size, and Duration: A retrospective study of SNP (Single Nucleotide Polymorphism) PGS outcome data from blastocysts biopsied on day 5 or day 6 was conducted to identify differences in euploidy and clinical pregnancy rates. Seven hundred and ninety four cycles of IVF treatment with PGS between January 2013 and January 2017 followed by 651 frozen embryo transfers were included in the study (506 patients, maternal age (y.o.) - 37.2 ± 4.31).Participants/materials, Setting, Methods: A total of 4034 embryos were analyzed (5.1 ± 3.76 per case) for euploidy status. All embryos were vitrified after biopsy, and selected embryos were subsequently thawed for a hormone replacement frozen embryo transfer cycle. All cycles were analyzed by total gonadotropin dosage (<3000 IU, 3000-5000 IU and >5000 IU), by number of eggs retrieved (1-5, 5-10, 10-15 and >15 eggs) and patient's age (<35, 35-37, 38-40 and ≥41 y.o.). Clinical pregnancy rate was defined by the presence of a fetal heartbeat at 6-7 weeks of gestation.Main Results and the Role Of Chance: Euploidy rates within the same age group were not statistically different regardless of the total dosage of gonadotropins used or the number of eggs retrieved. In the youngest group of patients (<35 y.o. - 187 IVF cycles) euploidy rates ranged from 62.3% (<3000 IU were used in the IVF cycle) to 67.5% (>5000 IU were used in the IVF cycle) (OR = 0.862, 95% CI 0.687-1.082, P = 0.2) and from 69.5% (1-5 eggs retrieved) to 60.0% (>15 eggs retrieved) (OR = 0.658, 95% CI 0.405-1.071, P = 0.09). Similar data were obtained in the oldest group of patients (≥41 y.o. - 189 IVF cycles): euploidy rates ranged from 30.7 to 26.4% (OR = 0.811, 95% CI 0.452-1.454, P = 0.481) when analyzed by total dosage of gonadotropins used in the IVF cycle and from 40.0 to 30.7% (OR = 0.531, 95% CI 0.204-1.384, P = 0.19), when assessed by the total number of eggs retrieved. Ongoing pregnancy rates were similar, not only within particular age groups, but also between different age groups regardless of the total dosage of gonadotropins used: ranging from to 63.6% (<3000 IU, < 35 y.o.) to 54.8% (>5000 IU, ≥41 y.o) (OR = 0.696, 95% CI 0.310-1.565, P = 0.38).Limitations, Reasons For Caution: Retrospective study and heterogeneity of patients included.Wider Implications Of the Findings: These data are reassuring for the common practice of increasing gonadotropin dosages in PGS cycles, particularly in older woman.Study Funding/competing Interest(s): No formal funding has been received for this study.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Conventionally, the search for carrier status was based on ethnicity and/or family history and targeted to a restricted number of genetic conditions and mutations. This is now being replaced by extended panels testing for hundreds of genetic disorders with a broad range of phenotypes, in what is called 'expanded carrier screening'. While the ultimate aim of these panels is to increase the reproductive autonomy of the individuals and couples by providing preconception knowledge that could lead to the broadest range of available options, including PGD, we argue that: (i) Given the number and heterogeneity of the conditions included in panels, it cannot be guaranteed that a couple who tests positive for one of those conditions will be eligible for PGD; patients should be informed of this potential limitation before undertaking screening. (ii) Family history is typically lacking in couples identified through panels as being at high-risk for certain disorders. This should promote a reflection on the inclusion of personal experience with a condition as a consideration for PGD in disorders with incomplete penetrance or for which treatment options are available. (iii) With the advent of next-generation sequencing panels, cases of couples in which one member carries a disease-causing variant and the other has a variant of uncertain significance found in the same gene are likely to become more common and need to be discussed from the PGD perspective. (iv) With comprehensive panels where healthy individuals are likely to be identified as carriers for several conditions, testing of carrier status for embryos and prioritisation of the embryos to transfer needs reassessing. We believe that these points should be included in the discussion on expanded carrier screening and that all stakeholders, patients included, must be aware of the challenges and limitations that may come with a positive result. [ABSTRACT FROM AUTHOR]

Study Question: How does the data collection XIV-XV of the European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium compare with the cumulative data for data collections I-XIII?Summary Answer: The 14th and 15th retrospective collection represents valuable data on PGD/PGS cycles, pregnancies and children: the main trend observed is the increased application of array technology at the cost of FISH testing in PGS cycles and in PGD cycles for chromosomal abnormalities.What Is Known Already: Since 1999, the PGD Consortium has collected, analysed and published 13 previous data sets and an overview of the first 10 years of data collections.Study Design, Size, Duration: Data were collected from each participating centre using a FileMaker Pro database (versions 5-12). Separate predesigned FileMaker Pro files were used for the cycles, pregnancies and baby records. The study documented cycles performed during the calendar years 2011 and 2012 and follow-up of the pregnancies and babies born which resulted from these cycles (until October 2013).Participants/materials, Settings, Method: Data were submitted by 71 centres (full PGD Consortium members). Records with incomplete or inconsistent data were excluded from the calculations. Corrections, calculations and tables were made by expert co-authors.Main Results and the Role Of Chance: For data collection XIV-XV, 71 centres reported data for 11 637 cycles with oocyte retrieval (OR), along with details of the follow-up on 2147 pregnancies and 1755 babies born. A total of 1953 cycles to OR were reported for chromosomal abnormalities, 144 cycles to OR for sexing for X-linked diseases, 3445 cycles to OR for monogenic diseases, 6095 cycles to OR for PGS and 38 cycles to OR for social sexing. From 2010 until 2012, the use of arrays for genetic testing increased from 4% to 20% in PGS and from 6% to 13% in PGD cycles for chromosomal abnormalities; the uptake of biopsy at the blastocyst stage (from <1% up to 7%) was only observed in cycles for structural chromosomal abnormalities, alongside the application of array comparative genomic hybridization.Limitations, Reasons For Caution: The findings apply to the 71 participating centres and may not represent worldwide trends in PGD.Wider Implications Of the Findings: The annual data collections provide an important resource for data mining and for following trends in PGD/PGS practice.Study Funding/competing Interest(s): None. [ABSTRACT FROM AUTHOR]

Study Question: What is the association between the ovarian response and the number of CGG repeats among full mutation and premutation carriers of fragile X (FMR1), undergoing controlled ovarian hyperstimulation (COH) for PGD?Summary Answer: Ovarian response was normal in full mutation patients but decreased in premutation carriers, although the number of repeats was not statistically significantly associated with the number of oocytes retrieved.What Is Known Already: There is inconsistent data in the literature regarding ovarian response in FMR1 carriers. Studies exploring the ovarian response of full mutation patients are lacking.Study Design, Size, Duration: Retrospective study, a university affiliated tertiary hospital, IVF unit, PGD referral center.Participants/materials, Setting, Methods: We examined the medical records of all women undergoing fresh IVF-PGD cycles due to fragile X. Data recorded included demography, duration of stimulation, amount of gonadotropins administered, number of dominant follicles, maximal E2 levels and number of oocytes retrieved. Data were analyzed using univariate and multivariate mixed models. P-values <0.05 were considered significant. Data were collected from the medical records of 21 patients with a full mutation on the FMR1 gene and 51 premutation carriers. Overall 309 fresh cycles were analyzed.Main Results and the Role Of Chance: Premutation carriers displayed reduced ovarian response, as demonstrated by fewer oocytes retrieved. In contrast, full mutation patients had a normal response. Comparison of premutation carriers and full mutation patients showed: mean oocytes retrieved per cycle (8.4 ± 1.1 versus 14.1 ± 1.7, P = 0.005), lower levels of estradiol (E2; 1756 ± 177, versus 2928 ± 263, P = 0.0004), respectively. There was no significant difference between premutation carriers and full mutation patients in regard to fertilization rate, cleavage rate or biopsy rate. No correlation was found between the number of repeats in the premutation carriers and the number of oocytes retrieved or E2 levels. Age and the type of protocol were the only factors found to be in correlation with the number of the oocyte retrieved (P = 0.037, and P = 0.003, respectively) among the premutation carriers. Similarly, no association was found between the number of repeats and the fertilization rate, cleavage rate or biopsy rate among premutation carriers.Limitations, Reasons For Caution: We had a relatively low number of premutation carriers with >100 repeats, which made it challenging to draw a firm conclusions from this group.Wider Implications Of the Findings: Physicians must address the increased risk for reduced ovarian response and  primary ovarian insufficiency (POI) among carriers and consider surveillance of ovarian reserve markers. The last, might expedite family plans completion or fertility preservation.Study Funding/competing Interest(s): None. [ABSTRACT FROM AUTHOR]

Study question: Do external factors affect euploidy in egg donor cycles?Summary answer: The study demonstrates that during human assisted reproduction, embryonic chromosome abnormalities may be partly iatrogenic.What is known already: Chromosome abnormalities have been linked in the past to culture conditions such as temperature and Ph variations, as well as hormonal stimulation. Those reports were performed with older screening techniques (FISH), or ART methods no longer in use, and the subjects studied were not a homogeneous group.Study design, size, duration: A total of 1645 donor oocyte cycles and 13 282 blastocyst biopsies from 42 fertility clinics were included in this retrospective cohort study. Samples from donor cycles with PGS attempted between September 2011 and July 2015 were included.Participants/materials, setting, methods: PGS cycles from multiple fertility clinics referred to Reprogenetics (Livingston, NJ) that involved only oocyte donation were included in this study. Testing was performed by array comparative genomic hybridization (aCGH). Ploidy data were analyzed using Generalized Linear Mixed Models with logistic regression using a logit link function considering a number of variables that represent fixed and random effects.Main results and the role of chance: Euploidy rate was associated with the referring center and independent of almost all the parameters examined except donor age and testing technology. Average euploidy rate per center ranged from 39.5 to 82.5%. The mean expected rate of euploidy was 68.4%, but there are variations in this rate associated with the center effect.Limitations, reasons for caution: Data set does not include details of the donor selection process, donor race or ethnic origin, ovarian reserve or ovarian responsiveness. Due to the retrospective nature of the study, associations are apparent, however, causality cannot be established. Discrepancies in regard to completeness and homogeneity of data exist due to data collection from over 40 different clinics.Wider implications of the findings: This is the first study to show a strong association between center-specific ART treatment practices and the incidence of chromosome abnormality in human embryos, although the meiotic or mitotic origin of these abnormalities could not be determined using these technologies. Given the widespread applications of ART in both subfertile and fertile populations, our findings should be of interest to the medical community in general as well as the ART community in particular.Study funding/competing interest(s): No external funds were used for this study. S. Munne is a founding principle of Reprogenetics/current employee of Cooper Genomics. M Alikani's spouse is a founding principle of Reprogenetics/current consultant for Cooper Genomics. The remaining authors have no conflicts to declare. [ABSTRACT FROM AUTHOR]