Comprehensive Chromosome Screening Research Articles

Does recurrent implantation failure exist? Prevalence and outcomes of five consecutive euploid blastocyst transfers in 123987 patients.

What are the clinical pregnancy and live birth rates in women who underwent up to two more euploid blastocyst transfers after three failures in the absence of another known factor that affects implantation? The fourth and fifth euploid blastocyst transfers resulted in similar live birth rates of 40% and 53.3%, respectively, culminating in a cumulative live birth rate of 98.1% (95% CI = 96.5-99.6%) after five euploid blastocyst transfers. The first three euploid blastocysts have similar implantation and live birth rates and provide a cumulative live birth rate of 92.6%. An international multi-center retrospective study was conducted at 25 individual clinics. The study period spanned between January 2012 and December 2022. A total of 123987 patients with a total of 64572 euploid blastocyst transfers were screened for inclusion. Patients with a history of any embryo transfer at another clinic, history of any unscreened embryo transfer at participating clinics, parental karyotype abnormalities, the use of donor oocytes or a gestational carrier, untreated intracavitary uterine pathology (e.g. polyp, leiomyoma), congenital uterine anomalies, adenomyosis, communicating hydrosalpinx, endometrial thickness <6 mm prior to initiating of progesterone, use of testicular sperm due to non-obstructive azoospermia in the male partner, transfer of an embryo with a reported intermediate chromosome copy number (i.e. mosaic), preimplantation genetic testing cycles for monogenic disorders, or structural chromosome rearrangements were excluded. Ovarian stimulation protocols and embryology laboratory procedures including trophectoderm biopsy followed the usual practice of each center. The ploidy status of blastocysts was determined with comprehensive chromosome screening. Endometrial preparation protocols followed the usual practice of participating centers and included programmed cycles, natural or modified natural cycles. A total of 105 (0.085% of the total population) patients met the criteria and underwent at least one additional euploid blastocyst transfer after failing to achieve a positive pregnancy test with three consecutive euploid blastocyst transfers. Outcomes of the fourth and fifth euploid blastocyst transfers were similar across participating centers. Overall, the live birth rate was similar with the fourth and fifth euploid blastocysts (40% vs 53.3%, relative risk = 1.33, 95% CI = 0.93-1.9, P value = 0.14). Sensitivity analyses excluding blastocysts biopsied on Day 7 postfertilization, women with a BMI >30 kg/m2, cycles using non-ejaculate or donor sperm, double-embryo transfer cycles, and cycles in which the day of embryo transfer was modified due to endometrial receptivity assay test result yielded similar results. Where data were available, the fourth euploid blastocyst had similar live birth rate with the first one (relative risk = 0.84, 95% CI = 0.58-1.21, P = 0.29). The cumulative live birth rate after five euploid blastocyst transfers was 98.1% (95% CI = 96.5-99.6%). Retrospective design has its own inherent limitations. Patients continuing with a further euploid embryo transfer and patients dropping out from treatment after three failed euploid transfers can be systematically different, perhaps with regard to ovarian reserve or economic status. Implantation failure seems to be mainly due to embryonic factors. Given the stable and high live birth rates up to five euploid blastocysts, unexplained recurrent implantation failure should have a prevalence of <2%. Proceeding with another embryo transfer can be the best next step once a known etiology for implantation failure is ruled out. None. N/A.

Clinical outcomes in carriers of insertional translocation: a retrospective analysis of comprehensive chromosome screening results

To evaluate the clinical outcomes in the carriers of insertional translocation (IT). Retrospective case series. University-affiliated reproductive medical center. Twenty-three couples with ITs. No direct interventions were involved; however, this study included patients who underwent preimplantation genetic testing for structural chromosomal rearrangements (PGT-SR). Outcome of preimplantation genetic testing for structural chromosomal rearrangements and percentage of blastocysts available for transfer. Among 23 IT carriers, 15 were simple interchromosome ITs (type A), 3 were intrachromosome IT carriers (type B), and 5 were interchromosome IT carriers combined with other translocations (type C). A total of 190 blastocysts from 30 cycles were biopsied, 187 embryos were tested successfully, and only 57 blastocysts (30.5%) from 21 patients were available for transfer (normal or balanced). The unbalanced rearrangement rate of type C was 79.2% (42/53), and the proportion of type A was 50.0% (57/114), which was significantly higher than that of type B (5%, 1/20). In type A, the probability of embryos harboring unbalanced rearrangement in female carriers was 56.0% (51/91), which was higher than that in male carriers (26.1%, 6/23). Furthermore, the haploid autosomal length value of the inserted fragment was correlated linearly with the incidence of abnormal embryos. In type A gametes, most gametes produced by 2:2 separation without crossover, and no 3:1 separation gamete was observed. The chance of identifying normal or balanced and mosaic blastocysts per mature oocytes in patients with ITs are 16.6% (67/404). Greater IT complexity results in fewer transferable embryos. For simple interchromosome ITs, female carriers and those with higher haploid autosomal length values have a higher risk of producing embryos with unbalanced rearrangement.

Comprehensive chromosomal screening for preimplantation genetic testing: A mini-review

Preimplantation genetic testing (PGT) consists of a group of genetic tests to evaluate preimplantation embryos before transfer to the uterus during in vitro fertilization (IVF). It effectively reduces the incidence of genetic defects at birth by preventing the transmission of inherited diseases to embryos. The use of PGT in IVF clinics has greatly improved clinical pregnancy outcomes for carriers of genetic abnormalities through the selection of embryos that are free from any genetic mutation/chromosomal anomalies. However, the accuracy of PGT in detecting aneuploidies and genetic mutations remains a point of contention due to the varied effectiveness of the techniques used. In recent years, a number of high-throughput assays have been developed to overcome the challenges associated with comprehensive chromosomal analysis. In this review, we will summaries the recent progress in using comprehensive chromosomal screening techniques, including array comparative genomic hybridization, single nucleotide polymorphism array, and next-generation sequencing, to evaluate chromosomal genetic defects.

Knowledge-embedded spatio-temporal analysis for euploidy embryos identification in couples with chromosomal rearrangements.

The goal of the assisted reproductive treatment is to transfer one euploid blastocyst and to help infertile women giving birth one healthy neonate. Some algorithms have been used to assess the ploidy status of embryos derived from couples with normal chromosome, who subjected to preimplantation genetic testing for aneuploidy (PGT-A) treatment. However, it is currently unknown whether artificial intelligence model can be used to assess the euploidy status of blastocyst derived from populations with chromosomal rearrangement. From February 2020 to May 2021, we collected the whole raw time-lapse videos at multiple focal planes from in vitro cultured embryos, the clinical information of couples, and the comprehensive chromosome screening results of those blastocysts that had received PGT treatment. Initially, we developed a novel deep learning model called the Attentive Multi-Focus Selection Network (AMSNet) to analyze time-lapse videos in real time and predict blastocyst formation. Building upon AMSNet, we integrated additional clinically predictive variables and created a second deep learning model, the Attentive Multi-Focus Video and Clinical Information Fusion Network (AMCFNet), to assess the euploidy status of embryos. The efficacy of the AMCFNet was further tested in embryos with parental chromosomal rearrangements. The receiver operating characteristic curve (ROC) was used to evaluate the superiority of the model. A total of 4112 embryos with complete time-lapse videos were enrolled for the blastocyst formation prediction task, and 1422 qualified blastocysts received PGT-A ( n = 589) or PGT for chromosomal structural rearrangement (PGT-SR, n = 833) were enrolled for the euploidy assessment task in this study. The AMSNet model using seven focal raw time-lapse videos has the best real-time accuracy. The real-time accuracy for AMSNet to predict blastocyst formation reached above 70% on the day 2 of embryo culture, and then increased to 80% on the day 4 of embryo culture. Combing with 4 clinical features of couples, the AUC of AMCFNet with 7 focal points increased to 0.729 in blastocysts derived from couples with chromosomal rearrangement. Integrating seven focal raw time-lapse images of embryos and parental clinical information, AMCFNet model have the capability of assessing euploidy status in blastocysts derived from couples with chromosomal rearrangement.

Current Applications and Controversies in Preimplantation Genetic Testing for Aneuploidies (PGT-A) in In Vitro Fertilization.

Preimplantation genetic testing for aneuploidy (PGT-A) has evolved over recent years, including improvements in embryo culture, biopsy, transfer, and genetic testing. The application of new comprehensive chromosome screening analysis has improved the accuracy in determining the chromosomal status of the analyzed sample, but it has brought new challenges such as the management of partial aneuploidies and mosaicisms. For the past two decades, PGT-A has been involved in a controversy regarding its efficiency in improving IVF outcomes, despite its widespread worldwide implementation. Understanding the impact of embryo aneuploidy in IVF (in vitro fertilization) should theoretically allow improving reproductive outcomes. This review of the literature aims to describe the impact of aneuploidy in human reproduction and how PGT-A was introduced to overcome this obstacle in IVF (in vitro fertilization). The article will try to analyze and summarize the evolution of the PGT-A in the recent years, and its current applications and limitations, as well as the controversy it generates. Conflicting published data could indicate the lacking value of a single biopsied sample to determine embryo chromosomal status and/or the lack of standardized methods for embryo culture and management and genetic analysis among other factors. It has to be considered that PGT-A may not be a universal test to improve the reproductive potential in IVF patients, rather each clinic should evaluate the efficacy of PGT-A in their IVF program based on their population, skills, and limitations.

Systematic review and meta-analysis: does pre-implantation genetic testing for aneuploidy at the blastocyst stage improve live birth rate?

PurposeTo establish if preimplantation genetic testing for aneuploidy (PGT-A) at the blastocyst stage improves the composite outcome of live birth rate and ongoing pregnancy rate per embryo transfer compared to conventional morphological assessment.MethodsA systematic literature search was conducted using PubMed, EMBASE and Cochrane database from 1st March 2000 until 1st March 2022. Studies comparing reproductive outcomes following in vitro fertilisation using comprehensive chromosome screening (CCS) at the blastocyst stage with traditional morphological methods were evaluated.ResultsOf the 1307 citations identified, six randomised control trials (RCTs) and ten cohort studies fulfilled the inclusion criteria. The pooled data identified a benefit between PGT-A and control groups in the composite outcome of live birth rate and ongoing pregnancy per embryo transfer in both the RCT (RR 1.09, 95% CI 1.02–1.16) and cohort studies (RR 1.50, 95% CI 1.28–1.76). Euploid embryos identified by CCS were more likely to be successfully implanted amongst the RCT (RR 1.20, 95% CI 1.10–1.31) and cohort (RR 1.69, 95% CI 1.29–2.21) studies. The rate of miscarriage per clinical pregnancy is also significantly lower when CCS is implemented (RCT: RR 0.73, 95% CI 0.56–0.96 and cohort: RR 0.48, 95% CI 0.32–0.72).ConclusionsCCS-based PGT-A at the blastocyst biopsy stage increases the composite outcome of live births and ongoing pregnancies per embryo transfer and reduces the rate of miscarriage compared to morphological assessment alone. In view of the limited number of studies included and the variation in methodology between studies, future reviews and analyses are required to confirm these findings.

P-747 Chromosomal mosaic characteristics among women under IVF treatment: multicentric study of 2282 mosaic embryos diagnosed by preimplantation genetic testing with trophectoderm biopsy

Abstract Study question Which chromosomal mosaic features do women under IVF treatment show? Summary answer Single aneuploidies were the most represented among mosaic embryos and their frequency as well as the level of mosaicism increased in older women. What is known already Chromosomal mosaic embryos are characterised by the presence of chromosomally different cell lines within the same embryo. We previously demonstrated that reproductive potential of mosaic embryos is affected by the complexity of and the number of aneuploid cells present in trophectoderm (TE) biopsy. Although with the introduction of next generation sequencing (NGS) chromosomal constitution of human embryos have been elucidated, only limited number of mosaic have been characterised. Therefore, we performed a larger-scale multicenter study on mosaic embryos to examine the patterns and prevalence of chromosome specific mosaicisms in TE samples. Study design, size, duration This is a retrospective cohort study from May 2019 to May 2021 of 20200 embryos obtained from 5770 women under IVF treatment. From this cohort, 2280 mosaic embryos were analysed. All embryos were cultured to blastocyst stage; TE biopsy was performed on Day-5 of development or on Day-6/7 for slow growing embryos. Participants/materials, setting, methods TE biopsies underwent comprehensive chromosome screening (CCS) utilizing validated NGS. TE biopsies were classified as mosaic if they had 20%-80% of abnormal cells. For statistical analysis, embryos were divided in: single whole-chromosome trisomy, double whole-chromosome trisomies, single whole-chromosome monosomy, double whole-chromosome monosomies and complex aneuploidy (more than two different aneuploidies) groups. In addition, the frequency of single, double and complex segmental aneuploidies was evaluated. For each group, the trend related to maternal age was examined. Main results and the role of chance Among 2282 mosaic embryos single (whole-chromosome and segmental) aneuploidies were the most represented(52%) followed by complex(36%) and double ones(14%). When embryos were divided based on different types of aneuploidies, complex aneuploidies were the most frequent(34%) followed by segmental(31%), single monosomies(14%), single trisomy(12%). The more affected chromosomes within trisomies were 16(7%), 19 (7%), 21 and 22(6%). Monosomy involved mainly chromosomes 7(8%), 21 and 22(7%). Chromosomes 1, 5(9%), and 9(8%) were the most targeted by segmental duplication while segmental deletion mostly involved chromosomes 1(11%), 2(13%) and X(8%). The frequency of single aneuploidies as well as the percentage of mosaicism increased in older women. Limitations, reasons for caution This study was retrospective and observational, demonstrating the relative frequency of mosaicisms but not offering any direct insight into the clinical relevance of the findings. More clinical data must be obtained before this approach can be evaluated as an additional tool to choose mosaic embryos for the transfer. Wider implications of the findings This study provides a large dataset of mosaic embryos and offers detailed information on the distribution of different types of mosaicism among a general population of women under IVF treatment. Trial registration number Not applicable

P-721 Nanopore sequencing for detecting reciprocal translocation carrier status in preimplantation genetic testing

Abstract Study question Analyze the concordance between nanopore sequencing and MaReCs to validate the feasibility of nanopore sequencing in distinguishing normal embryos from carrier embryos in PGT-SR cycles. Summary answer Nanopore sequencing is a powerful strategy for accurately distinguishing nontranslocation embryos from translocation carrier embryos and precisely localizing translocation breakpoints. What is known already Balanced reciprocal translocation (BRT) is one of the most common chromosomal abnormalities that causes infertility, recurrent miscarriage, and birth defects. Preimplantation genetic testing (PGT) is widely used to select euploid embryos for BRT carriers to increase the chance of a healthy live birth. Several strategies can be used to distinguish reciprocal translocation carrier embryos from those with a normal karyotype; however, these techniques are time-consuming and difficult to implement in clinical laboratories. Study design, size, duration The Nanopore Sequencing was performed on two pregnant patients conducted on Mapping Allele with Resolved Carrier Status” (MaReCs) in PGT-SR cycles to identify translocation breakpoints for a year. Combined conventional MaReCs ’results, amniocentesis was performed to identify karyotypes at 18–20 weeks of gestation to verify the correctness of the Nanopore Sequencing. Participants/materials, setting, methods A total of 11 embryos from 2 couple1 and 12 embryos from couple 2 were tested simultaneously in PGT-SR cycles with a combination strategy of mate pair sequencing and PCR breakpoint analysis, SNP array-based comprehensive chromosome screening (CCS), NGS following micro-dissecting junction region (Micro-Seq) to distinguish between normal and carrier embryos. The Nanopore Sequencing based on TGS was performed on 2 couples. Lastly, amniocentesis was performed to test karyotypes. Main results and the role of chance The translocation breakpoints in both reciprocal translocation carriers were accurately identifed by nanopore sequencing and were in accordance with the results obtained using MaReCs. More than one euploid non-balanced translocation carrier embryo was identified in both patients. Amniocentesis results revealed normal karyotypes, consistent with the findings by MaReCs and nanopore sequencing. Limitations, reasons for caution One limitation is that it is not possible to identify the translocation breakpoints of Robertsonian translocation carriers or those located in the gap regions of the human genome. In addition, high-molecular-weight genomic DNA and large amounts of peripheral blood samples would be a challenge most PGT centers. Wider implications of the findings The nanopore sequencing accurately detects translocation breakpoints in BRT carriers and distinguishes translocation-free embryos from balanced diploid embryos in clinical PGT-SR cycles. It is superior to short reads (NGS) for the detection of translocation breakpoints, which shows broad prospects for clinical applications in blocking translocation propagation in the population. Trial registration number Not applicable

O-077 Time-lapse imaging analysis of segmental aneuploid embryos: a multicenter study identifies morpho-kinetic patterns associated with chromosomal mosaicism

Abstract Study question Do segmental aneuploid (SA) embryos display unique morpho-kinetic patterns at early developmental stages? Summary answer The first three cell division cycles of embryos carrying segmental aneuploidies are significantly slower than euploid and whole-chromosome aneuploid (WCA) embryos. What is known already Segmental aneuploidies in blastocyst-stage human embryos prevalently originate (i.e., 70% of cases) from mitotic errors. Their clinical management is challenging because it is impossible to establish their meiotic/mitotic origin from PGT-A analysis of a single trophectoderm biopsy. Morpho-kinetic analysis based on Time-lapse microscopy (TLM) has been proposed as a valuable tool for systematic embryo evaluation, although it is unclear whether this approach alone can predict chromosomal abnormalities. This study aims to pinpoint morphokinetic patterns associated with the occurrence of segmental aneuploidies and/or mitotic errors, possibly improving DNA-based diagnostic interpretation and their clinical management. Study design, size, duration This is a retrospective multicenter study including a total of 7,693 embryos from 2,370 IVF cycles and 2,127 couples, cultured between 2016 and 2021 in 4 European IVF centers. Embryos with no more than 4 chromosomal alterations were considered in the analysis, resulting in 3,288 euploids, 3,155 WCA, 715 SA, and 535 complex aneuploids (CA). Overall, the dataset contained 3,742 distinct euploid-SA embryo sibling pairs. Participants/materials, setting, methods Standard morpho-kinetic features were annotated using various TLM systems. Blastocysts were subjected to comprehensive chromosomal screening via PGT-A. Morpho-kinetic timings across different embryo groups were compared using Kolmogorov-Smirnov (KS) test and binomial test, and associations with cleavage features were assessed via two-sided Fisher Exact (FS) test. Multi-center and center-specific logistic regression models were calibrated using a subset of data (70%), and their predictive performance was evaluated on independent test data using Area-Under-ROC curve (AUROC) metrics. Main results and the role of chance SA embryos cleaved at significantly slower rate than their euploid siblings in the first 3 cell cycles, from the appearance of the second blastomere t2 (average delay delta=0.49h, KS p = 0.006, fraction of pairs in which SA was delayed compared to euploid sibling F = 54.2%, binomial p = 0.002) up to t8 (delta=2.16h, KS p = 4e-4, F = 57.9%, binomial p = 1e-8). The developmental delay was partially compensated at full blastocyst stage tB (delta=1.71h, KS = 0.0008, F = 57.5%, binomial p = 5e-8). SA timings also differed from WCA ones: SA embryos were significantly slower than their WCA siblings during early cell cycles, with maximum delay reached at t8 (delta=1.67h, KS p = 0.009, F = 55.4%, binomial p = 0.003), but they fully caught-up at tB, with no significant difference observed. These morpho-kinetic patterns were consistently observed in each center separately, but with different effect sizes. The presence of segmental aneuploidies was significantly associated with multinucleation (OR = 1.99, FS test p = 0.05) and morula cell exclusion (OR = 2.69, p = 0.006). A logistic model based on morpho-kinetic data and cleavage features from a single center dataset and regressed against embryo aneuploidy class (euploid vs. SA embryos) displayed adequate predictive performance on independent data from the same center (AUROC=0.70), although predictivity diminished when tested on data from other centers (AUROC=0.52-0.55). Limitations, reasons for caution We cannot rule out inter-center variability in the annotation procedures of morpho-kinetic parameters. Standardized and automated annotation would increase the performance of predictive models and their transferability. Further work is needed to assess whether the morpho-kinetic signal is driven by mitotic errors and if an association with clinical outcomes exists. Wider implications of the findings These findings might help uncovering biological events contributing to preimplantation mosaicism. Moreover, the developed predictive framework might help improving decision-making in PGT-A cycles, providing interpretation for segmental aneuploidies and possibly helping in the evaluation of embryos showing intermediate copy number values for whole chromosomes that are suggestive of mosaicism. Trial registration number Not applicable

The Fischer protocol for assisted reproductive technology treatment: Real-world data experience comparing elective single versus double embryo transfer with or without comprehensive chromosome screening.

High rates of multiple implantation after assisted reproductive technology (ART) treatment represent one of the major problems for both mothers and their fetuses. Given the availability of techniques intended to identify embryos with the highest chance for development to term, such as comprehensive chromosome screening (CCS) and blastocyst transfer, the decision on the number of embryos to transfer deserves careful consideration. This report presents real-life data from two clinics using the Fischer protocol for cycle programming in patients undergoing ART. Our data indicate that ovarian stimulation using the Fischer protocol provides consistent and optimal ART outcomes in centers following strict quality management standards. However, high multiple implantation rates were observed in fresh and frozen transfer cycles after transferring two embryos - even in patients aged over 39 years. The live birth rates after CCS were superior to those using untested embryos. These findings were held for the three age groups irrespective of the CCS culture day (D1=PN stages, or D5=blastocysts). Our results support a single embryo transfer policy, particularly in women under 34 years of age with favorable conditions during ART treatment, i.e., a high number of available fertilized oocytes.

Preimplantation Genetic Testing for Aneuploidy with Comprehensive Chromosome Screening in Patients Undergoing In Vitro Fertilization: A Systematic Review and Meta-analysis.

Next Fertility IVF Prof. Zech, Bregenz, Austria Next Fertility IVF Prof. Zech, Salzburg, Austria Financial Disclosure The authors did not report any potential conflicts of interest.

Majority of transferred mosaic embryos developed healthy live births revealed by a preclinical study using embryonic morphology assessment and noninvasive PGT-A on cell-free DNA in blastocoel fluid

This preclinical study aimed to evaluate whether using transferred mosaic embryos (primarily selected by embryonic morphology assessment (EMA) and compared by the noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) on cell-free DNA in blastocoel fluid (BF)) increases the rates of clinical pregnancies (CPs) and healthy live births (HLBs) and to investigate whether niPGT-A could provide valuable genetic information for the EMA-selected transferred mosaic embryos. This study collected 215 blastocyst culture samples and 182 BF samples. Cell-free DNA from the BF was amplified and examined by next-generation sequencing-based niPGT-A. All 182 patients underwent EMA. However, only 147 underwent in vitro fertilization and embryo transfer, and only 113 clinical outcomes were followed up. Comprehensive chromosome screening for the chorionic villus sampling of spontaneous miscarriages and noninvasive prenatal testing for ongoing pregnancies were also performed. The implantation rate was 77.55% in 147 transferred high-quality embryos selected by EMA. Among 113 CPs, 16 led to spontaneous miscarriage (14.16%), and 97 resulted in HLBs (85.84%). According to the niPGT-A results for 113 patients with clinical outcomes, 80.4% had CP (euploid, 20.54%; single aneuploid, 1.79%; mosaic chromosome aneuploid and/or segmental aneuploid, 58.04%). Of all the mosaic aneuploids, 90.76% were false positive, transforming to euploid. Transferred EMA-selected embryos showed higher implantation rates. The niPGT-A of BF provided valuable genetic status ("-ploid") information, which helped reduce aneuploid-induced implantation failure and miscarriage, thereby increasing the CP and HLB rates. Additionally, majority of the transferred embryos with complex/chaotic mosaic aneuploid would likely develop HLBs.

Preimplantation Genetic Testing for Aneuploidy With Comprehensive Chromosome Screening in Patients Undergoing In Vitro Fertilization: A Systematic Review and Meta-analysis.

To review the effect of comprehensive chromosome screening-based preimplantation genetic testing for aneuploidy (PGT-A) in women undergoing in vitro fertilization (IVF) treatment, we conducted this meta-analysis to compare pregnancy outcomes of women who did and did not undergo such testing. We searched Medline, EMBASE, the Cochrane Central Register of Controlled Trials, and ClinicalTrials.gov from their inception until February 28, 2022, for randomized controlled trials focusing on PGT-A treatment without any language restrictions. Randomized controlled trials involving women undergoing IVF with or without PGT-A and comprehensive chromosome testing. Pooled relative risks (RRs) with 95% CIs were calculated for the primary outcome using a random-effects model with the Mantel-Haenszel method. A total of nine trials with 3,334 participants were included. Overall, PGT-A was not associated with an increased live-birth rate (RR 1.13, 95% CI 0.96-1.34, I 2 =79%). However, PGT-A raised the live-birth rate in women of advanced maternal age (RR 1.34, 95% CI 1.02-1.77, I 2 =50%) but not in women of nonadvanced age (RR 0.94, 95% CI 0.89-0.99, I 2 =0%). Preimplantation genetic testing for aneuploidy increases the live-birth rate in women of advanced maternal age. PROSPERO, CRD42022311540.

Update on preimplantation genetic testing for aneuploidy and outcomes of embryos with mosaic results.

Preimplantation genetic testing for aneuploidy (PGT-A) is used as a frequent add-on for in-vitro fertilization (IVF) to improve clinical outcomes. The purpose is to select a euploid embryo following chromosomal testing on embryo biopsies. The current practice includes comprehensive chromosome screening (CCS) technology applied on trophectoderm (TE) biopsies. Despite its widespread use, PGT-A remains a controversial topic mainly because all of the RCTs comprised only good prognosis patients with 2 or more blastocysts available; hence the results are not generalizable to all groups of patients. Furthermore, with the introduction of the highly-sensitive platforms into clinical practice (i.e. next-generation sequencing [NGS]), a result consistent with intermediate copy number surfaced and is termed "Mosaic," consistent with a mixture of euploid and aneuploid cells within the biopsy sample. The optimal disposition and management of embryos with mosaic results is still an open question, as many 'mosaics' generated healthy live births with no identifiable congenital anomalies. The present article provides a complete and comprehensive up-to-date review on PGT-A. It discusses in detail the findings of all the published RCTs on PGT-A with CCS, comments on the subject of "mosaicism" and its current management, and describes the latest technique of non-invasive PGT-A.

ESHRE survey results and good practice recommendations on managing chromosomal mosaicism.

How should ART/preimplantation genetic testing (PGT) centres manage the detection of chromosomal mosaicism following PGT? Thirty good practice recommendations were formulated that can be used by ART/PGT centres as a basis for their own policy with regards to the management of 'mosaic' embryos. The use of comprehensive chromosome screening technologies has provided a variety of data on the incidence of chromosomal mosaicism at the preimplantation stage of development and evidence is accumulating that clarifies the clinical outcomes after transfer of embryos with putative mosaic results, with regards to implantation, miscarriage and live birth rates, and neonatal outcomes. This document was developed according to a predefined methodology for ESHRE good practice recommendations. Recommendations are supported by data from the literature, a large survey evaluating current practice and published guidance documents. The literature search was performed using PubMed and focused on studies published between 2010 and 2022. The survey was performed through a web-based questionnaire distributed to members of the ESHRE special interest groups (SIG) Reproductive Genetics and Embryology, and the ESHRE PGT Consortium members. It included questions on ART and PGT, reporting, embryo transfer policy and follow-up of transfers. The final dataset represents 239 centres. The working group (WG) included 16 members with expertise on the ART/PGT process and chromosomal mosaicism. The recommendations for clinical practice were formulated based on the expert opinion of the WG, while taking into consideration the published data and results of the survey. Eighty percent of centres that biopsy three or more cells report mosaicism, even though only 66.9% of all centres have validated their technology and only 61.8% of these have validated specifically for the calling of chromosomal mosaicism. The criteria for designating mosaicism, reporting and transfer policies vary significantly across the centres replying to the survey. The WG formulated recommendations on how to manage the detection of chromosomal mosaicism in clinical practice, considering validation, risk assessment, designating and reporting mosaicism, embryo transfer policies, prenatal testing and follow-up. Guidance is also provided on the essential elements that should constitute the consent forms and the genetic report, and that should be covered in genetic counselling. As there are several unknowns in chromosomal mosaicism, it is recommended that PGT centres monitor emerging data on the topic and adapt or refine their policy whenever new insights are available from evidence. Rather than providing instant standardized advice, the recommendations should help ART/PGT centres in developing their own policy towards the management of putative mosaic embryos in clinical practice. This document will help facilitate a more knowledge-based approach for dealing with chromosomal mosaicism in different centres. In addition to recommendations for clinical practice, recommendations for future research were formulated. Following up on these will direct research towards existing research gaps with direct translation to clinical practice. Emerging data will help in improving guidance, and a more evidence-based approach of managing chromosomal mosaicism. The WG received technical support from ESHRE. M.D.R. participated in the EQA special advisory group, outside the submitted work, and is the chair of the PGT WG of the Belgian society for human genetics. D.W. declared receiving salary from Juno Genetics, UK. A.C. is an employee of Igenomix, Italy and C.R. is an employee of Igenomix, Spain. C.S. received a research grant from FWO, Belgium, not related to the submitted work. I.S. declared being a Co-founder of IVFvision Ltd, UK. J.R.V. declared patents related to 'Methods for haplotyping single-cells' and 'Haplotyping and copy number typing using polymorphic variant allelic frequencies', and being a board member of Preimplantation Genetic Diagnosis International Society (PGDIS) and International Society for Prenatal Diagnosis (ISPD). K.S. reported being Chair-elect of ESHRE. The other authors had nothing to disclose. This Good Practice Recommendations (GPR) document represents the views of ESHRE, which are the result of consensus between the relevant ESHRE stakeholders and are based on the scientific evidence available at the time of preparation. ESHRE GPRs should be used for information and educational purposes. They should not be interpreted as setting a standard of care or be deemed inclusive of all proper methods of care, or be exclusive of other methods of care reasonably directed to obtaining the same results. They do not replace the need for application of clinical judgement to each individual presentation, or variations based on locality and facility type. Furthermore, ESHRE GPRs do not constitute or imply the endorsement, or favouring, of any of the included technologies by ESHRE.

The aneuploidy testing of blastocysts developing from 0PN and 1PN zygotes in conventional IVF through TE-biopsy PGT-A and minimally invasive PGT-A.

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 TE-biopsy 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 2PN-derived 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 1PN-derived blastocysts in conventional IVF.

Case Report: Successful Pregnancy and Birth of a Healthy Baby with PGT-A for Woman with Advanced Maternal Age

Introduction: The most common cause of failed implantation and miscarriage is embryonic aneuploidy and the risk of aneuploidy increases significantly with increasing maternal age. Pre-implantation genetic testing for aneuploidy (PGT-A) can be an option to improve the prognosis of women with advanced maternal age by extracting 3-5 trophectoderm cells from Day 5 and Day 6 blastocysts for genetic testing. Case History: A 37 years old patient tried to conceive for 5 years was recruited into our PGS research study initiated by Ministry of Health in Singapore. She had 3 previous implantation failure and one miscarriage between 2015-2018. The patient had regular menses every 30-35 days. Antral follicle count was 48 and her husband’s semen analysis was normal with Density 87 million/ml, motility 34% and morphology 4%. Results: Patient underwent an IVF cycle with PGT-A in December 2018 at our IVF Centre. 23 eggs were retrieved, 21 eggs were mature to undergo ICSI and 11 fertilized. The eggs were cultured in EmbryoGen immediately after ICSI and the embryos were transferred to BlastGen on Day 3 for extended culture. A total of 5 embryos were suitable for biopsy, 3 on Day 5 and 2 on Day 6. Embryos were vitrified while waiting for the genetic results. Chromosomal analysis of the biopsied trophectoderm cells was performed using NGS platform. Out of the 5 embryos tested, 2 embryos were euploid, giving 60% aneuploidy rate. One euploid embryo was thawed and transferred in a regulated frozen-thawed cycle in March 2020. The patient became pregnant and delivered a healthy baby boy. She returned in January 2022 to put back the remaining euploid embryo, she was again pregnant. Conclusion: PGT-A for comprehensive chromosome screening in selecting and transferring euploid embryos in assisted reproduction has an immediate impact on implantation, reduces the rate of clinical miscarriages and results in higher live birth rates.

P-532 Embryo aneuploidy risk is increased in couples with unexplained infertility

Abstract Study question Are chromosomal abnormalities (aneuploidy and mosaicism) increased in couples with unexplained infertility? Summary answer Couples with unexplained infertility show higher rates of embryo aneuploidy compared to IVF with donor sperm cycles. What is known already Aneuploidy is the most common genetic abnormality found in early embryos. Although there is strong evidence about the correlation between oocyte age and aneuploidy rates, the potential contribution of additional female and male factor warrants further elucidation. Unexplained infertility is a diagnosed of exclusion affecting ∼30% of couples seeking infertility care. Recent medical evidence suggests that these couples have lower odds of having a live birth compared to their counterparts using donor sperm. Therefore, the aim of this study was to elucidate whether chromosomal abnormalities (aneuploidy and mosaicism) were increased in couples with unexplained infertility. Study design, size, duration Retrospective analysis of the chromosomic evaluation of 700 embryos coming from 220 IVF/PGT-A cycles (March 2017 - September 2021) performed in a private fertility center. The comprehensive chromosome screening included testing for aneuploidy and mosaicism. In addition, whole and segmental chromosomal alterations were also identified and reported. Participants/materials, setting, methods Participants were divided into two groups: couples with unexplained infertility (study group) and women undergoing IVF using donated sperm (control group). Only normospermic and normal sperm FISH samples were included. The Veriseq NGS (Illumina) platform was employed for PGT-A testing. An SPSSv20.0 logistic regression analysis was performed for aneuploidy and mosaicism rate. The resulted odds ratio was then adjusted to account for: female/male age, previous implantation failure, recurrent pregnancy loss, previous chromosomopathies and embryo quality. Main results and the role of chance A total of 220 PGT-A cycles were included in the analysis (study group n = 94 vs control group n = 126) comprising 700 embryos (study group n = 313 vs control group n = 387). The female age was 38.17 ± 3.6 vs 40.08 ± 2.4 and the male age was 40.57 ± 5.0 and 25.37 ± 3.8, in the study vs control groups, respectively. Unexplained infertility couples showed a higher risk for generating aneuploid embryos compared to IVF with donor sperm cycles [OR = 2.33 IC 95% (1.11-5.00)]. In addition, whole chromosomal alterations [OR = 2.11 IC 95% (1.00-4.44)] as well as segmental chromosomal alterations rate [OR = 1.38 IC 95% (0.49-3.86)], were higher in unexplained infertility couples, but without reaching statistical significance. Regarding to mosaicism rate, no significant differences were found between groups: overall [OR = 0.74 IC 95% (0.30-1.83)], whole chromosome [OR = 0.92 IC 95% (0.35-2.46)] and segmental chromosomal [OR = 0.36 IC95% (0.10-1.34)] mosaicism. Limitations, reasons for caution The inherent limitations of a retrospective analysis. Additional studies are warranted to clarify the potential factors explaining the higher embryo aneuploidy risk in this sub-set of patients. Wider implications of the findings Our data suggest an increased risk for embryo aneuploidy in unexplained infertility couples undergoing ART. Since the treatment for these couples is largely empirical, our results emphasize the importance of PGT-A analysis as a potential strategy for unexplained infertility patients. Trial registration number Not applicable

New insights regarding origin of monosomy occurrence in early developing embryos as demonstrated in preimplantation genetic testing

IntroductionAnalyses of miscarriage products indicate that the majority of aneuploidies in early developing embryos derive from errors occurring during maternal meiosis and the paternal contribution is less than 10%. Our aim was to assess the aneuploidy (mainly monosmies) frequencies at the earliest stages of embryo development, 3 days following fertilization during In vitro fertilization (IVF) treatments and to elucidate their parental origin. Later, we compared monosomies rates of day 3 to those of day 5 as demonstrated from Preimplantation Genetic Testing for Structural chromosomal Rearrangement (PGT-SR) results.MethodsFor a retrospective study, we collected data of 210 Preimplantation Genetic Testing for Monogenic Disorder (PGT-M) cycles performed between years 2008 and 2019.This study includes 2083 embryos, of 113 couples. It also included 432 embryos from 90 PGT-SR cycles of other 45 patients, carriers of balanced translocations. Defining the parental origin of aneuploidy in cleavage stage embryos was based on haplotypes analysis of at least six informative markers flanking the analyzed gene. For comprehensive chromosomal screening (CCS), chromosomal microarray (CMA) and next generation sequencing (NGS) was used.ResultsWe inspected haplotype data of 40 genomic regions, flanking analyzed genes located on 9 different chromosomes.151 (7.2%) embryos presented numerical alterations in the tested chromosomes. We found similar paternal and maternal contribution to monosomy at cleavage stage. We demonstrated paternal origin in 51.5% of the monosomy, and maternal origin in 48.5% of the monosomies cases.ConclusionIn our study, we found equal parental contribution to monosomies in cleavage-stage embryos. Comparison to CCS analyses of PGT-SR patients revealed a lower rate of monosomy per chromosome in embryos at day 5 of development. This is in contrast to the maternal dominancy described in studies of early miscarriage. Mitotic errors and paternal involvement in chemical pregnancies and IVF failure should be re-evaluated. Our results show monosomies are relatively common and may play a role in early development of ART embryos.

The Relationship between Human Embryo Parameters and De Novo Chromosomal Abnormalities in Preimplantation Genetic Testing Cycles.

Design In total, 456 PGT cycles, including 283 PGT-SR cycles and 173 PGT-A cycles, were assessed through comprehensive chromosome screening (CCS) from January 2017 to June 2020 at the Department of Reproductive Medicine of the Third Affiliated Hospital of Zhengzhou University. Trophectoderm (TE) biopsies were sequenced using next-generation sequencing (NGS). The incidence of de novo chromosome abnormalities was calculated, and the relationships between de novo chromosome abnormality rates and maternal age, number of oocytes retrieved, and parameters of cleavage-stage embryos and blastocyst-stage embryos were investigated. Results The incidence of de novo chromosome abnormalities was 28.0% (318/1,135) in the PGT-SR cycles and 36.3% (214/590) in the PGT-A cycles, which increased with maternal age in both PGT-SR cycles (P = 0.018) and PGT-A cycles (P < 0.001). The incidence of de novo chromosome abnormalities was related to TE grade (P < 0.001), internal cell mass grade (P = 0.002), and development speed (day 5 vs. day 7: P < 0.001) of blastocyst-stage embryos. The incidence of de novo chromosomal abnormalities was irrelevant to the number of oocytes retrieved and the parameters of the embryo at the cleavage stage. Conclusion Blastocysts with higher morphology scores and faster progression had a lower incidence of de novo chromosome abnormalities, especially complex chromosome abnormalities. De novo chromosome abnormalities may negatively affect the morphological grading of blastocysts. Our findings will provide valuable information to the fertility doctor for embryo selection in non-PGT cycles.