Human embryo live imaging reveals nuclear DNA shedding during blastocyst expansion and biopsy.

Proper preimplantation development is essential to assemble a blastocyst capable of implantation. Live imaging has uncovered major events driving early development in mouse embryos; yet, studies in humans have been limited by restrictions on genetic manipulation and lack of imaging approaches. We have overcome this barrier by combining fluorescent dyes with live imaging to reveal the dynamics of chromosome segregation, compaction, polarization, blastocyst formation, and hatching in the human embryo. We also show that blastocyst expansion mechanically constrains trophectoderm cells, causing nuclear budding and DNA shedding into the cytoplasm. Furthermore, cells with lower perinuclear keratin levels are more prone to undergo DNA loss. Moreover, applying trophectoderm biopsy, a mechanical procedure performed clinically for genetic testing, increases DNA shedding. Thus, our work reveals distinct processes underlying human development compared with mouse and suggests that aneuploidies in human embryos may not only originate from chromosome segregation errors during mitosis but also from nuclear DNA shedding.

Trophectoderm biopsy of blastocysts following IVF and embryo culture increases epigenetic dysregulation in a mouse model.

STUDY QUESTION: Does trophectoderm biopsy (TEBx) of blastocysts for preimplantation genetic testing in the clinic affect normal placental and embryo development and offspring metabolic outcomes in a mouse model? SUMMARY ANSWER: TEBx impacts placental and embryonic health during early development, with some alterations resolving and others worsening later in development and triggering metabolic changes in adult offspring. WHAT IS KNOWN ALREADY: Previous studies have not assessed the epigenetic and morphological impacts of TEBx either in human populations or in animal models. STUDY DESIGN, SIZE, DURATION: We employed a mouse model to identify the effects of TEBx during IVF. Three groups were assessed: naturally conceived (Naturals), IVF, and IVF + TEBx, at two developmental timepoints: embryonic day (E)12.5 (n = 40/Naturals, n = 36/IVF, and n = 36/IVF + TEBx) and E18.5 (n = 42/Naturals, n = 30/IVF, and n = 35/IVF + TEBx). Additionally, to mimic clinical practice, we assessed a fourth group: IVF + TEBx + Vitrification (Vit) at E12.5 (n = 29) that combines TEBx and vitrification. To assess the effect of TEBx in offspring health, we characterized a 12-week-old cohort (n = 24/Naturals, n = 25/IVF and n = 25/IVF + TEBx). PARTICIPANTS/MATERIALS, SETTING, METHODS: Our mouse model used CF-1 females as egg donors and SJL/B6 males as sperm donors. IVF, TEBx, and vitrification were performed using standardized methods. Placenta morphology was evaluated by hematoxylin-eosin staining, in situ hybridization using Tpbpa as a junctional zone marker and immunohistochemistry using CD34 fetal endothelial cell markers. For molecular analysis of placentas and embryos, DNA methylation was analyzed using pyrosequencing, luminometric methylation assay, and chip array technology. Expression patterns were ascertained by RNA sequencing. Triglycerides, total cholesterol, high-, low-, and very low-density lipoprotein, insulin, and glucose were determined in the 12-week-old cohort using commercially available kits. MAIN RESULTS AND THE ROLE OF CHANCE: We observed that at E12.5, IVF + TEBx had a worse outcome in terms of changes in DNA methylation and differential gene expression in placentas and whole embryos compared with IVF alone and compared with Naturals. These changes were reflected in alterations in placental morphology and blood vessel density. At E18.5, early molecular changes in fetuses were maintained or exacerbated. With respect to placentas, the molecular and morphological changes, although different compared to Naturals, were equivalent to the IVF group, except for changes in blood vessel density, which persisted. Of note is that most differences were sex specific. We conclude that TEBx has more detrimental effects in mid-gestation placental and embryonic tissues, with alterations in embryonic tissues persisting or worsening in later developmental stages compared to IVF alone, and the addition of vitrification after TEBx results in more pronounced and potentially detrimental epigenetic effects: these changes are significantly different compared to Naturals. Finally, we observed that 12-week IVF + TEBx offspring, regardless of sex, showed higher glucose, insulin, triglycerides, lower total cholesterol, and lower high-density lipoprotein compared to IVF and Naturals, with only males having higher body weight compared to IVF and Naturals. Our findings in a mouse model additionally support the need for more studies to assess the impact of new procedures in ART to ensure healthy pregnancies and offspring outcomes. LARGE SCALE DATA: Data reported in this work have been deposited in the NCBI Gene Expression Omnibus under accession number GSE225318. LIMITATIONS, REASONS FOR CAUTION: This study was performed using a mouse model that mimics many clinical IVF procedures and outcomes observed in humans, where studies on early embryos are not possible. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights the importance of assaying new procedures used in ART to assess their impact on placenta and embryo development, and offspring metabolic outcomes. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by a National Centers for Translational Research in Reproduction and Infertility grant P50 HD068157-06A1 (M.S.B., C.C., M.M.), Ruth L. Kirschstein National Service Award Individual Postdoctoral Fellowship F32 HD107914 (E.A.R.-C.) and F32 HD089623 (L.A.V.), and National Institutes of Health Training program in Cell and Molecular Biology T32 GM007229 (C.N.H.). No conflict of interest.

Impact of trophectoderm biopsy for preimplantation genetic testing on obstetric and neonatal outcomes: a meta-analysis.

OBJECTIVE: This study aimed to investigate whether trophectoderm biopsy for preimplantation genetic testing is associated with an increased risk of adverse obstetrical and neonatal outcomes compared with conventional in vitro fertilization or intracytoplasmic sperm injection without preimplantation genetic testing. DATA SOURCES: Entries between January 1990 and August 2022 were searched using MEDLINE, Embase, Web of Science, the Cochrane Library, and Google Scholar. STUDY ELIGIBILITY CRITERIA: Publications comparing the outcomes of pregnancies after preimplantation genetic testing using trophectoderm biopsy and in vitro fertilization or intracytoplasmic sperm injection were included. Only human studies with a cohort or case-control design or randomized controlled trials were eligible for inclusion. METHODS: The study selection process was performed independently by 2 investigators. The quality of the observational studies was assessed using the Newcastle-Ottawa Scale, and the Cochrane risk-of-bias tool version 2 was used to grade the level of bias in randomized controlled trials. The pooled odds ratio and 95% confidence interval were calculated using a random-effects model when substantial heterogeneity occurred (indicated by I2 of >50% and P<.1). Otherwise, a fixed-effects model was used. RESULTS: This meta-analysis included 13 studies involving 11,469 live births after preimplantation genetic testing treatment with trophectoderm biopsy before embryo transfer and 20,438 live births after in vitro fertilization or intracytoplasmic sperm injection only. The odds ratio of preterm delivery was higher in the trophectoderm-biopsied group than in the routine in vitro fertilization or intracytoplasmic sperm injection group (pooled odds ratio, 1.12; 95% confidence interval, 1.03-1.21); however, the difference did not exist after sensitivity analysis (odds ratio, 0.97; 95% confidence interval, 0.84-1.11). The risk of low birthweight did not increase among the biopsied pregnancies (pooled odds ratio, 1.01; 95% confidence interval, 0.85-1.20). No marked difference was observed in the risk of other obstetrical or neonatal outcomes between the biopsy and control groups. Furthermore, no difference was noted in the perinatal outcomes between trophectoderm-biopsied and nonbiopsied groups in the subgroup analyses by intracytoplasmic sperm injection, frozen-thawed transfer, or single embryo transfer. CONCLUSION: Trophectoderm biopsy for preimplantation genetic testing treatment did not alter the risk of obstetrical or neonatal outcomes compared with conventional in vitro fertilization or intracytoplasmic sperm injection without preimplantation genetic testing. However, this study was limited by the large observational evidence base, and more randomized controlled trials are needed to further confirm these findings.

Trophectoderm biopsy is associated with lower risks of moderate to extreme prematurity and low birthweights: a national registry cohort study of singleton livebirths from frozen-thawed blastocyst transfers.

BACKGROUND: Trophectoderm biopsy has become the mainstay assisted reproductive technique performed for preimplantation genetic testing, accounting for 43.8% of embryo transfer cycles in the United States in 2019 alone. Despite its prevalence, data on the obstetric and perinatal outcomes post-trophectoderm biopsy remains sparse and mixed. OBJECTIVE: This study aimed to examine the risks of adverse perinatal outcomes in birthweights and prematurity after transfers of the vitrified-thawed blastocyst with trophectoderm biopsy for preimplantation genetic testing. STUDY DESIGN: This was a retrospective observational cohort study of 45,712 singleton livebirths resulting from autologous vitrified-thawed blastocyst transfer cycles with or without trophectoderm biopsy for preimplantation genetic testing, reported by participating member clinics to the Society for Assisted Reproductive Technology national registry between 2014 and 2017. Adverse perinatal outcomes of preterm births and low birthweights were analyzed. Multivariable regression analyses were performed to control for covariates. Comparing the trophectoderm biopsy (n=21,584) and no trophectoderm biopsy (n=24,128) groups, adjusted odds ratios were calculated for the outcomes of small-for-gestational-age, large-for-gestational-age, low birthweight <2500 g, very low birthweight <1500 g, extremely low birthweight <1000 g, late preterm births <37 weeks, moderate preterm births <34 weeks, and extremely preterm births <28 weeks. RESULTS: Women in the trophectoderm biopsy group were older and more likely to have prior pregnancies, deliveries, and a history of spontaneous abortions. Tobacco use, diminished ovarian reserve, and recurrent pregnancy loss were also more prevalent in the trophectoderm biopsy group. Trophectoderm biopsy was not associated with small-for-gestational-age (adjusted odds ratio, 0.97; 95% confidence interval, 0.85-1.12; P=.72) or large-for-gestational-age newborns (adjusted odds ratio, 1.10; 95% confidence interval, 0.99-1.22; P=.09). Risks of preterm births <37 weeks gestation were similar between the biopsy and nonbiopsy groups (adjusted odds ratio, 0.93; 95% confidence interval, 0.85-1.02; P=.11). Trophectoderm biopsy was associated with a significantly lower risk of low birthweight <2500 g (adjusted odds ratio, 0.80; 95% confidence interval, 0.70-0.92; P<.001), very low birthweight <1500 g (adjusted odds ratio, 0.62; 95% confidence interval, 0.46-0.83; P<.001), extremely low birthweight <1000 g (adjusted odds ratio, 0.48; 95% confidence interval, 0.31-0.74; P<.001), moderate preterm birth <34 weeks (adjusted odds ratio, 0.76; 95% confidence interval, 0.64-0.91; P=.003), and extreme preterm birth <28 weeks (adjusted odds ratio, 0.63; 95% confidence interval, 0.43-0.92; P=.02). CONCLUSION: Trophectoderm biopsy is not associated with increased risks of small-for-gestational-age, large-for-gestational-age, or late preterm birth. Risks of low birthweight, very low birthweight, and extremely low birthweight from moderate and extreme preterm births are lower after trophectoderm biopsy, possibly by selecting against confined placental mosaicism or inducing placental epigenetic changes, the mechanisms of which warrant further investigation.

Embryo drop-out rates in preimplantation genetic testing for aneuploidy (PGT-A): a retrospective data analysis from the DoLoRes study.

PURPOSE: To evaluate the decline in transferable embryos in preimplantation genetic testing for aneuploidy (PGT-A) cycles due to (a) non-biopsable blastocyst quality, (b) failure of genetic analysis, (c) diagnosis of uniform numerical or structural chromosomal aberrations, and/or (d) chromosomal aberrations in mosaic constitution. METHODS: This retrospective multicenter study comprised outcomes of 1562 blastocysts originating from 363 controlled ovarian stimulation cycles, respectively, 226 IVF couples in the period between January 2016 and December 2018. Inclusion criteria were PGT-A cycles with trophectoderm biopsy (TB) and next generation sequencing (NGS). RESULTS: Out of 1562 blastocysts, 25.8% were lost due to non-biopsable and/or non-freezable embryo quality. In 10.3% of all biopsied blastocysts, genetic analysis failed. After exclusion of embryos with uniform or chromosomal aberrations in mosaic, only 18.1% of those originally yielded remained as diagnosed euploid embryos suitable for transfer. This translates into 50.4% of patients and 57.6% of stimulated cycles with no euploid embryo left for transfer. The risk that no transfer can take place rose significantly with a lower number of oocytes and with increasing maternal age. The chance for at least one euploid blastocyst/cycle in advanced maternal age (AMA)-patients was 33.3% compared to 52.1% in recurrent miscarriage (RM), 59.8% in recurrent implantation failure (RIF), and 60.0% in severe male factor (SMF). CONCLUSIONS: The present study demonstrates that PGT-A is accompanied by high embryo drop-out rates. IVF-practitioners should be aware that their patients run a high risk of ending up without any embryo suitable for transfer after (several) stimulation cycles, especially in AMA patients. Patients should be informed in detail about the frequency of inconclusive or mosaic results, with the associated risk of not having an euploid embryo available for transfer after PGT-A, as well as the high cost involved in this type of testing.

Single-cell multi-omics sequencing reveals chromosome copy number inconsistency between trophectoderm and inner cell mass in human reconstituted embryos after spindle transfer.

STUDY QUESTION: Is the chromosome copy number of the trophectoderm (TE) of a human reconstituted embryos after spindle transfer (ST) representative of the inner cell mass (ICM)? SUMMARY ANSWER: Single-cell multi-omics sequencing revealed that ST blastocysts have a higher proportion of cell lineages exhibiting intermediate mosaicism than conventional ICSI blastocysts, and that the TE of ST blastocysts does not represent the chromosome copy number of ICM. WHAT IS KNOWN ALREADY: Preimplantation genetic testing for aneuploidy (PGT-A) assumes that TE biopsies are representative of the ICM, but the TE and ICM originate from different cell lineages, and concordance between TE and ICM is not well-studied, especially in ST embryos. STUDY DESIGN, SIZE, DURATION: We recruited 30 infertile women who received treatment at our clinic and obtained 45 usable blastocysts (22 from conventional ICSI and 23 reconstituted embryos after ST). We performed single-cell multi-omics sequencing on all blastocysts to predict and verify copy number variations (CNVs) in each cell. We determined the chromosome copy number of each embryo by analysing the proportion of abnormal cells in each blastocyst. We used the Bland-Altman concordance and the Kappa test to evaluate the concordance between TE and ICM in the both groups. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted at a public tertiary hospital in China, where all the embryo operations, including oocytes retrieval, ST, and ICSI, were performed in the embryo laboratory. We utilized single-cell multi-omics sequencing technology at the Biomedical Pioneering Innovation Center, School of Life Sciences, Peking University, to analyse the blastocysts. Transcriptome sequencing was used to predict the CNV of each cell through bioinformatics analysis, and the results were validated using the DNA methylation library of each cell to confirm chromosomal normalcy. We conducted statistical analysis and graphical plotting using R 4.2.1, SPSS 27, and GraphPad Prism 9.3. MAIN RESULTS AND THE ROLE OF CHANCE: Mean age of the volunteers, the blastocyst morphology, and the developmental ratewere similar in ST and ICSI groups. The blastocysts in the ST group had some additional chromosomal types that were prone to variations beyond those enriched in the blastocysts of the ICSI group. Finally, both Bland-Altman concordance test and kappa concordancetest showed good chromosomal concordance between TE and ICM in the ICSI blastocysts (kappa = 0.659, P < 0.05), but not in ST blastocysts (P = 1.000), suggesting that the TE in reconstituted embryos is not representative of ICM. Gene functional annotation (GO and KEGG analyses) suggests that there may be new or additional pathways for CNV generation in ST embryos compared to ICSI embryos. LIMITATIONS, REASONS FOR CAUTION: This study was mainly limited by the small sample size and the limitations of single-cell multi-omics sequencing technology. To select eligible single cells, some cells of the embryos were eliminated or not labelled, resulting in a loss of information about them. The findings of this study are innovative and exploratory. A larger sample size of human embryos (especially ST embryos) and more accurate molecular genetics techniques for detecting CNV in single cells are needed to validate our results. WIDER IMPLICATIONS OF THE FINDINGS: Our study justifies the routine clinical use of PGT-A in ICSI blastocysts, as we found that the TE is a good substitute for ICM in predicting chromosomal abnormalities. While PGT-A is not entirely accurate, our data demonstrate good clinical feasibility. This trial was able to provide correct genetic counselling to patients regarding the reliability of PGT-A. Regarding ST blastocysts, the increased mosaicism rate and the inability of the TE to represent the chromosomal copy number of the ICM are both biological characteristics that differentiate them from ICSI blastocysts. Currently, ST is not used clinically on a large scale to produce blastocysts. However, if ST becomes more widely used in the future, our study will be the first to demonstrate that the use of PGT-A in ST blastocysts may not be as accurate as PGT-A for ICSI blastocysts. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the National Key R&D Program of China (2018YFA0107601) and the National Key R&D Program of China (2018YFC1003003). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

PGT-HLA programmes for the cure of a sick sibling: clinical strategies for this challenging search.

The ultimate goal of a preimplantation genetic testing and human leukocyte antigen (PGT-HLA) matching programme is the birth of a healthy, HLA-compatible child for the treatment or cure of a sick sibling. Several authors have published successful cases of the births of children HLA-matched to siblings affected by different conditions and diseases. However, there are many reports of failed attempts. Couples seeking an HLA-matched sibling for their affected child look for positive outcomes in the shortest possible time. Nevertheless, there is no published consensus or guidelines with recommendations for these cases. Here, the authors aimed to analyse different approaches for these programmes, highlighting the most promising strategies for the families and fertility units. Furthermore, the authors mention a successful case of a PGT-HLA matching programme after a previous failed attempt following the strategies proposed. Which is the most cost-effective and time-efficient approach in a PGT-HLA matching programme?

Polygenic embryo screening: are there potential maternal and fetal harms?

Polygenic embryo screening (PES) and its derivate the Embryo Health Score (EHS) have generated interest in both infertile and fertile populations due to their potential ability to select embryos with a reduced risk of disease and improved long-term health outcomes. Concerns have been raised regarding the potential harms of IVF itself, including possible epigenetic changes that may affect the health of the offspring in late adulthood, which are not fully captured in the EHS calculation. Knowledge of the potential impacts of the trophectoderm biopsy, which is a key component of the PES procedure, on the offsprings' health is limited by the heterogeneity of the population characteristics used in the published studies. Nonetheless, the literature suggests a possible increased risk of preterm delivery, birth defects and pre-eclampsia after trophectoderm biopsy. Overall, the risks of PES for prenatal and postnatal health remain uncertain, and further research is needed. Counselling patients regarding these risks before considering PES is important, to provide an understanding of the risks and benefits. This review aims to highlight some of these issues, the need for continued investigation in this area, and the importance of informed decision-making in the context of PES.

Concordance of PGT for aneuploidies between blastocyst biopsies and spent blastocyst culture medium.

RESEARCH QUESTION: Non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) avoids the possible detrimental impact of invasive PGT-A on embryo development and clinical outcomes. Does cell-free DNA (cfDNA) from spent blastocyst culture medium (BCM) reflect embryonic chromosome status better than trophectoderm (TE) biopsy? DESIGN: In this study, 35 donated embryos were used for research and the BCM, TE biopsy, inner cell mass (ICM) and residual blastocyst (RB) were individually picked up from these embryos. Whole genome amplification (WGA) was performed and amplified DNA was subject to next-generation sequencing. Chromosome status concordance was compared among the groups of samples. RESULTS: The WGA success rates were 97.0% (TE biopsy), 100% (ICM), 97.0% (RB) and 88.6% (BCM). Using ICM as the gold standard, the chromosomal ploidy concordance rates for BCM, TE biopsy and RB were 58.33% (14/24), 68.75% (22/32) and 78.57% (22/28); the diagnostic concordance rates were 83.33% (20/24), 87.50% (28/32) and 92.86% (26/28); and the sex concordance rates were 92.31% (24/26), 100% (32/32) and 100% (28/28), respectively. Considering RB the gold standard, the chromosome ploidy concordance rates for BCM and TE biopsy were 61.90% (13/21) and 81.48% (22/27); the diagnostic concordance rates were 71.43% (15/21) and 88.89% (24/27); and the sex concordance rates were 91.30% (21/23) and 100% (27/27), respectively. CONCLUSIONS: The results of niPGT-A of cfDNA of spent BCM are comparable to those of invasive PGT-A of TE biopsies. Modifications of embryo culture conditions and testing methods will help reduce maternal DNA contamination and improve the reliability of niPGT-A.

Trophectoderm biopsy is associated with adverse obstetric outcomes rather than neonatal outcomes.

BACKGROUND: With the wide application of preimplantation genetic testing (PGT) with trophectoderm (TE) biopsy, the safety of PGT has always been a concern. Since TE subsequently forms the placenta, it is speculated that the removal of these cells was associated with adverse obstetrical or neonatal outcomes after single frozen-thawed blastocyst transfer (FBT). Previous studies report contradictory findings with respect to TE biopsy and obstetric and neonatal outcomes. METHODS: We conducted a retrospective cohort study including 720 patients with singleton pregnancies from single FBT cycles who delivered at the same university-affiliated hospital between January 2019 and March 2022. The cohorts were divided into two groups: the PGT group (blastocysts with TE biopsy, n = 223) and the control group (blastocysts without biopsy, n = 497). The PGT group was matched with the control group by propensity score matching (PSM) analysis at a ratio of 1:2. The enrolled sample sizes in the two groups were 215 and 385, respectively. RESULTS: Patient demographic characteristics were comparable between the groups after PSM except for the proportion of recurrent pregnancy loss, which was significantly higher in the PGT cohort (31.2 vs. 4.2%, P < 0.001). Patients in the PGT group had significantly higher rates of gestational hypertension (6.0 vs. 2.6%, adjusted odds ratio (aOR) 2.91, 95% confidence interval (CI) 1.18-7.18, P = 0.020) and abnormal umbilical cord (13.0 vs. 7.8%, aOR 1.94, 95% CI 1.08-3.48, P = 0.026). However, the occurrence of premature rupture of membranes (PROM) (12.1 vs. 19.7%, aOR 0.59, 95% CI 0.35-0.99, P = 0.047) was significantly lower in biopsied blastocysts than in unbiopsied embryos. There were no significant differences in regard to other obstetric and neonatal outcomes between the two groups. CONCLUSIONS: Trophectoderm biopsy is a safe approach, as the neonatal outcomes from biopsied and unbiopsied embryos were comparable. Furthermore, PGT is associated with higher risks of gestational hypertension and abnormal umbilical cord but may have a protective effect on PROM.

Should embryo rebiopsy be considered a regular strategy to increase the number of embryos available for transfer?

PURPOSE: To investigate whether embryo rebiopsy increases the yield of in vitro fertilization (IVF) cycles. METHODS: Retrospective study including 18,028 blastocysts submitted for trophectoderm biopsy and preimplantation genetic testing for aneuploidy (PGT-A) between January 2016 and December 2021 in a private IVF center. Out of the 517 embryos categorized as inconclusive, 400 survived intact to the warming procedure, re-expanded, and were suitable for rebiopsy. Of them, 71 rebiopsied blastocysts were transferred. Factors affecting the probability of obtaining an undiagnosed blastocyst and clinical outcomes from blastocysts biopsied once and twice were investigated. RESULTS: The overall diagnostic rate was 97.1%, with 517 blastocysts receiving inconclusive reports. Several blastocyst and laboratory features, such as the day of the biopsy, the stage of development, and the biopsy methodology, were related to the risk of obtaining an inconclusive diagnosis after PGT-A. A successful diagnosis was obtained in 384 of the rebiopsied blastocysts, 238 of which were chromosomally transferable. A total of 71 rebiopsied blastocysts were transferred, resulting in 32 clinical pregnancies [(clinical pregnancy rate (CPR)=45.1%], 16 miscarriages [(miscarriage rate (MR)=41%], and, until September 2020, 12 live births [(live birth rate (LBR)=23.1%]. A significantly lower LBR and higher MR were obtained after transferring rebiopsied blastocysts compared to those biopsied once. CONCLUSION: Although an extra round of biopsy and vitrification may cause a detrimental effect on embryo viability, re-analyzing the test-failure blastocysts contributes to increasing the number of euploid blastocysts available for transfer and the LBR.

A novel embryo biopsy morphological analysis and genetic integrality criterion system significantly improves the outcome of preimplantation genetic testing.

PURPOSE: While efforts have been made to establish blastocyst grading systems in the past decades, little research has examined the quality of biopsy specimens. This study is the first to correlate the morphology of biopsied trophectoderm (TE) cells to their quality and subsequent genetic testing results of preimplantation genetic testing (PGT), through an innovative Morphological Analysis and Genetic Integrality Criterion (MAGIC) system. METHODS: Biopsied TE cells were first evaluated according to the MAGIC procedure, followed by whole-genome amplification (WGA) and library construction, and then sequenced using the Illumina X Ten Platform. Copy number variation (CNV) and allele drop-out (ADO) rates as well as test failure rates were compared and analyzed. RESULTS: Our data explores the relationship between TE cell morphology and its quality and final genetic testing outcome, which is established based on the MAGIC system. MAGIC guarantees that only high- or good-quality TE cells are used for genetic testing to generate excellent data uniformity and lower ADO rates. Low-quality cells containing biopsied TE cell mass are responsible for the "background noise" of CNV analysis. CONCLUSION: The MAGIC application has effectively decreased the false-positive mosaicism, hence to ensure the stability and veracity of detection results, to avoid misdiagnoses, and to improve accuracy, as well as to avoid re-biopsy procedures. The study also contributes to understand how the IVF laboratory and the molecular biology laboratory depend on each other to achieve good-quality PGT results, which are clinically relevant for the patients.

Quality assurance (QA) for monitoring the performance of assisted reproductive technology (ART) staff using artificial intelligence (AI).

PURPOSE: Deep learning neural networks have been used to predict the developmental fate and implantation potential of embryos with high accuracy. Such networks have been used as an assistive quality assurance (QA) tool to identify perturbations in the embryo culture environment which may impact clinical outcomes. The present study aimed to evaluate the utility of an AI-QA tool to consistently monitor ART staff performance (MD and embryologist) in embryo transfer (ET), embryo vitrification (EV), embryo warming (EW), and trophectoderm biopsy (TBx). METHODS: Pregnancy outcomes from groups of 20 consecutive elective single day 5 blastocyst transfers were evaluated for the following procedures: MD performed ET (N = 160 transfers), embryologist performed ET (N = 160 transfers), embryologist performed EV (N = 160 vitrification procedures), embryologist performed EW (N = 160 warming procedures), and embryologist performed TBx (N = 120 biopsies). AI-generated implantation probabilities for the same embryo cohorts were estimated, as were mean AI-predicted and actual implantation rates for each provider and compared using Wilcoxon singed-rank test. RESULTS: Actual implantation rates following ET performed by one MD provider: "H" was significantly lower than AI-predicted (20% vs. 61%, p = 0.001). Similar results were observed for one embryologist, "H" (30% vs. 60%, p = 0.011). Embryos thawed by embryologist "H" had lower implantation rates compared to AI prediction (25% vs. 60%, p = 0.004). There were no significant differences between actual and AI-predicted implantation rates for EV, TBx, or for the rest of the clinical staff performing ET or EW. CONCLUSIONS: AI-based QA tools could provide accurate, reproducible, and efficient staff performance monitoring in an ART practice.

PGT-A: Houston, we have a problem.

Preimplantation genetic testing for aneuploidy (PGT-A) is a common add-on to IVF cycles. As it is presently performed, PGT-A relies on whole genome amplification of small amounts of DNA from cells removed from the trophectoderm (TE) of a blastocyst for determination of gain or loss of chromosomal material by next-generation sequencing. Whole genome amplification may introduce artifacts such as allele dropout and loss of heterozygosity in up to 25% of cases. In addition, the high prevalence of mosaicism in human embryos is a complicating factor in interpreting the results of PGT-A screening. In the presence of mosaicism, biopsy of TE cells cannot provide accurate results regarding the chromosomal make-up of the inner cell mass. The available clinical data suggest that PGT-A is probably harmful when IVF outcomes are analyzed by intention to treat or by live birth rate per cycle started rather than per embryo transfer, especially in women with three or fewer blastocysts. In addition, hypothesized advantages of reduced spontaneous abortion rate and reduced time to conception may be modest at best.

Evaluation of non-invasive gene detection in preimplantation embryos: a systematic review and meta-analysis.

BACKGROUND: Genetic abnormalities in embryos are responsible for most miscarriages and repeated embryo implantation failures, so a reliable preimplantation genetic screening method is urgently needed. Non-invasive preimplantation genetic testing (niPGT) is a potential method for embryo genetic diagnosis. However, the value of its application is controversial. This meta-analysis aimed to investigate and validate the diagnostic value of niPGT in patients undergoing in vitro fertilization (IVF). METHODS: This review used the "Preferred Reporting Items" as a systematic review and meta-analysis of the diagnostic test accuracy (PRISMA-DTA) statement. We searched PubMed, Embase, Web of Science Core Collection, and Cochrane Library up to May 2022 to retrieve non-invasive preimplantation gene detection studies. The eligible research quality was evaluated following the quality assessment study-2 system for diagnostic accuracy. The pooled receiver operator characteristic curve (SROC) and the area under SROC (AUC) were used to evaluate diagnostic performance quantitatively. Threshold effect, subgroup analysis, and meta-regression analysis were used to explore the source of heterogeneity. Deeks' funnel plots and sensitivity analyses were used to test the publication bias and stability of the meta-analysis, respectively. FINDINGS: Twenty studies met the inclusion criteria. The pooled sensitivity, specificity, and AUC were 0.84 (95% CI 0.72-0.91), 0.85 (95% CI 0.74-0.92), and 0.91 (95% CI 0.88-0.93), respectively. Subgroup analysis showed that the spent culture medium (SCM) subgroup had higher sensitivity and lower specificity than the SCM combined with the blastocoel fluid (BF) subgroup. Subgroup analysis showed that the study sensitivity and specificity of < 100 cases were higher than those of ≥ 100. Heterogeneity (chi-square) analysis revealed that sample size might be a potential source of heterogeneity. Sensitivity analysis and Deeks' funnel plots indicated that our results were relatively robust and free from publication bias. INTERPRETATION: The present meta-analysis indicated that the pooled sensitivity, specificity, and AUC of niPGT in preimplantation genetic testing were 0.84, 0.85, and 0.91, respectively. niPGT may have high detection accuracy and may serve as an alternative model for embryonic analysis. Additionally, by subgroup analysis, we found that BF did not improve the accuracy of niPGT in embryos. In the future, large-scale studies are needed to determine the detection value of niPGT.

Adverse maternal and neonatal outcomes of preimplantation genetic testing with trophectoderm biopsy: a retrospective cohort study of 3373 intracytoplasmic sperm injection single frozen-thawed blastocyst transfer cycles.

PURPOSE: To investigate whether trophectoderm biopsy increases the risk of adverse maternal and neonatal outcomes in intracytoplasmic sperm injection (ICSI) single frozen-thawed blastocyst transfer cycles. METHODS: This respective cohort study enrolled 3373 ICSI single frozen-thawed blastocyst transfer cycles with and without trophectoderm biopsy. Statistical methods including univariate logistic regression analysis, multivariate logistic regression analysis, and stratified analyses were performed to explore the impact of trophectoderm biopsy on adverse maternal and neonatal outcomes. RESULTS: The rates of adverse maternal and neonatal outcomes were comparable between the two groups. Univariate analysis showed that the live birth rate (45.15% vs. 40.75%; P = 0.010) in the biopsied group was statistically higher than that in the unbiopsied group, and the rates of miscarriage (15.40% vs. 20.00%; P = 0.011) and birth defects (0.58% vs. 2.16%; P = 0.007) were statistically lower in the biopsied group. After adjusting for confounding factors, the rates of miscarriage (aOR = 0.74; 95% CI = 0.57-0.96; P = 0.022) and birth defects (aOR = 0.24, 95% CI = 0.08-0.70, P = 0.009) in the biopsied group were significantly lower than those in the unbiopsied group. Stratified analyses showed that the birth defects rate after biopsy was significantly reduced in the subgroups of age < 35 years old, BMI ≥ 24 kg/m2, artificial cycle with downregulation, poor-quality blastocysts, and Day 5 poor-quality blastocysts. CONCLUSION: Preimplantation genetic testing (PGT) with trophectoderm biopsy does not increase the risk of adverse maternal and neonatal outcomes in ICSI single frozen-thawed blastocyst transfer cycles, and PGT can effectively reduce the rates of miscarriage and birth defects.

Segmental aneuploid hotspots identified across the genome concordant on reanalysis.

The aim of this study was to characterize a large set of full segmental aneuploidies identified in trophectoderm (TE) biopsies and evaluate concordance in human blastocysts. Full segmental aneuploid errors were identified in TE biopsies (n = 2766) from preimplantation genetic testing for aneuploid (PGT-A) cycles. Full segmental deletions (n = 1872; 66.1%) presented twice as many times as duplications (n = 939; 33.9%), mapped more often to the q-arm (n = 1696; 61.3%) than the p-arm (n = 847; 31.0%) or both arms (n = 223; 8.1%; P < 0.05), and were eight times more likely to include the distal end of a chromosome than not (P < 0.05). Additionally, 37 recurring coordinates (each ≥ 10 events) were discovered across 17 different chromosomes, which were also significantly enriched for distal regions (P = 4.1 × 10-56). Blinded concordance analysis of 162 dissected blastocysts validated the original TE PGT-A full segmental result for a concordance of 96.3% (n = 156); remaining dissected blastocysts were identified as mosaic (n = 6; 3.7%). Origin of aneuploid analysis revealed full segmental aneuploid errors were mostly paternally derived (67%) in contrast to whole chromosome aneuploid errors (5.8% paternally derived). Errors from both parental gametes were observed in 6.5% of aneuploid embryos when multiple whole chromosomes were affected. The average number of recombination events was significantly less in paternally derived (1.81) compared to maternally derived (3.81) segmental aneuploidies (P < 0.0001). In summary, full segmental aneuploidies were identified at hotspots across the genome and were highly concordant upon blinded analysis. Nevertheless, future studies assessing the reproductive potential of full (non-mosaic) segmental aneuploid embryos are critical to rule out potential harmful reproductive risks.

Effect of parental origin and predictors for obtaining a euploid embryo in balanced translocation carriers.

RESEARCH QUESTION: What is the effect of parental origin of translocation and predictors for obtaining a euploid embryo in preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) for balanced translocation carriers? DESIGN: A total of 179 PGT-SR cycles and 614 blastocysts from 123 couples carrying a balanced translocation were retrospectively analysed. Next-generation sequencing (NGS) was performed after trophectoderm biopsy. RESULTS: There were no differences in ovarian stimulation parameters or PGT-SR outcomes regarding the number of oocytes retrieved (11.95 ± 5.71 versus 11.82 ± 6.26), blastulation rate (0.42 ± 0.27 versus 0.45 ± 0.28), biopsy cancellation rate (11.7% versus 12.9%), the number of blastocysts for biopsy (3.70 ± 2.58 versus 4.04 ± 3.51), or the proportion of euploid embryos (23.80% versus 25.42%), aneuploid embryos (58.10% versus 57.52%) and mosaic embryos (18.10% versus 17.06%) between female carriers and male partner carriers. In a multivariate logistic regression model, the number of blastocysts for biopsy (adjusted odds ratio 1.752; 95% confidence interval 1.359-2.259; P < 0.001) was significantly associated with the chance of obtaining at least one euploid embryo. Receiver operating characteristic analysis with a threshold of 3.5 was conducted to calculate the number of blastocysts required for biopsy to obtain at least one euploid embryo. CONCLUSIONS: The parental origin of translocation does not significantly affect the PGT-SR outcomes for young balanced translocation carriers. At least 3.5 blastocysts are required to obtain one euploid embryo. Couples should be informed that the probability of obtaining one euploid embryo is low when fewer than 4 blastocysts are obtained in one PGT cycle.

Neonatal and clinical outcomes after transfer of a mosaic embryo identified by preimplantation genetic testing for aneuploidies.

RESEARCH QUESTION: Do clinical and neonatal outcomes differ between mosaic embryo transfers (MET) and euploid embryo transfers (EET)? DESIGN: This retrospective cohort study compared the implantation rate, live birth rate (LBR) and miscarriage rate between 513 euploid embryos and 118 mosaic embryos (72 whole chromosome mosaic [WCM], 40 segmental mosaic and six complex mosaic). Blastocysts were analysed using preimplantation genetic testing for aneuploidies with next-generation sequencing, followed by a single vitrified-warmed embryo transfer. Trophectoderm biopsies were classified as mosaic if they had 20-80% abnormal cells. RESULTS: Overall, EET resulted in a significantly higher implantation rate (47.0%) and LBR (40.7%) than MET (implantation rate 39.0%, P = 0.005; LBR 28.8%, P = 0.008) and WCM embryos (implantation rate 37.5%, P = 0.01; LBR 22.2%, P = 0.007) after covariate adjustment. Segmental mosaic embryos had an implantation rate (47.5%) and LBR (45.0%) comparable to those of euploid embryos. Mosaic embryos with a high percentage of aneuploid cells (≥60%) showed a significantly lower LBR (10.5% versus 40.7%, P = 0.03) than euploid embryos after covariate adjustment, with three of the five implantations of mosaic embryos resulting in miscarriage. Neonatal outcomes did not differ significantly between the mosaic and euploid groups. Of the 34 women with a live birth after MET, 13 had a prenatal or postnatal genetic testing result, and no abnormalities were found. CONCLUSIONS: Mosaic embryos were associated with a lower LBR, while segmental mosaic embryos had similar clinical outcomes to euploid embryos. Mosaic embryos with a high aneuploidy percentage (≥60%) should be assigned a low transfer priority. Neonatal outcomes did not differ significantly between the euploid and mosaic groups.

The effects of the day of trophectoderm biopsy and blastocyst grade on the clinical and neonatal outcomes of preimplantation genetic testing-frozen embryo transfer cycles.

This study analyzed the effects of the day of trophectoderm (TE) biopsy and blastocyst grade on clinical and neonatal outcomes. The results showed that the implantation and live birth rates of day 5 (D5) TE biopsy were significantly higher compared with those of D6 TE biopsy. The miscarriage rate of the former was lower than that of the latter, but there was no statistically significant difference. Higher quality blastocysts can achieve better implantation and live birth rates. Among good quality blastocysts, the implantation and live birth rates of D5 and D6 TE biopsy were not significantly different. Among fair quality and poor quality blastocysts, the implantation and live birth rates of D5 TE biopsy were significantly higher compared with those of D6 TE biopsy. Neither blastocyst grade nor the day of TE biopsy significantly affected the miscarriage rate. Neonatal outcomes, including newborn sex, gestational age, preterm birth, birth weight and low birth weight in the D5 and D6 TE biopsies were not significantly different. Both blastocyst grade and the day of TE biopsy must be considered at the same time when performing preimplantation genetic testing-frozen embryo transfer.