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Following early studies showing no adverse effects, cleavage stage biopsy by zona drilling using acid Tyrode's solution, and removal of single blastomeres for preimplantation genetic testing (PGT) and identification of sex in couples at risk of X-linked disease, was performed by Handyside and colleagues in late 1989, and pregnancies reported in 1990. This method was later used for specific diagnosis of monogenic conditions, and a few years later also for chromosomal structural and/or numerical impairments, thereby establishing a valuable alternative option to prenatal diagnosis. This revolutionary approach in clinical embryology spread worldwide, and several other embryo biopsy strategies developed over three decades in a process that is still ongoing. The rationale of this narrative review is to outline the different biopsy approaches implemented across the years in the workflow of the IVF clinics that provided PGT: their establishment, the first clinical experiences, their downsides, evolution, improvement and standardization. The history ends with a glimpse of the future: minimally/non-invasive PGT and experimental embryo micromanipulation protocols. This grand theme review outlines a timeline of the evolution of embryo biopsy protocols, whose implementation is increasing worldwide together with the increasing application of PGT techniques in IVF. It represents a vade mecum especially for the past, present and upcoming operators and experts in this field to (re)live this history from its dawn to its most likely future. [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: 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]

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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]

Background: The unbalanced transmission of chromosomes in human gametes and early preimplantation embryos causes aneuploidy, which is a major cause of infertility and pregnancy failure. A baseline of 20% of human oocytes are estimated to be aneuploid and this increases exponentially from 30 to 35 years, reaching on average 80% by 42 years. As a result, reproductive senescence in human females is predominantly determined by the accelerated decline in genetic quality of oocytes from 30 years of age.Objective and Rationale: Understanding mechanisms of chromosome segregation and aneuploidies in the female germline is a crucial step towards the development of new diagnostic approaches and, possibly, for the development of therapeutic targets and molecules. Here, we have reviewed emerging mechanisms that may drive human aneuploidy, in particular the maternal age effect.Search Methods: We conducted a systematic search in PubMed Central of the primary literature from 1990 through 2016 following the PRISMA guidelines, using MeSH terms related to human aneuploidy. For model organism research, we conducted a literature review based on references in human oocytes manuscripts and general reviews related to chromosome segregation in meiosis and mitosis.Outcomes: Advances in genomic and imaging technologies are allowing unprecedented insight into chromosome segregation in human oocytes. This includes the identification of a novel chromosome segregation error, termed reverse segregation, as well as sister kinetochore configurations that were not predicted based on murine models.Wider Implications: Elucidation of mechanisms that result in errors in chromosome segregation in meiosis may lead to therapeutic developments that could improve reproductive outcomes by reducing aneuploidy. [ABSTRACT FROM AUTHOR]

Defining the actual incidence and prevalence of mosaicism in human blastocysts still remains a difficult task. The small amount of evidence generated by animal and human studies does not support the existence of mechanisms involved in developmental arrest, clonal depletion, or aneuploidy rescue for abnormal cells in euploid/aneuploid embryos during preimplantation development. However, studies in humans are mainly descriptive and lack functional evidence. Understanding the biological mechanisms that beset preimplantation differentiation holds the potential to reveal the role of aneuploidies and gene dosage imbalances in cell fate decision, providing important clues on the origin and evolution of embryonic mosaicism. The evidence on human blastocysts suggests that a mosaic euploid/aneuploid configuration is detected in around 5% of embryos. This figure supports the extremely low level of mosaicism reported in natural and IVF pregnancies. Similarly, the clinical management of patterns consistent with the presence of mosaicism in a trophectoderm biopsy during preimplantation genetic diagnosis cycles (PGD-A) is still a controversial issue. Despite the facts that some contemporary comprehensive chromosomal screening platforms can detect mosaic samples in cell mixture models with variable accuracy and many reproductive genetics laboratories are now routinely including embryonic mosaicism on their genetic reports, a diagnosis of certainty for mosaicism in PGD-A cycles is conceptually impracticable. Indeed, several technical and biological sources of errors clearly exist when trying to estimate mosaicism from a single trophectoderm biopsy in PGD-A cycles and must be understood to adequately guide patients during clinical care. [ABSTRACT FROM AUTHOR]

Embryonic mosaicism, defined as the presence of karyotypically distinct cell lines within an embryo, has been frequently reported with a high incidence in preimplantation embryos derived from IVF and is thought to be one of the major biological limitations for the routine application of PGD for aneuploidies (PGD-A). The incidence of mosaicism in preimplantation embryos is in fact reported to be between 4 and 90%. However, these data are in sharp contrast with what is known from clinical pregnancies, where true foetal mosaicism is observed in less than 0.5% of cases. Here, we challenge these previous observations in preimplantation embryos, presenting an alternative perspective, which also considers the impact of technical variation to diagnose mosaicism as one possible cause contributing to overestimation of the incidence of mosaicism in embryos. Although euploid/aneuploid mosaicism may be present in blastocysts, the possibility of detecting this phenomenon within a single trophectoderm biopsy represents a contemporary challenge to bring about improvement to the practice of PGD-A. The purpose of this opinion paper is to provide a critical review of the literature, provide a possible alternative interpretation of the data, and discuss future challenges with diagnosing mosaicism in PGD-A cycles. [ABSTRACT FROM AUTHOR]

Chromosome deletions and duplications-copy number variations (CNVs)-are a major contribution to the genome variability and can be either pathogenic or not. A particular class, the microdeletions and microduplications, which alter <5 Mb, have been extensively associated with developmental delay and intellectual disability. Although their prevalence in pregnancies and newborn is relatively low, their estimates in preimplantation embryos are poorly defined. The introduction of novel technologies for preimplantation genetic diagnosis of aneuploidies (PGD-A) caused new possibilities and challenges associated with diagnosis of subchromosomal CNVs. Both technical aspects of performing genomewide microarray or next generation sequencing analysis on single cells and interpretation issues are subject of debate. The latter include the reliability of detection of CNVs from embryonic biopsies, their clinical classification based on reproductive outcomes, as well as how before and after test counseling should be organized. It is also important to consider that the current resolution of these technologies from single cells is usually >10 Mb, thus ruling out the possibility to diagnose the most important recurrent microdeletion and microduplication syndromes. Furthermore, at present we face with a lack of well-designed studies addressing the actual resolution and accuracy of CNVs detection in PGD-A and no reference databases is available to evaluate their pathogenicity. Accordingly, it seems reasonable at the moment to avoid the reporting of subchromosomal CNVs in PGD-A. However, although these issues require proper handling, they should not lead us away from providing an improved preimplantation genetic diagnosis. [ABSTRACT FROM AUTHOR]

For an IVF clinic that wishes to implement preimplantation genetic diagnosis for monogenic diseases (PGD) and for aneuploidy testing (PGD-A), a global improvement is required through all the steps of an IVF treatment and patient care. At present, CCS (Comprehensive Chromosome Screening)-based trophectoderm (TE) biopsy has been demonstrated as a safe, accurate and reproducible approach to conduct PGD-A and possibly also PGD from the same biopsy. Key challenges in PGD/PGD-A implementation cover genetic and reproductive counselling, selection of the most efficient approach for blastocyst biopsy as well as of the best performing molecular technique to conduct CCS and monogenic disease analysis. Three different approaches for TE biopsy can be compared. However, among them, the application of TE biopsy approaches, entailing the zona opening when the expanded blastocyst stage is reached, represent the only biopsy methods suited with a totally undisturbed embryo culture strategy (time lapse-based incubation in a single media). Moreover, contemporary CCS technologies show a different spectrum of capabilities and limits that potentially impact the clinical outcomes, the management and the applicability of the PGD-A itself. In general, CCS approaches that avoid the use of whole genome amplification (WGA) can provide higher reliability of results with lower costs and turnaround time of analysis. The future perspectives are focused on the scrupulous and rigorous clinical validations of novel CCS methods based on targeted approaches that avoid the use of WGA, such as targeted next-generation sequencing technology, to further improve the throughput of analysis and the overall cost-effectiveness of PGD/PGD-A. [ABSTRACT FROM AUTHOR]

Background: Preimplantation genetic screening (PGS) provides an opportunity to eliminate a potential implantation failure due to aneuploidy in infertile couples. Some studies clearly show that twins following single embryo transfer (SET) can be the result of a concurrent natural conception and an incidence as high as 1 in 5 twins has been reported. In our case PGS was performed on trophectoderm (TE) biopsies by quantitative polymerase chain reaction (qPCR). The product of conception (POC) was cytogenetically investigated after selection of the placental villi by means of the direct method. Molecular cytogenetic characterization of the POC was performed by fluorescence in situ hybridization (FISH) and array-comparative genomic hybridization (a-CGH) analyses. To investigate the possibility of a spontaneous conception, a panel of 40 single nucleotide polymorphisms (SNPs) was used to compare genetic similarity between the DNA of the POC and the DNA leftover of the TE biopsy. Findings: We describe a 36-year old infertile woman undergoing PGS who had a spontaneous abortion after a single euploid embryo transfer on a spontaneous cycle. The POC showed a 45,X karyotype confirmed by FISH and a-CGH. DNA fingerprinting demonstrated a genetic similarity of 75 % between the DNA of the POC and TE biopsy, consistent with a sibling status. All supernumerary euploid embryos were also tested showing a non-self relationship with the POC, excluding a mix-up event at the time of fetal embryo transfer. Conclusions: DNA fingerprinting of the transferred blastocyst and POC, confirmed the occurrence of a spontaneous conception. This case challenges the assumption that a pregnancy after assisted reproductive technology (ART) is always a result of ART, and strengthens the importance to avoid intercourses during PGS and natural transfer cycles. Moreover, cytogenetic analysis of the POCs is strongly recommended along with fingerprinting children born after PGS to see what the concordance is between the embryo transferred and the resultant child. [ABSTRACT FROM AUTHOR]

Preimplantation Genetic Diagnosis and Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential. [ABSTRACT FROM AUTHOR]

Study Question: Is blastocyst biopsy and quantitative real-time PCR based comprehensive chromosome screening a consistent and reproducible approach across different biopsy practitioners?Summary Answer: The blastocyst biopsy approach provides highly consistent and reproducible laboratory and clinical outcomes across multiple practitioners from different IVF centres when all of the embryologists received identical training and use similar equipment.What Is Known Already: Recently there has been a trend towards trophectoderm (TE) biopsy in preimplantation genetic screening (PGS)/preimplantation genetic diagnosis (PGD) programmes. However, there is still a lack of knowledge about the reproducibility that can be obtained from multiple biopsy practitioners in different IVF centres in relation also to blastocysts of different morphology. Although it has been demonstrated that biopsy at the blastocyst stage has no impact on embryo viability, it remains a possibility that less experienced individual biopsy practitioners or laboratories performing TE biopsy may affect certain outcomes. We investigated whether TE biopsy practitioners can have an impact on the quality of the genetic test and the subsequent clinical outcomes.Study Design, Size, Duration: This longitudinal cohort study, between April 2013 and December 2014, involved 2586 consecutive blastocyst biopsies performed at three different IVF centres and the analysis of 494 single frozen euploid embryo transfer cycles (FEET).Participants/materials, Setting, Methods: Seven biopsy practitioners performed the blastocyst biopsies in the study period and quantitative PCR was used for comprehensive chromosome screening (CCS). The same practitioner performed both the biopsy and tubing procedures for each blastocyst they biopsied. To investigate the quality of the biopsied samples, the diagnostic rate, sample-specific concurrence and the cell number retrieved in the biopsy were evaluated for each biopsy operator. Clinical outcomes following FEET cycles were stratified by biopsy operator and compared. Cellularity of the biopsy sample was also correlated with clinical outcomes.Main Results and the Role Of Chance: The seven practitioners performed 2586 biopsies, five in centre IVF-1 and one in each of the other two IVF centres (IVF-2 and IVF-3). Overall, 2437 out of 2586 (94.2%) blastocyst biopsies resulted in a conclusive diagnosis, 119 (4.6%) showed a nonconcurrent result and 30 (1.2%) failed to amplify, suggesting the absence of TE cells in the test tube or presence of degenerated/lysed cells only. Among the samples producing a conclusive diagnosis, a mean concurrence value of 0.253 (95% CI = 0.250-0.257) was observed. Logistic regression analysis adjusted for confounding factors showed no differences in the diagnosis rate and in the concurrence of the genetic analysis between different biopsy practitioners. An overall mean number of 7.32 cells (95% CI = 6.82-7.81; range 2-15) were predicted from all biopsies. Higher cellularity was significantly associated with a better quality of the CCS diagnosis (P < 0.01) and with the conclusive diagnosis rate, with nonconcurrent samples showing significantly lower numbers of cells (2.1; 95% CI=1.5-2.7) compared with samples resulting in a conclusive diagnosis (mean cells number 7.5; 95% CI = 7.1-7.9, P < 0.01). However, no differences were recorded between different biopsy practitioners regarding cellularity of the biopsy. Finally, logistic analysis showed no impact of the biopsy practitioners on the observed ongoing rates of implantation, biochemical pregnancy loss and miscarriage after the FEET cycles.Limitations, Reasons For Caution: These data come from a restricted set of laboratories where all of the embryologists received identical training and use identical equipment. A single TE biopsy method and CCS technology was used and these data particularly apply to PGS programmes using blastocyst biopsy without zona opening at the cleavage stage and using qPCR-based CCS. To make firm conclusions on the potential impact of biopsy on biochemical pregnancy loss and miscarriages according to practitioner and biopsy cellularity, a larger sample size is needed.Wider Implications Of the Findings: We reported a very high consistency and reproducibility of the blastocyst biopsy approach coupled with qPCR-based CSS for both genetic and clinical outcomes across different practitioners working in different IVF centres when appropriate training is provided and when the same laboratory setting is used. These data are important considering the trend towards the use of blastocyst biopsy worldwide for PGD/PGS applications.Study Funding/competing Interests: None. [ABSTRACT FROM AUTHOR]

Background: The recent identification of embryonic cell-free DNA in spent blastocyst media has opened a new era of possibilities for noninvasive embryo aneuploidy testing in assisted reproductive technologies. Yet, previous studies assessing a limited number of embryos reported variable concordance between embryonic cell-free DNA and trophectoderm biopsies, thus questioning the validity of this approach.Objective: This study aimed to evaluate the concordance and reproducibility of testing embryonic cell-free DNA vs trophectoderm DNA obtained from the same embryo in a large sample of human blastocysts and to assess the contribution of the inner cell mass and trophectoderm to embryonic cell-free DNA released to the culture media.Study Design: This is an interim analysis of a prospective, observational study among 8 in vitro fertilization centers in 4 continents to assess consistency between noninvasive embryo aneuploidy testing of embryonic cell-free DNA and conventional trophectoderm biopsy. The analysis included 1301 day-6/7 blastocysts obtained in 406 in vitro fertilization cycles from 371 patients aged 20-44 years undergoing preimplantation genetic testing for aneuploidy. Fresh oocytes underwent intracytoplasmic sperm injection or in vitro fertilization. No previous assisted hatching or vitrification was allowed before media collection. Individual spent blastocyst medium was collected from embryos cultured at least 40 hours from day 4. After media collection, conventional preimplantation genetic testing for aneuploidy, comprising trophectoderm biopsy and blastocyst vitrification, was performed. Embryonic cell-free DNA was analyzed blindly after embryo transfer. Inner cell mass and trophectoderm biopsies were also performed in a subset of 81 aneuploid blastocysts donated for research.Results: Embryonic cell-free DNA analyses were 78.2% (866/1108) concordant with the corresponding trophectoderm biopsies. No significant differences were detected among centers ranging from 72.5% to 86.3%. Concordance rates exceeded 86% when all defined steps in the culture laboratory were controlled to minimize the impact of maternal and operator contamination. Sensitivity per center ranged from 76.5% to 91.3% and specificity from 64.7% to 93.3%. The false-negative rate was 8.3% (92/1108), and false-positive rate was 12.4% (137/1108). The 2 fertilization techniques provided similar sensitivity (80.9% vs 87.9%) and specificity (78.6% vs 69.9%). Multivariate analysis did not reveal any bias from patient clinical background, ovarian stimulation protocols, culture conditions, or embryo quality on testing accuracy of concordance. Moreover, concordances of embryonic cell-free DNA with trophectoderm and inner cell mass suggest that the embryonic cell-free DNA originates from both compartments of the human embryo.Conclusion: Noninvasive analysis of embryonic cell-free DNA in spent blastocyst culture media demonstrates high concordance with trophectoderm biopsy results in this large multicenter series. A noninvasive approach for prioritizing embryo euploidy offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population. [ABSTRACT FROM AUTHOR]

Objective: To report laboratory and clinical outcomes in preimplantation genetic diagnosis for aneuploidies (PGD-A) cycles for women 44 to 47 years old.Design: Multicenter, longitudinal, observational study.Setting: In vitro fertilization (IVF) centers.Patient(s): One hundred and thirty-seven women aged 44.7 ± 0.7 years (range: 44.0-46.7) undergoing 150 PGD-A cycles during April 2013 to January 2016.Intervention(s): Quantitative polymerase chain reaction-based PGD-A on trophectoderm biopsies and cryopreserved euploid single-embryo transfer (SET).Main Outcomes Measure(s): Primary outcome measure: delivery rate per cycle; secondary outcome measures: miscarriage rate, and the rate and reasons for cycle cancelation with subanalyses for female age and number of metaphase 2 oocytes retrieved.Result(s): In 102 (68.0%) of 150 cycles blastocyst development was obtained, but only 21 (14.0%) were euploid blastocysts. The overall euploidy rate was 11.8% (22 of 187). Twenty-one SET procedures were performed, resulting in 13 clinical pregnancies, of which 1 miscarried and 12 delivered. The delivery rate was 57.1% per transfer, 8.0% per cycle, and 8.8% per patient. The logistic regression analysis found that only female age (odds ratio 0.78) and number of metaphase 2 oocytes retrieved (odds ratio 1.25) statistically significantly correlated with the likelihood of delivery. The delivery rate per cycle was 10.6% (11 of 104) in patients aged 44.0 to 44.9 years and 2.6% in patients aged 45.0 to 45.9 years (n = 1 of 38). No euploid blastocysts were found for patients older than 45.0 years.Conclusion(s): Extensive counseling based on biological and clinical data should be provided to women older than 43 years who are requesting IVF because of their very low odds of success and high risk for embryonic aneuploidies. Nevertheless, the low miscarriage and good delivery rates reported in this study in women with good ovarian reserve aged 44 should encourage the use of PGD-A in this population. [ABSTRACT FROM AUTHOR]

Preimplantation genetic diagnosis and aneuploidy testing (PGD/PGS) use is constantly growing in IVF, and embryo/biopsy traceability during the additional laboratory procedures needed is pivotal. An electronic witnessing system (EWS), which showed a significant value in decreasing mismatch occurrence and increasing detection possibilities during standard care IVF, still does not guarantee the same level of efficiency during PGD/PGS cycles. Specifically, EWS cannot follow single embryos throughout the procedure. This is however critical when an unambiguous diagnosis corresponds to each embryo. Failure Mode and Effects Analysis (FMEA) is a proactive method generally adopted to define tools ensuring safety along a procedure. Due to the implementation of a large quantitative PCR (qPCR)-based blastocyst stage PGD/PGS programme in our centre, and to evaluate the potential procedural risks, a FMEA was performed in September 2014. Forty-four failure modes were identified, among which six were given a moderate risk priority number (>15) (RPN; product of estimated occurrence, severity and detection). Specific corrective measures were then introduced and implemented, and a second evaluation performed six months later. The meticulous and careful application of such measures allowed the risks to be decreased along the whole protocol, by reducing their estimated occurrence and/or increasing detection possibilities. [ABSTRACT FROM AUTHOR]

[Display omitted] Hereditary cancer syndromes are a heterogeneous group of genetic conditions that are associated with an increased risk of developing cancer during lifespan. In affected women, parenthood may be accompanied by concerns for the offspring, considering the common autosomal dominant inheritance. Moreover, fertility preservation to prevent the detrimental effects of cancer treatments differs compared to other clinical contexts. The necessity to preserve gametes is indeed predictable and expected to be common. For these reasons, we advocate a personalized and early fertility counseling. Carriers should be aware of the risk of transmission. The possibility to perform elective oocytes cryopreservation, either before (previvors) or after (survivors) cancer diagnosis should be discussed. Finally, they should be informed about the options of preimplantation genetic test (PGT) and oocytes donation. In conclusion, physicians engaged in oncofertility should personalize the counseling for women with hereditary cancer syndromes, being aware of their peculiar needs. [ABSTRACT FROM AUTHOR]

Objective: To determine whether blastocoel fluid (BF) or spent blastocyst medium (SBM) is a suitable template for genotype and/or karyotype assessment of in vitro fertilization-generated embryos.Design: Prospective blinded study.Setting: Genetic laboratory.Patient(s): From 26 patients undergoing preimplantation genetic testing (PGT) treatments, 103 trophectoderms (TE), 92 BF samples, and 72 SBM samples.Intervention(s): The BF and SBM were retrieved at the time of TE biopsy. Two DNA extraction strategies were evaluated on independent BF and SBM samples. Further enrolled samples were processed using next-generation sequencing and quantitative polymerase chain reaction for assessment of monogenic disorders (PGT-M) or aneuploidy (PGT-A).Main Outcome Measure(s): DNA amplification and concordance rates across BF, SBM, and TE to assess diagnostic efficiency.Result(s): No differences were detected among the DNA extraction methods tested. In PGT-M tests, for BF and SBM, 2.9% and 20.8% of all samples, respectively, produced a diagnosis concordant with the corresponding TE (n = 2 of 69 and 15 of 72, respectively). The SBM samples were associated with higher discordance rates and higher artifacts/contamination detection compared with BF. In multiple occasions, the maternal mutated variant was detected in the SBM of homozygous wild-type embryos, showing evidence of maternal DNA persistence in culture medium. In PGT-A tests, BF analysis showed high amplification failure rates (65.2%) and an overall concordance rate of 37.5% among amplified samples.Conclusion(s): Based on current methodologies, BF and SBM genetic analyses do not provide sufficiently reliable results to be employed clinically. Until the risk of maternal contamination can be properly prevented, SBM should not be used for PGT-M purposes. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the impact of the male factor on the outcomes of intracytoplasmic sperm injection (ICSI) cycles combined with preimplantation genetic testing for aneuploidies (PGT-A).Design: Observational longitudinal cohort study.Setting: Private in vitro fertilization (IVF) center.Patient(s): A total of 1,219 oocyte retrievals divided into five study groups according to sperm parameters: normozoospermia (N), moderate male factor (MMF), severe oligoasthenoteratozoospermia (OAT-S), obstructive azoospermia (OA), and nonobstructive azoospermia (NOA).Intervention(s): ICSI with ejaculated/surgically retrieved sperm, blastocyst culture, trophectoderm-based quantitative polymerase chain reaction PGT-A, and frozen-warmed euploid embryo transfer (ET).Main Outcomes Measure(s): The primary outcome measures were fertilization, blastocyst development, and euploidy rates; the secondary outcome measures were live birth and miscarriage rates. Perinatal and obstetrical outcomes were monitored as well.Result(s): A total of 9,042 metaphase II oocytes were inseminated. The fertilization rate was significantly reduced in MMF, OAT-S, OA, and NOA compared with N (74.8%, 68.7%, 67.3%, and 53.1% vs. 77.2%). The blastocyst rate per fertilized oocyte was significantly reduced in MMF and NOA compared with N (48.6% and 40.6% vs. 49.3%). The timing of blastocyst development also was affected in OA and NOA. Logistic regression analysis adjusted for confounders highlighted NOA as a negative predictor of obtaining an euploid blastocyst per OPU (odds ratio 0.5). When the analysis was performed per obtained blastocyst, however, no correlation between male factor and euploidy rate was observed. Embryo transfers also resulted in similar live birth and miscarriage rates. No impact of sperm factor on obstetrical/perinatal outcomes was observed.Conclusion(s): Severe male factor impairs early embryonic competence in terms of fertilization rate and developmental potential. However, the euploidy rate and implantation potential of the obtained blastocysts are independent from sperm quality. [ABSTRACT FROM AUTHOR]

Objective: To test whether abnormally fertilized oocyte (AFO)-derived blastocysts are diploid and can be rescued for clinical use.Design: Longitudinal-cohort study from January 2015 to September 2016 involving IVF cycles with preimplantation genetic testing for aneuploidy (PGT-A). Ploidy assessment was incorporated whenever a blastocyst from a monopronuclear (1PN) or tripronuclear zygote (2PN + 1 smaller PN; 2.1 PN) was obtained.Setting: Private IVF clinics and genetics laboratories.Patient(s): A total of 556 women undergoing 719 PGT-A cycles.Intervention(s): Conventional chromosome analysis was performed on trophectoderm biopsies by quantitative polymerase chain reaction. For AFO-derived blastocysts, ploidy assessment was performed on the same biopsy with the use of allele ratios for hetorozygous SNPs analyzed by means of next-generation sequencing (1:1 = diploid; 2:1 = triploid; loss of heterozygosity = haploid). Balanced-diploid 1PN- and 2.1PN-derived blastocysts were transferred in the absence of normally fertilized transferable embryos.Main Outcome Measure(s): Ploidy constitution and clinical value of AFO-derived blastocysts in IVF PGT-A cycles.Result(s): Of the 5,026 metaphase II oocytes injected, 5.2% and 0.7% showed 1PN and 2.1PN, respectively. AFOs showed compromised embryo development (P<.01). Twenty-seven AFO-derived blastocysts were analyzed for ploidy constitution. The 1PN-derived blastocysts were mostly diploid (n = 9/13; 69.2%), a few were haploid (n = 3/13; 23.1%), and one was triploid (n = 1/13; 7.7%). The 2.1PN-derived blastocysts were also mostly diploid (n = 12/14; 85.7%), and the remainder were triploid. Twenty-six PGT-A cycles resulted in one or more AFO-derived blastocysts (n = 26/719; 3.6%). Overall, eight additional balanced-diploid transferable embryos were obtained from AFOs. In three cycles, the only balanced-diploid blastocyst produced was from an AFO (n = 3/719; 0.4%). Three AFO-derived live births were achieved: one from a 1PN zygote and two from 2.1PN zygotes.Conclusion(s): Enhanced PGT-A technologies incorporating reliable ploidy assessment provide an effective tool to rescue AFO-derived blastocysts for clinical use. [ABSTRACT FROM AUTHOR]