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• Embryo rebiopsy significantly reduces live birth rate. • Embryo rebiopsy does not affect birthweight/sex ratio. • Longterm follow-up of children born following double embryo biopsy is lacking. • Subfertility background/embryo characteristics are predictors for live birth. To study the effect of double trophectoderm biopsy on clinical outcomes following single euploid blastocyst transfer. Retrospective cohort study of 2046 single euploid frozen-thawed blastocyst transfers from January 2015 to June 2022 in a single centre. All patients undergoing a frozen-thawed embryo transfer (FTET) cycle with euploid blastocysts, biopsied for any indication, were included. The outcomes were compared for blastocysts which were biopsied and vitrified once (Group 1, n = 1684), biopsied once but vitrified twice (Group 2, n = 312) and biopsied and vitrified twice (Group 3n = 50). We adjusted for confounders and performed subgroup analysis for PGT-A, PGT-M and PGT-SR cycles. The primary outcome was live birth rate. Secondary outcomes included pregnancy, clinical pregnancy, birthweight and sex ratio. After adjusting for confounders (previous failed euploid implantations, embryo quality and day of biopsy), embryos which were biopsied twice had lower OR for clinical pregnancy (0.48, CI 0.26–0.88, p = 0.019) and for live birth (0.50 CI 0.27–0.92, p = 0.025) compared to controls. Embryos which were biopsied once but vitrified twice had no different ORs for all reproductive outcomes compared to controls. No significant difference was observed for neonatal birthweight or sex ratio amongst the three groups. This is a retrospective single centre study with inherent bias and results may not be transferable to all settings. This study is the largest to date assessing the outcomes of FTET cycles following double trophectoderm biopsy. The results are in keeping with the existing literature and can be incorporated into patient counselling. Whilst double biopsy seems to adversely impact LBR, it is only one of the many factors that can affect success rates. The subfertility background and embryo characteristics should not be overlooked. This study provides reassuring evidence since double biopsied embryos still result in live births with no difference in sex ratio or birthweight. However, long term follow up of the off-springs is lacking and should be reported in future studies. [ABSTRACT FROM AUTHOR]

• The results demonstrate a clear pattern of increasing aneuploidy with donor age. • Younger donors exhibited a reduced rate of aneuploid embryos. • The rate of embryonic mosaicism seems comparable irrespective of the age of the donor. Conflicting data exists regarding whether a younger age of donors has a negative influence on the outcomes of oocyte donation cycles. Is there any correlation between a younger age of donors and the rate of embryonic aneuploidy in oocyte donation cycles? Retrospective study including 515 oocyte donation cycles carried out between February 2017 and November 2022. Comprehensive chromosomal screening was performed on 1831 blastocysts. 1793 had a result which were categorised into groups based on the age of the donor: 18–22 (n = 415), 23–25 (n = 600), 26–30 (n = 488), and 31–35 years (n = 290). The analysis aimed to determine the percentage of biopsy samples that were euploid and the number that were aneuploid, relative to the age group of the oocyte donor. Additionally, linear regression was employed to examine the relationship between age and the proportion of aneuploid embryos, while controlling for relevant variables. Aneuploidy increased predictably with donor age: 18–22 years: 27.5 %; 23–25 years: 31.2 %; 26–30 years: 31.8 %; and 31–35 years: 38.6 %. In the donor group aged 31–35 years, a higher percentage of aneuploid embryos was observed compared to younger donors in univariate analysis (OR: 1.66, 95 % CI: 1.21–2.29, p = 0.002) and multivariate logistic analysis (OR: 2.65, 95 % CI: 1.67–4.23, p < 0.001). The rates of embryonic mosaicism revealed no significant differences. The lowest risk of embryonic aneuploidy was found among donors aged <22 years. Conversely, an elevated prevalence was evident within the donor group aged 31–35 years, in contrast to the younger cohorts. The incidence of mosaic embryos remained consistent across all age groups. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The implementation of next generation sequencing (NGS) in preimplantation genetic testing for aneuploidy (PGT-A) has led to a higher prevalence of mosaic diagnosis within the trophectoderm (TE) sample. Regardless, mosaicism could potentially increase the rate of live-born children with chromosomic syndromes, though available data from the transfer of embryos with putative PGT-A mosaicism are scarce but reassuring. Even with lower implantation and higher miscarriage rates, mosaic embryos can develop into healthy live births. Therefore, this urges an explanation for the disappearance of aneuploid cells throughout development, to provide guidance in the management of mosaicism in clinical practice. Technical overestimation of mosaicism, together with some sort of "self-correction" mechanisms during the early post-implantation stages, emerged as potential explanations. Unlike the animal model, in which the elimination of genetically abnormal cells from the future fetal lineage has been demonstrated, in human embryos this capability remains unverified even though the germ layer displays an aneuploidy-induced cell death lineage preference with higher rates of apoptosis in the inner cell mass (ICM) than in the TE cells. Moreover, the reported differential dynamics of cell proliferation and apoptosis between euploid, mosaic, and aneuploid embryos, together with pro-apoptosis gene products (cfDNA and mRNA) and extracellular vesicles identified in the blastocoel fluid, may support the hypothesis of apoptosis as a mechanism to purge the preimplantation embryo of aneuploid cells. Alternative hypotheses, like correction of aneuploidy by extrusion of a trisomy chromosome or by monosomic chromosome duplication, are even, though they represent an extremely rare phenomenon. On the other hand, the technical limitations of PGT-A analysis may lead to inaccuracy in embryo diagnoses, identifying as "mosaic" those embryos that are uniformly euploid or aneuploid. NGS assumption of "intermediate copy number profiles" as evidence of a mixture of euploid and aneuploid cells in a single biopsy has been reported to be poorly predictive in cases of mosaicism diagnosis. Additionally, the concordance found between the TE and the ICM in cases of TE biopsies displaying mosaicism is lower than expected, and it correlates differently depending on the type (whole chromosome versus segmental) and the level of mosaicism reported. Thus, in cases of low-/medium-level mosaicism (<50%), aneuploid cells would rarely involve the ICM and other regions. However, in high-level mosaics (≥50%), abnormal cells in the ICM should display higher prevalence, revealing more uniform aneuploidy in most embryos, representing a technical variation in the uniform aneuploidy range, and therefore might impair the live birth rate. [ABSTRACT FROM AUTHOR]

ICSI is widely recommended for patients undergoing preimplantation genetic testing (PGT), but are sperm a potential source of paternal cell contamination in PGT? Semen samples were obtained from five normozoospermic men consenting to research. From each sample 1, 2, 4, 8 and 10 sperm were collected in PCR tubes and whole genome amplification according to PGT-A and PGT-SR processing protocols was undertaken. None of the 25 samples submitted (a total of 125 sperm) showed evidence of DNA amplification. Thus, paternal cell contamination resulting from using conventional in vitro fertilization (IVF) as the insemination method, carries a low risk of an adverse event or misdiagnosis in PGT-A. Due to the higher risk incurred with PGT-SR, clinics may wish to exercise increased caution and continue using ICSI, while PGT-M involves different processing protocols, presenting a different risk profile. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

BACKGROUND A normal chromosomal constitution defined through PGT-A assessing all chromosomes on trophectoderm (TE) biopsies represents the strongest predictor of embryo implantation. Yet, its positive predictive value is not higher than 50–60%. This gap of knowledge on the causes of euploid blastocysts' reproductive failure is known as 'the black box of implantation'. OBJECTIVE AND RATIONALE Several embryonic, maternal, paternal, clinical, and IVF laboratory features were scrutinized for their putative association with reproductive success or implantation failure of euploid blastocysts. SEARCH METHODS A systematic bibliographical search was conducted without temporal limits up to August 2021. The keywords were '(blastocyst OR day5 embryo OR day6 embryo OR day7 embryo) AND (euploid OR chromosomally normal OR preimplantation genetic testing) AND (implantation OR implantation failure OR miscarriage OR abortion OR live birth OR biochemical pregnancy OR recurrent implantation failure)'. Overall, 1608 items were identified and screened. We included all prospective or retrospective clinical studies and randomized-controlled-trials (RCTs) that assessed any feature associated with live-birth rates (LBR) and/or miscarriage rates (MR) among non-mosaic euploid blastocyst transfer after TE biopsy and PGT-A. In total, 41 reviews and 372 papers were selected, clustered according to a common focus, and thoroughly reviewed. The PRISMA guideline was followed, the PICO model was adopted, and ROBINS-I and ROB 2.0 scoring were used to assess putative bias. Bias across studies regarding the LBR was also assessed using visual inspection of funnel plots and the trim and fill method. Categorical data were combined with a pooled-OR. The random-effect model was used to conduct the meta-analysis. Between-study heterogeneity was addressed using I 2. Whenever not suitable for the meta-analysis, the included studies were simply described for their results. The study protocol was registered at http://www.crd.york.ac.uk/PROSPERO/ (registration number CRD42021275329). OUTCOMES We included 372 original papers (335 retrospective studies, 30 prospective studies and 7 RCTs) and 41 reviews. However, most of the studies were retrospective, or characterized by small sample sizes, thus prone to bias, which reduces the quality of the evidence to low or very low. Reduced inner cell mass (7 studies, OR: 0.37, 95% CI: 0.27–0.52, I 2 = 53%), or TE quality (9 studies, OR: 0.53, 95% CI: 0.43–0.67, I 2 = 70%), overall blastocyst quality worse than Gardner's BB-grade (8 studies, OR: 0.40, 95% CI: 0.24–0.67, I 2 = 83%), developmental delay (18 studies, OR: 0.56, 95% CI: 0.49–0.63, I 2 = 47%), and (by qualitative analysis) some morphodynamic abnormalities pinpointed through time-lapse microscopy (abnormal cleavage patterns, spontaneous blastocyst collapse, longer time of morula formation I, time of blastulation (tB), and duration of blastulation) were all associated with poorer reproductive outcomes. Slightly lower LBR, even in the context of PGT-A, was reported among women ≥38 years (7 studies, OR: 0.87, 95% CI: 0.75–1.00, I 2 = 31%), while obesity was associated with both lower LBR (2 studies, OR: 0.66, 95% CI: 0.55–0.79, I 2 = 0%) and higher MR (2 studies, OR: 1.8, 95% CI: 1.08–2.99, I 2 = 52%). The experience of previous repeated implantation failures (RIF) was also associated with lower LBR (3 studies, OR: 0.72, 95% CI: 0.55–0.93, I 2 = 0%). By qualitative analysis, among hormonal assessments, only abnormal progesterone levels prior to transfer were associated with LBR and MR after PGT-A. Among the clinical protocols used, vitrified-warmed embryo transfer was more effective than fresh transfer (2 studies, OR: 1.56, 95% CI: 1.05–2.33, I 2 = 23%) after PGT-A. Lastly, multiple vitrification-warming cycles (2 studies, OR: 0.41, 95% CI: 0.22–0.77, I 2 = 50%) or (by qualitative analysis) a high number of cells biopsied may slightly reduce the LBR, while simultaneous zona-pellucida opening and TE biopsy allowed better results than the Day 3 hatching-based protocol (3 studies, OR: 1.41, 95% CI: 1.18–1.69, I 2 = 0%). WIDER IMPLICATIONS Embryo selection aims at shortening the time-to-pregnancy, while minimizing the reproductive risks. Knowing which features are associated with the reproductive competence of euploid blastocysts is therefore critical to define, implement, and validate safer and more efficient clinical workflows. Future research should be directed towards: (i) systematic investigations of the mechanisms involved in reproductive aging beyond de novo chromosomal abnormalities, and how lifestyle and nutrition may accelerate or exacerbate their consequences; (ii) improved evaluation of the uterine and blastocyst-endometrial dialogue, both of which represent black boxes themselves; (iii) standardization/automation of embryo assessment and IVF protocols; (iv) additional invasive or preferably non-invasive tools for embryo selection. Only by filling these gaps we may finally crack the riddle behind 'the black box of implantation'. [ABSTRACT FROM AUTHOR]

Abstract 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

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. [ABSTRACT FROM AUTHOR]

Arrested cells/ cellular debris is component left in the zona pellucida after blastocyst hatching. To identify whether expelling arrested cells/cellular debris from blastocysts is a process of human embryo self-correction by eliminating abnormal cells, 21 pairs of trophectoderm (TE) biopsies and the corresponding arrested cells/cellular debris expelled from the blastocysts from July to December 2020 were collected and analyzed using next-generation sequencing (NGS). Then, the NGS results of TE biopsies and the corresponding arrested cells/cellular debris were compared. We identified that 47.6% of blastocysts (10/21) were aneuploidies and mosaicism. A total of 18 groups of arrested cells/cellular debris (85.7%) expelled from blastocysts were abnormal, including nine aneuploid embryos and nine euploid embryos. In the arrested cells/cellular debris, all the chromosomes were affected. In conclusion, mosaicism and aneuploidies are common features of early embryonic development, and the arrested cells/cellular debris expelled from blastocysts provides evidence of early embryonic self-correction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

BACKGROUND Preimplantation genetic testing (PGT) of embryos developed in vitro requires a biopsy for obtaining cellular samples for the analysis. Signs of cell injury have been described in association with this procedure. Thus, the consequences of the biopsy on obstetric and neonatal outcomes have been the subject of some quantitative analyses, although the reliability of data pooling may be limited by important issues in the various reports. OBJECTIVE AND RATIONALE The present review identifies evidence for whether pregnancies conceived after embryo biopsy are associated with a higher risk of adverse obstetric, neonatal, and long-term outcomes. Available evidence has been summarized considering manipulation at various stages of embryo development. SEARCH METHODS We used the scoping review methodology. Searches of article databases were performed with keywords pertaining to the embryo biopsy technique and obstetric, neonatal, and postnatal outcomes. Studies in which embryos were biopsied at different stages (i.e. both at the cleavage and blastocyst stages) were excluded. We included data on fresh and frozen embryo transfers. The final sample of 31 documents was subjected to qualitative thematic analysis. OUTCOMES Sound evidence is lacking to fully address the issues on the potential obstetric, neonatal or long-term consequences of embryo biopsy. For polar body biopsy, the literature is too scant to draw any conclusion. Some data, although limited and controversial, suggest a possible association of embryo biopsy at the cleavage stage with an increased risk of low birthweight and small for gestational age neonates compared to babies derived from non-biopsied embryos. An increase in preterm deliveries and birth defects in cases of trophectoderm biopsy was suggested. For both biopsy methods (at the cleavage and blastocyst stages), an increased risk for hypertensive disorders of pregnancy was found. However, these findings may be explained by confounders such as other embryo manipulation procedures or by intrinsic patient or population characteristics. WIDER IMPLICATIONS Since there is inadequate evidence to assess obstetric, neonatal, and long-term health outcomes following embryo biopsy, an invasive PGT strategy should be developed with a cautious approach. A non-invasive approach, based on the analysis of embryo cell-free DNA, needs to be pursued to overcome the potential limitations of embryo biopsy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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? 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. 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. 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. [ABSTRACT FROM AUTHOR]

Contributed to the development of next-generation sequencing (NGS) technology, more and more chromosomally mosaic and aneuploid embryos are discovered during the preimplantation genetic testing for aneuploidy (PGT-A) cycles. Because mosaicism and aneuploidy are routine phenomena throughout human pre- and post-implantation development. The benefit of implanting such mosaicism or aneuploidies detected by precise NGS remains controversial. This study aimed to investigate chromosomal concordance between babies produced by PGT-A and trophectoderm (TE) biopsies, and whether precise NGS resolution would reduce the development of an abnormal embryo in PGT cycles. Peripheral blood samples from 17 PGT-A babies were collected to compare with TE biopsy results at different NGS resolutions. 16 euploid embryos diagnosed by 10 Mb resolution developed into 16 healthy babies with normal copy number variations (CNVs). One mosaic embryo diagnosed by both 10 Mb and 4 Mb resolution also produced a euploid baby finally. Among them, four euploid embryos diagnosed by 10 Mb NGS, showed segmental aneuploidy at 4 Mb NGS resolution. Four of them developed into euploid babies with normal CNVs finally. NGS at 10 Mb resolution is accurate enough to diagnose viable embryos. A more precise NGS resolution (e.g., 4 Mb resolution) results in discard of some potentially viable embryos. It is suggested to analyze the TE biopsy at both 10 Mb and 4 Mb resolutions to identify embryos with adverse chromosomal aberrations, but using 10 Mb resolution for guide transfer to increase a development chance of an embryo. Trial registration: www.ClinicalTrials.gov , identifier ChiCTR2100042522. [ABSTRACT FROM AUTHOR]

To present a novel trophectoderm biopsy method, independent of laser pulses, using innovatively designed micropipettes on blastocysts at different stages and show variable characteristics. A step-by-step demonstration of this method with narrated video. In vitro laboratory fertilization. Individuals whose embryos underwent preimplantation genetic testing. Trophectoderm biopsy is accomplished using micropipettes that contain a set of innovative designs. Both biopsy and holding pipettes are characterized by sharp, flat opening ends. The holding pipette is designed with an inclined plane on the outer wall surface of its opening end. It is used to help the biopsy pipette make contact with the holding pipette with increased stability, preventing slipping during the detachment of the trophectoderm cells. There is a narrow structure inside the biopsy pipette, which is designed to trap released fragments and prevent sample loss. A trophectoderm biopsy for fully expanded blastocysts starts from artificial shrinkage, followed by zona pellucida drilling. Then, 5–10 trophectoderm cells are aspirated into a biopsy pipette. The blastocyst is released from the holding pipette, the edge of the opening end of the biopsy pipette is tightly pressed onto the inclined plane of the holding pipette, and the biopsy pipette is flicked directly without laser pulses or pulling off the trophectoderm cells. The aspirated trophectoderm cells are subsequently detached by mechanical friction between the edges of the biopsy and holding pipettes. Apart from drilling the zona pellucida for fully expanded blastocysts, the remaining steps do not require lasers. For hatching (including peanut-shaped and 8-shaped) and hatched blastocysts, a trophectoderm biopsy is accomplished by aspirating the cells without securing the blastocyst with a holding pipette, followed by detachment using the direct flicking method. The biopsy time, sample loss rate, successful DNA amplification rate, and survival rate. The innovatively designed micropipettes facilitate the successful detachment of trophectoderm cells through a single direct flicking procedure. This eliminates thermal damage caused by laser pulses, notably simplifying operational steps and shortening the biopsy time. Significant differences were noted between the direct flicking and conventional methods, wherein laser pulses and pulling of trophectoderm cells are prerequisites for cell detachment. When comparing the average biopsy time of fully expanded blastocyst (61 ± 8s vs. 104 ± 9s, P <.05), peanut-shaped hatching blastocyst (35 ± 6s vs. 113 ± 13s, P <.05), 8-shaped hatching blastocyst (32 ± 4s vs. 59 ± 6s, P <.05), and hatched blastocyst (34 ± 4s vs. 67 ± 8s, P <.05), the direct flicking method shows a significantly decreased biopsy time. The narrow structure inside the biopsy pipette effectively prevents sample loss, showing a significantly reduced sample loss rate (0%) compared with the conventional biopsy method (18%) for trainees. Moreover, a satisfactory survival rate (100%) and successful DNA amplification rate (99.5%) were achieved using the direct flicking method. This innovative trophectoderm biopsy method, independent of laser pulses, has wide applicability and a satisfactory, stable performance. Moreover, the simplicity of the method makes it easy to master. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: To investigate whether the noninvasive preimplantation genetic testing (niPGT) complement conventional preimplantation genetic testing (PGT) in the embryos for aneuploidy., Results: 40 spent culture medium (SCM) samples from routine embryo culture were collected, and half of each SCM (10 µL) sample was used for whole genome amplification, while the other half was stored at -80 °C for 3-6 months. Thirty-six out of 40 fresh SCM samples were successfully amplified and sequenced. Thirty-six paired frozen-thawed SCM samples showed 100% concordance with the freshly amplified SCM samples. Then, SCM and trophectoderm (TE) samples from 149 blastocysts from 51 couples were collected. A 98.0% successful SCM sample amplification rate (146/149) was achieved. For the 146 paired TE biopsy and SCM samples, the overall concordance rate was 82.9% (121/146). Ten embryos with aneuploid TE results but euploid niPGT results were donated. A 70.0% (7/10) true negative rate was achieved by niPGT with respect to the inner cell mass (ICM) results (TE-positive embryos)., Conclusions: These results suggested that SCM stored at -80 °C for 6 months without affecting niPGT results based on NICSInst amplification., Competing Interests: Declarations. Ethics approval and consent to participate: The study was conducted in accordance with the Declaration of Helsinki, and approved by the ethics committee of the International Peace Maternity & Child Health Hospital (IPMCH). Informed consent was obtained from all patients included in this study as required by the ethics committee of IPMCH. All experiments were performed following the relevant guidelines and regulations. Consent for publication: Not Applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Study Question: Does the number of biopsied trophectoderm cells sampled for preimplantation genetic testing for monogenic disease (PGT-M) affect subsequent clinical outcomes for those selected embryos?, Summary Answer: The number of biopsied cells does not affect the pregnancy outcome of preimplantation genetically tested embryos., What Is Known Already: The successful execution of PGT relies on the availability of a certain number of high-quality biopsied cells. Evidence in the literature has reported that blastocyst biopsies may have a negative impact on clinical outcomes., Study Design, Size, Duration: A retrospective cohort study including 850 single-blastocyst transfer cycles from 605 couples between May 2014 and August 2024 was conducted at Peking University Third Hospital. The primary clinical outcome measure was the biochemical pregnancy rate, while other indicators such as the live birth rate, the clinical pregnancy rate, and the miscarriage rate were also recorded., Participants/materials, Setting, Methods: This study included 850 blastocysts obtained from routine PGT-M cycles. Based on biopsied cell numbers, data were categorized into four groups: Group 1 (1-5 cells) (n = 234), Group 2 (6-10 cells) (n = 328), Group 3 (11-15 cells) (n = 192), and Group 4 (>15 cells) (n = 96)., Main Results and the Role of Chance: The number of cells biopsied from the embryo did not significantly affect either the biochemical pregnancy rate or the live birth rate in the routine PGT process (P > 0.05). There were 129 of 234 embryos (55.1%) in the 1-5 biopsied cell group, 183 of the 328 embryos (55.8%) with 6-10 biopsied cells, 92 of 192 embryos (47.9%) with 11-15 biopsied cells, and 48 of 96 (50.0%) embryos with more than 15 biopsied cells which achieved successful pregnancies. The live birth rates were 42.7%, 49.7%, 43.2%, and 43.8% for each of the biopsy groups, respectively., Limitations, Reasons for Caution: Data for this study were collected from one center only, therefore multicenter, large-scale cohort studies are essential to confirm the accuracy and the reliability of this study., Wider Implications of the Findings: The number of biopsied cells in a blastocyst is associated with the embryo quality and hatching status. The conclusion of this study emphasizes that routine procedures during the biopsy process do not affect pregnancy outcomes. It is crucial to strike a balance between minimizing damage to the blastocyst's developmental potential and achieving the highest possible detection efficiency for PGT-M., Study Funding/competing Interest(s): This project is funded by the National Key Research and Development Program of China (2019YFA0801401, 2019YFA0110001) and the National Natural Science Foundation of China (82125013). The authors declare that they have no competing interests., Trial Registration Number: N/A., (© The Author(s) 2025. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., (Copyright © 2024. Published by Elsevier Inc.)

Abstract Non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) aiming to assess cell-free embryonic DNA in spent culturemedia is promising, especially because it might overcome the diminished rates of implantation caused by the inadequate performance of trophectoderm (TE) biopsy. Our center is part of the largest study to date assessing the concordance between conventional PGT-A and niPGT-A, and we report here the delivery of the first baby born in Brazil using niPGT-A. The parents of the baby were admitted to our center in 2018. They did not present history of infertility, and they were interested in using in vitro fertilization (IVF) and PGT-A in order to avoid congenital anomalies in the offspring. A total of 11 (3 day-5 and 8 day-6) expanded blastocysts were biopsied, and the spent culture media (culture from day-4 to day-6) from 8 day-6 blastocysts were collected for niPGT-A. Overall, 7 embryos yielded informative results for trophectoderm (TE) and media samples. Among the embryos with informative results, 5 presented concordant diagnosis between conventional PGTA and niPGT-A, and 2 presented discordant diagnosis (1 false-positive and one falsenegative). The Blastocyst 4, diagnosed as 46, XY by both niPGT-A and conventional PGTA, was warmed up and transferred, resulting in the birth of a healthy 3.8 kg boy in February 2020. Based on our results and the recent literature, we believe that the safest current application of niPGT-A would be as a method of embryo selection for patients without an indication for conventional PGT-A. The approximate 80% of reliability of niPGT-A in the diagnosis of ploidy is superior to predictions provided by other noninvasive approaches like morphology and morphokinetics selection.

The worldwide demand of preimplantation genetic testing for aneuploidy (PGT-A) is still growing. However, chromosomal mosaic results greatly challenge the clinical practice. The recently published PGDIS Position Statement on the Transfer of Mosaic Embryos is the third PGDIS position statement on how to deal with embryos diagnosed as chromosomal mosaics (CM) and, one of many attempts of different societies and working groups to provide a guideline for clinicians, laboratories, clinics, and genetic counselors. But still, as in previous statements, many issues remained unresolved. Moreover, from our point of view, the question how to deal with embryos diagnosed as CM, consisting of two or more karyological cell lines cannot be separated from all the other aspects of PGT-A including its accuracy. The paucity of clearcut indications for PGT-A and evidence of benefit as well as an overall cost–benefit assessment is given below. [ABSTRACT FROM AUTHOR]

Purpose: To determine if blastocyst trophectoderm biopsy for PGT-A is associated with an increased rate of live birth per embryo in good prognosis IVF patients at a single center.Methods: We performed a retrospective cohort study of good prognosis embryo transfer cycles at a single center from 1/1/2017 to 12/31/2019. We evaluated the rate of live birth per embryo with and without PGT-A for transfer of embryos in two groups of good prognosis patients: embryos from donor oocytes and embryos from autologous oocytes with maternal age less than 35 years at oocyte retrieval. Two-sided Fisher's exact tests were used for comparisons between groups.Results: After transfer of embryos created from donor oocytes the live birth rate per euploid embryo was 70.6% (24/34) compared to 34.3% (35/102) for untested embryos for a rate difference of 36.3% (95% CI 18.4-54.1%, p < 0.01). After transfer of embryos created from autologous oocytes with maternal age less than 35 years at oocyte retrieval the live birth rate per euploid embryo was 70.0% (49/70) compared to 52.5% (53/101) for untested embryos for a rate difference of 17.5% (95% CI 3.0-32.0%, p = 0.03).Conclusions: In good prognosis patients at our center the live birth rate per euploid blastocyst was higher than for untested blastocysts. [ABSTRACT FROM AUTHOR]

Zygotes without a pronuclear (0PN) or with one pronuclear (1PN) were defined as abnormal fertilization in conventional in vitro fertilization (IVF). The removal of 0PN and 1PN zygotes from conventional IVF cycles has always been controversial. This study aimed to investigate the chromosomal aneuploidy rates of 0PN-and 1PN-derived blastocysts in conventional IVF cycles and to assess the concordance rate between TE-biopsy PGT-A and miPGT-A. TE biopsies and culture media with blastocoel fluid (CM-BF) samples were whole-genome amplified by multiple annealing and looping-based amplification cycle-based single-cell ChromInst method. Next generation sequencing was performed for comprehensive chromosomal screening on a NextSeq550 sequencer using the NextSeq 500/550 High Output kit v2. The aneuploidy rates of 0PN-derived blastocysts were 19.7% for TE-biopsy PGT-A, and 36.1% for miPGT-A; the concordance rate for ploidy was 77.0%; and the sensitivity and specificity were 83.3% and 75.5%, respectively. The aneuploidy rates of 1PN-derived blastocysts were 37.5% and 37.5% by TEbiopsy PGT-A and miPGT-A, respectively; the concordance rate between TE biopsies and CM-BF samples was 83.3%; and the sensitivity and specificity were 77.8% and 86.7%, respectively. Regarding TE-biopsy PGT-A, there were no significant differences in aneuploidy rates among 0PN-, 1PN- and 2PNderived blastocysts (PGT-M cycles) (19.7% vs. 37.5% vs. 24.3%, P = 0.226), but the aneuploidy rate of 1PN-derived blastocysts was slightly higher than the other two groups. An increase in aneuploidy rates was observed for 0PN/1PN-derived day 6 blastocysts compared to 0PN/1PN-derived day 5 blastocysts (TE-biopsy PGT-A: 35.7% vs. 19.3%, P = 0.099; miPGT-A: 39.3% vs. 35.1%, P = 0.705). The present study is the first that contributes to understanding the chromosomal aneuploidies in 0PN- and 1PN-derived blastocysts in conventional IVF cycles using TE-biopsy PGT-A and miPGT-A. The clinical application value of 0PN- and 1PN-derived blastocysts in conventional IVF should be assessed using TE-biopsy PGT-A or miPGT-A due to the existence of chromosomal aneuploidies. In terms of consistency, the miPGT-A using blastocoel fluid enriched culture medium is promising as an alternative to TE-biopsy PGT-A for aneuploidy testing of 0PN- or 1PNderived blastocysts in conventional IVF. [ABSTRACT FROM AUTHOR]

Abstract Background Although preimplantation genetic test (PGT) has been used worldwide, few studies investigated the effect of trophectoderm biopsy of blastocysts on early embryo development. This study aimed to investigate whether trophectoderm (TE) biopsy of blastocysts for a PGT affected serum β-human chorionic gonadotropin (hCG) levels 14 days after transfer. Methods This was a retrospective cohort study conducted at the Third Affiliated Hospital of Guangzhou Medical University. The study population comprised pregnant women undergoing the transfer of single vitrified-warmed blastocysts after PGT between January 1, 2018, and July 30, 2020. The control group had non-PGT cycles with other inclusion criteria identical to those for the study group. Propensity score matching was used to screen a group of patients so that the baseline characteristics were similar between the two groups. Serum β-hCG levels were compared between the PGT and non-PGT cycles. Multiple linear regression was used to analyze the influence of PGT on serum β-hCG levels, while receiver operating characteristic curves (ROC curves) were plotted to predict pregnancy outcomes using serum β-hCG levels. Results Serum β-hCG levels were comparable between the PGT and non-PGT patients: live birth: 2503 ± 1702 mIU/mL vs 2266 ± 1289 mIU/mL (P = 0.219); clinical pregnancy: 2261 ± 1564 mIU/mL vs 2148 ± 1348 mIU/mL (P = 0.461); and ongoing pregnancy: 2412 ± 1589 mIU/mL vs 2278 ± 1308 mIU/mL (P = 0.422). Multiple linear regression analysis indicated no impact of PGT on the serum β-hCG level (standardized coefficient = − 0.001, P = 0.989). For clinical pregnancy, the cutoff value was 482 mIU/mL and 302 mIU/mL for PGT and non-PGT patients, respectively. The threshold to predict live birth was 1345 mIU/mL and 1621 mIU/mL in the PGT and non-PGT cycles, respectively. Conclusion Trophectoderm biopsy of blastocysts for PGT did not affect the serum β-hCG level 14 days after transfer.

Do clinical and neonatal outcomes differ between mosaic embryo transfers (MET) and euploid embryo transfers (EET)? 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. 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. 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. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the diagnostic efficiency of spent blastocyst culture medium (BCM) in noninvasive preimplantation genetic testing (niPGT) by comparing the karyotype concordance with corresponding inner cell mass (ICM) among initial trophectoderm (TE) biopsy, TE re-biopsy, and BCM sampling. Design: Re-analysis aneuploid/mosaic blastocysts donated for research by couples. Setting: Institutional in vitro fertilization center. Patient(s): A total of 12 couples donated their blastocysts, which had previously been diagnosed as aneuploid or mosaic by initial TE-biopsy preimplantation genetic testing for aneuploidy (PGT-A) for research. Intervention(s): A total of 26 frozen−thawed blastocysts were re-analyzed by TE re-biopsy, ICM biopsy, and the collection of spent BCM. Main Outcome Measure(s): Karyotype concordance rates. Result(s): For 23 embryos diagnosed as aneuploid by initial TE biopsy, 78.3% of initial TE samples, 87.0% of TE re-biopsies samples, and 78.3% of BCM samples were concordant with corresponding ICM samples, and for three mosaic embryos, the concordance rates with ICM of these three groups were 0%, 100%, and 100%, respectively. With the corresponding ICM result as the true result, sensitivity of both niPGT-A and initial TE were 100%; however, the false-positive rate (FPR) of initial TE was higher than that of niPGT-A (100% vs. 0). Conclusion(s): niPGT-A using BCM had diagnostic efficiency similar to that of TE-biopsy PGT-A. In the case of mosaic embryos, niPGT-A using BCM may be more reliable for predicting karyotypes of ICM than initial TE biopsy.

Abstract Background Spent culture medium (SCM) as a source of DNA for preimplantation genetic tests aneuploidy (PGT-A) has been widely discussed. Methods Seventy-five blastocysts that were donated for research provided a unique possibility in which multiple specimens, including trophectoderm (TE) biopsy, SCM, and paired corresponding whole blastocyst (WB) specimens from the same blastocyst source, could be utilized for the purpose of this preclinical validation. Results To conduct a validation ploidy concordance assessment, we evaluated the full chromosomal concordance rates between SCM and WB (SCM-to-WB), and between TE and WB (TE-to-WB) as well as sensitivity, specificity and overall diagnostic accuracy. 78.67% (59/75) of NGS results in the SCM group were interpretable, a significantly lower percentage than their corresponding TE and WB groups. This discrepancy manifests itself in intrinsically low quantity and poor integrity DNA from SCM. Subsequently, remarkable differences in full concordance rates (including mosaicism, and segmental aneuploidies) are seen as follows: 32.2% (SCM-to-WB, 19/59) and 69.33% (TE-to-WB, 52/75), (p

Abstract Background Recently, the Japan Society of Obstetrics and Gynecology initiated a clinical study of preimplantation genetic test for aneuploidy. There will be a great need for a standardized embryo biopsy technique in Japan. However, the gold standard trophectoderm (TE) biopsy procedure has not been established, and this review outlines the clinical use of TE biopsy. Methods Based on literature, the method and associated techniques for TE biopsy, a dissection method of TE cells from blastocysts, were investigated. Main findings Two TE biopsy methods are used, namely assisted hatching (herniating) and non‐assisted hatching (direct suction); however, it is not clear which of these methods is superior. It is critical to understand whether the flicking or pulling method is beneficial. Conclusion Non‐assisted hatching biopsy method may cause blastocyst collapse with a higher probability, and it may extend the biopsy time. The biopsy procedure should be performed within 3 minutes, and thus direct TE suction may have greater disadvantages. It is a fact that pulling method of TE dissection with laser pulse is simple; however, excess laser shots may induce a higher frequency of mosaicism. It is important to understand that each technique of TE biopsy has benefits and disadvantages.

Preimplantation genetic testing (PGT) involves taking a biopsy of an early embryo created through in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). Genetic testing is performed on the biopsy, in order to select which embryo to transfer. PGT began as an experimental procedure in the 1990s, but is now an integral part of assisted human reproduction (AHR). PGT allows for embryo selection which can reduce the risk of transmission of inherited disease and may reduce the chance of implantation failure and pregnancy loss. This is a rapidly evolving area, which raises important ethical issues. This review article aims to give a brief history of PGT, an overview of the current evidence in PGT along with highlighting exciting areas of research to advance this technology., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

ObjectiveTo evaluate whether trophectoderm (TE) biopsy differentially influence the level of serum β-human chorionic gonadotropin (β-hCG) with different TE-scored blastocysts transferred in early pregnancy.MethodsThis retrospective cohort study contained 7847 single-blastocyst transfer cycles executed between January 2019 and June 2020, including 2657 preimplantation genetic testing (PGT) cycles and 5190 in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycles. All cycles were classified into biopsy and control groups, and further stratified based on the TE morphological scores into three subgroups: grades A, B, and C for TE scores, respectively. Intra-group and inter-group analyses were performed on serum β-hCG levels on the 12th day after blastocyst transfer (HCG12), and obstetric and neonatal outcomes.ResultsFor cycles with a live birth, in grade A TE score subgroups, the HCG12 level did not exhibit statistical significance between the control and biopsy groups after adjustment (769 mIU/mL vs. 753 mIU/mL, P=0.631). In contrast, in grade B and C TE score subgroups, the control group showed a significantly higher level of HCG12 relative to the biopsy group (690 mIU/mL vs. 649 mIU/mL, P=0.001; 586 mIU/mL vs. 509 mIU/mL, P

Abstract Purpose To examine how differences in trophectoderm biopsy techniques affect the frequency of mosaic embryos and sequencing results. Methods We examined differences in next‐generation sequencing (NGS) analysis results among operators or according to biopsy technique. Additionally, we determined the cut‐off for the number of collected cells to predict the occurrence of mosaicism. We collected cells according to the cut‐off value and examined whether there was a difference in the NGS analysis results between the pulling and flicking methods. Results There was no difference in the NGS analysis results among the operators. Regarding re‐biopsy, changes in the mosaic were observed in all specimens. The cut‐off value for the number of collected cells was five, and when more than five cells were collected, there was no difference in the NGS analysis results between the two methods. Conclusions We demonstrated that if trophectoderm biopsy techniques and NGS are stable, the cell collection location has a greater effect on NGS results than the biopsy technique.

Abstract Purpose This study was conducted to assess the effectiveness of a new trophectoderm (TE) biopsy method that does not require prior opening of the zona pellucida at the blastocyst stage. Methods TE biopsy was conducted using a modified extrusion method for embryos during the cleavage stage. In this method, culture medium was injected into the perivitelline space to help extrude TE cells from the zona pellucida before TE biopsy. Results Our extrusion method preserves the embryo culture environment until immediately before biopsy because it does not require opening of the zona pellucida prior to TE biopsy. Furthermore, this method does not require a waiting time for blastocyst hatching after laser irradiation, thereby minimizing damage to the embryos and maintaining the time schedule of culture operations. Conclusions TE biopsy using this novel extrusion method may be useful in various applications, including the collection of TE cells for next‐generation sequencing analysis.

Abstract Purpose The present study aimed to examine the correlations of the time interval from trophectoderm (TE) biopsy to vitrification with the blastocyst survival rate and blastocyst outgrowth ability. Methods A total of 1,202 mouse blastocysts were randomly divided into control (non‐biopsy) and TE biopsy groups. The biopsied blastocysts were vitrified at various time points. The survival rate after warming, blastocyst adhesion rate, and outgrowth area was investigated. Several biopsied blastocysts were cultured in a time‐lapse incubator, and the time required for re‐expansion was measured. Results Blastocyst survival rates after warming and blastocyst adhesion rates were comparable between the control and biopsy groups. The area of trophoblast outgrowth in the 1‐h biopsy group was significantly smaller than that in the control, 0‐h biopsy, and 4‐h biopsy groups (p = 0.0304, p = 0.0058, and p = 0.0029, respectively). Re‐expansion of blastocysts was observed at a high incidence 1–2 h after TE biopsy. Conclusions The vitrification of biopsied blastocysts in the process of re‐expansion impairs outgrowth competence; therefore, blastocyst vitrification should be performed immediately after TE biopsy and before initiation of re‐expansion.

Objective: To evaluate whether trophectoderm (TE) biopsy differentially influence the level of serum β-human chorionic gonadotropin (β-hCG) with different TE-scored blastocysts transferred in early pregnancy. Methods: This retrospective cohort study contained 7847 single-blastocyst transfer cycles executed between January 2019 and June 2020, including 2657 preimplantation genetic testing (PGT) cycles and 5190 in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycles. All cycles were classified into biopsy and control groups, and further stratified based on the TE morphological scores into three subgroups: grades A, B, and C for TE scores, respectively. Intra-group and inter-group analyses were performed on serum β-hCG levels on the 12th day after blastocyst transfer (HCG12), and obstetric and neonatal outcomes. Results: For cycles with a live birth, in grade A TE score subgroups, the HCG12 level did not exhibit statistical significance between the control and biopsy groups after adjustment (769 mIU/mL vs. 753 mIU/mL, P=0.631). In contrast, in grade B and C TE score subgroups, the control group showed a significantly higher level of HCG12 relative to the biopsy group (690 mIU/mL vs. 649 mIU/mL, P=0.001; 586 mIU/mL vs. 509 mIU/mL, P<0.001, respectively). We observed no statistically significant differences in obvious adverse obstetric and neonatal outcomes between the same TE-score subgroups of the biopsy group and control group. Conclusions: While blastocysts with higher TE grades produced higher serum β-hCG levels in early pregnancy, TE biopsy might exert a negative impact on serum β-hCG levels by blastocysts with a grade-B TE score and below. TE biopsy did not increase the risk for adverse obstetric and neonatal outcomes. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

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? 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. 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. 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. [ABSTRACT FROM AUTHOR]

Purpose: To examine how differences in trophectoderm biopsy techniques affect the frequency of mosaic embryos and sequencing results. Methods: We examined differences in next‐generation sequencing (NGS) analysis results among operators or according to biopsy technique. Additionally, we determined the cut‐off for the number of collected cells to predict the occurrence of mosaicism. We collected cells according to the cut‐off value and examined whether there was a difference in the NGS analysis results between the pulling and flicking methods. Results: There was no difference in the NGS analysis results among the operators. Regarding re‐biopsy, changes in the mosaic were observed in all specimens. The cut‐off value for the number of collected cells was five, and when more than five cells were collected, there was no difference in the NGS analysis results between the two methods. Conclusions: We demonstrated that if trophectoderm biopsy techniques and NGS are stable, the cell collection location has a greater effect on NGS results than the biopsy technique. [ABSTRACT FROM AUTHOR]

Purpose: The present study aimed to examine the correlations of the time interval from trophectoderm (TE) biopsy to vitrification with the blastocyst survival rate and blastocyst outgrowth ability. Methods: A total of 1,202 mouse blastocysts were randomly divided into control (non‐biopsy) and TE biopsy groups. The biopsied blastocysts were vitrified at various time points. The survival rate after warming, blastocyst adhesion rate, and outgrowth area was investigated. Several biopsied blastocysts were cultured in a time‐lapse incubator, and the time required for re‐expansion was measured. Results: Blastocyst survival rates after warming and blastocyst adhesion rates were comparable between the control and biopsy groups. The area of trophoblast outgrowth in the 1‐h biopsy group was significantly smaller than that in the control, 0‐h biopsy, and 4‐h biopsy groups (p = 0.0304, p = 0.0058, and p = 0.0029, respectively). Re‐expansion of blastocysts was observed at a high incidence 1–2 h after TE biopsy. Conclusions: The vitrification of biopsied blastocysts in the process of re‐expansion impairs outgrowth competence; therefore, blastocyst vitrification should be performed immediately after TE biopsy and before initiation of re‐expansion. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) and intrauterine growth retardation (IUGR) are the results of defective placentation associated with the downregulation of different genes in the human trophoblast including the Placental Growth Factor (PGF). TrophEctoderm (TE) biopsy is increasingly performed for Pre-implantation Genetic Testing of Aneuploidies and it involves the traumatical removal of an unpredictable number of mural TE cells from the human blastocyst. We observed strikingly similar obstetrical and neonatal complications in pregnancies where the placenta bears PGF downmodulation or a TE biopsy has been done. In both groups, the risk of PE, IUGR, congenital cardiac ventricular septal defects, caesarean section, sex ratio in favour of males and preterm birth is significantly increased compared to controls. Given the high degree of correlation, the observation may not be a casual one. We postulate herein that the TE biopsy may induce persistent dysregulation of different genes in the placenta including PGF. The mechanism proposed is the disruption of tight junctions caused by the TE biopsy. [ABSTRACT FROM AUTHOR]

Vitrification and slow freezing are the two commonly used embryo cryopreservation methods. In most studies, vitrification of intact embryos has proven superior in several respects, including cell and embryo survival and pregnancy rate. However, there is a lack of data for comparing these two methods in in vitro produced (IVP) bovine blastocysts, which have been subjected to the retrieval of trophectoderm (TE) biopsy. Day 7 IVP blastocysts were pooled and randomized into four groups: 1) non-biopsy (NB), 2) biopsy (B), 3) biopsy-vitrification (BV), 4) biopsy-slow freeze (BSF). The blastocysts in the B, BV, and BSF groups were subjected to TE biopsy. For the B group, this was followed by 5 hours (h) incubation and subsequent scoring of the biopsy-survival (re-expansion) rate before processing for further analyses. For the BV and BSF groups, the biopsy procedure was followed by 2 h incubation, allowing for a quick re-expansion, after which the blastocysts were subjected to vitrification and slow freezing, respectively. After warming and thawing, respectively, they were then incubated for 5 h followed by scoring the cryo-survival (re-expansion) rates before processing for further analyses. These included quantification of ICM and TE cells, cleaved caspase-3- and TUNEL-positive cells, quantitative PCR on cellular stress markers (SOD1 and PRDX1), and ultrastructural analysis. The biopsy-survival rate in the B group was 94% (307/326). The cryo-survival rate in BV (86%, 138/161) was higher than that in BSF (57%, 81/142; P < 0.001). No differences were noted between the average ICM, TE, and total cell numbers of the groups. The percentages of cleaved caspase-3-positive cells were higher in BV vs. NB (P < 0.05), in BSF vs. NB (P < 0.001), and in BSF vs. B (P < 0.001). The percentages of TUNEL-positive cells were higher in BV vs. NB (P < 0.05) and in BSF vs. NB (P < 0.001). The levels of mRNA abundance for SOD1 and PRDX1 in B, BV, and BSF were not different from that in NB. The ultrastructural analysis of blastocysts in the BV and BSF groups showed distension of extracellular spaces and appearance of intracellular vacuoles in the ICM, distension of mitochondria, and disorganization of mitochondrial cristae in both ICM and TE, and weakened tight junctions between adjacent TE cells. In summary, our findings demonstrate that vitrification yields a higher cryo-survival rate than slow freezing in biopsied bovine IVP blastocysts. However, biopsy-vitrification and biopsy-slow-freeze values are comparable in terms of ICM, TE, and total blastocyst cell numbers, as well as cleaved caspase-3- and TUNEL-positive cell rates. Moreover, biopsy and cryopreservation performed alone had no effect on ICM, TE, total blastocyst cell numbers, or TUNEL-positive cell rates. Biopsy and vitrification performed alone had no effect on the cleaved caspase-3 positive cell rates, whereas slow freezing resulted in an increased rate. Furthermore, double traumatization with a combination of biopsy and cryopreservation, either vitrification or slow freezing, resulted in increased rates of cleaved caspase-3- and TUNEL-positive cells. • Vitrification (V) yielded a higher cryo-survival rate than slow freezing (SF) in biopsied (B) bovine IVP blastocysts. • ICM, TE, total cell numbers, and ICM:TE ratios were comparable between the BV and BSF blastocysts. • Vitrification and slow freezing produced comparable apoptotic and TUNEL-positive cell rates in biopsied IVP blastocysts. • TEM revealed extracellular space expansion and intracellular vacuoles appearance in the ICMs of the BV and BSF blastocysts. • TEMs of BV and BSF showed mitochondrial distension and cristae disorganization in ICM and TE, and weakened TE-TE junctions. [ABSTRACT FROM AUTHOR]

Recurrent implantation failure (RIF) is an intrigue condition during in vitro fertilization (IVF) cycles or intracytoplasmic sperm injection (ICSI) treatments. The purpose of this retrospective study is to explore the value of next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) of trophectoderm biopsy in the clinical outcomes for RIF patients with advanced age. A total of 265 RIF patients, who underwent 346 oocyte retrieval cycles and 250 PGT-A cycles, were classified as two groups according to the female age, including < 38 and ≥ 38 years old groups. The two groups were statistically comparable in baseline characteristics. The component of aneuploid embryos was significantly higher in advanced age group than in younger age group (68.9 vs 39.9%, P < 0.001). But there were no statistically significant differences in pregnancy rate (43.5 vs 64.7%), clinical pregnancy rate (39.1 vs 48.0%), implantation rate (39.1 vs 51.0%), and miscarriage rate (4.3 vs 7.8%) per embryo transfer (ET) between the two groups. Results suggest that the embryo-related factor plays a crucial role in RIF. Maternal age does not influence the implantation potential of euploid blastocysts. The NGS-based PGT-A involving trophectoderm biopsy is valuable for RIF patients of advanced age by improving their clinical outcomes. In conclusion, the NGS-based PGT-A involving trophectoderm biopsy may represent a valuable supplement to the current RIF management. Nonetheless, these findings should be further validated in a well-designed randomized controlled trial. [ABSTRACT FROM AUTHOR]

Background: Although preimplantation genetic test (PGT) has been used worldwide, few studies investigated the effect of trophectoderm biopsy of blastocysts on early embryo development. This study aimed to investigate whether trophectoderm (TE) biopsy of blastocysts for a PGT affected serum β-human chorionic gonadotropin (hCG) levels 14 days after transfer. Methods: This was a retrospective cohort study conducted at the Third Affiliated Hospital of Guangzhou Medical University. The study population comprised pregnant women undergoing the transfer of single vitrified-warmed blastocysts after PGT between January 1, 2018, and July 30, 2020. The control group had non-PGT cycles with other inclusion criteria identical to those for the study group. Propensity score matching was used to screen a group of patients so that the baseline characteristics were similar between the two groups. Serum β-hCG levels were compared between the PGT and non-PGT cycles. Multiple linear regression was used to analyze the influence of PGT on serum β-hCG levels, while receiver operating characteristic curves (ROC curves) were plotted to predict pregnancy outcomes using serum β-hCG levels. Results: Serum β-hCG levels were comparable between the PGT and non-PGT patients: live birth: 2503 ± 1702 mIU/mL vs 2266 ± 1289 mIU/mL (P = 0.219); clinical pregnancy: 2261 ± 1564 mIU/mL vs 2148 ± 1348 mIU/mL (P = 0.461); and ongoing pregnancy: 2412 ± 1589 mIU/mL vs 2278 ± 1308 mIU/mL (P = 0.422). Multiple linear regression analysis indicated no impact of PGT on the serum β-hCG level (standardized coefficient = − 0.001, P = 0.989). For clinical pregnancy, the cutoff value was 482 mIU/mL and 302 mIU/mL for PGT and non-PGT patients, respectively. The threshold to predict live birth was 1345 mIU/mL and 1621 mIU/mL in the PGT and non-PGT cycles, respectively. Conclusion: Trophectoderm biopsy of blastocysts for PGT did not affect the serum β-hCG level 14 days after transfer. [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]