Embryo Aneuploidy Research Articles

P-711 Female age at embryo creation and embryo transfer do not impact the ongoing pregnancy rates in single euploid frozen embryo transfers

Abstract Study question Which factors influence the ongoing pregnancy rate in single euploid frozen embryo transfer (seFET) cycles? Summary answer Ongoing pregnancy rates in seFET cycles are significantly influenced by parity, BMI, embryo quality, trophectoderm biopsy day and endometrial preparation, but not by female age. What is known already The probability of a pregnancy is influenced by several factors and female age is the major determinant as the ovarian reserve decreases and embryo aneuploidy increases with age. Preimplantation genetic testing for aneuploidy and embryo selection was considered to mitigate the effect of female age on ART treatment outcomes by selecting euploid embryo(s) for embryo transfer (ET). Previously published data point to a decline in pregnancy rates with increased maternal age even when a euploid embryo is transferred. However, other factors besides embryo ploidy might play a role in the chance of achieving an ongoing pregnancy. Study design, size, duration Retrospective study, including 1923 women who underwent a seFET between 04/2017 and 08/2023. Patients’ (age at oocyte-pick-up and seFET, BMI, parity, endometrial preparation type) and embryo characteristics (Day of biopsy, embryo quality (EQ)) were considered for the analysis. Ongoing pregnancy was defined as viable pregnancy beyond 12 weeks of gestation. Participants/materials, setting, methods Women, undergoing a seFET either in a true natural cycle (NC) t or in a hormonal replacement cycle (HRT). Only cycles with embryos being biopsied on day 5 or day 6 and a known outcome of the cycle were included. Main results and the role of chance There were 990 (51.48%) ongoing pregnancies among 1923 included transfers. The ongoing pregnancy rates were 51.8%, 47.4%, 52.3% and 52.8% for women aged ≤35, >35 to ≤ 37, >37 to ≤ 40, and >40 years-old at seFET, respectively. Cochrane Armitage test did not show a significant trend for decreasing pregnancy rates with increasing female age at OPU (P = 0.679) or at transfer (P = 0.609). Multivariable regression analyses showed primiparous, and multiparous women were more likely to have an ongoing pregnancy compared to nulliparous women (OR: 2.96, 95%CI:2.22-3.94 and 2.73, 95%CI:2.10-3.55, respectively, P < 0.001 for both) and those who had a natural endometrial preparation (OR: 1.53, 95%CI: 1.24-1.88, P < 0.001). Transfers with good quality embryo had higher ongoing pregnancy rates compared to those who had a fair quality embryo (OR: 2.0, 95%CI: 1.37-2.91, P < 0.001). Being overweight or obese at the time of transfer were associated with lower ongoing pregnancy rates compared to normal weight (OR: 0.71, 95%CI: 0.56-0.89, and 0.68, 95%CI: 0.52-0.88, respectively, P = 0.003 for both). Age categories at the time of transfer did not show a significant effect on ongoing pregnancy rates in multivariable analyses (P > 0.05 for all) and there was no significant interaction between age at OPU and embryo quality categories either (P = 0.881 and 0.601). Limitations, reasons for caution The retrospective design of the study is a limitation. Wider implications of the findings This analysis did not confirm the otherwise reported negative impact of female age on the outcome of seFET cycles. Outcomes are significantly influenced by BMI, parity, EQ-related factors as well as the endometrial preparation. As BMI and endometrial preparation can be influenced, the adequate choice might improve seFET outcomes. Trial registration number not applicable

O-239 Automation in the embryology laboratory

Abstract Most processes in an IVF laboratory are performed manually by technicians and embryologists, such as tracking and prepare dishes, pipetting of gametes and embryos from one dish to another, loading freezing straws, and operating micromanipulators for ICSI or biopsy. In most centres doctors manage hormonal stimulation based on their own learned experience or at most of their team. This results in significant variability in pregnancy outcomes from centre to centre and even operator to operator. For example, centres can be compared based on egg donor cycles, which are all young and fertile patients selected in similar ways in most centres. But even In egg donor cycles, embryo aneuploidy rates vary 20% to 60% (1) while pregnancy rates fluctuate from 10% to 80% depending on the clinic (2). Furthermore, although large amounts of data are generated from >2M procedures of IVF a year worldwide, most of it is siloed in each centre in a myriad of EMRs or worse, spreadsheets, impossible to query globally. The lack of a massive dataset to query and the absence of a constant baseline in pregnancy outcomes makes introducing new innovations harder to determine if they work or not. Automation should allow constant results with similar results clinic to clinic, especially desirable for networks to offer a consistent service across centres. By reducing the reliance on highly skilled manual labour, which is facing a global scarcity, automation has the potential to offer more people affordable treatment. Moreover, the utilization of automation generates vast amounts of data, with machines sharing information to inform medical decisions based on millions of standardized treatments, surpassing the insights of individual physicians. This data-driven approach also establishes a stable foundation for innovation by minimizing human variables. Advancements in Affordable Robotics, coupled with high precision and miniaturization, alongside the evolution of software, are transforming robots into versatile tools akin to smartphones. This transformation encompasses various fields, including microfluidics, miniaturized imaging systems, Artificial Intelligence, and the Internet of Things (IoT). Some automation has already occurred, such time-lapse incubators with imaging allowing for AI classification of embryos, or witness systems. Although historical lack of government funding and current trends of consolidation of clinics into networks, may slow innovation, numerous startup companies are actively developing automation solutions for IVF laboratory operations and patient management. These solutions range from tools for remote patient monitoring to automating processes such as egg denudation, sperm analysis, oocyte and embryo vitrification and warming, as well as procedures like automated ICSI and in vitro fertilization. Furthermore, artificial intelligence is being harnessed for tasks like embryo selection and hormonal stimulation, with groundbreaking concepts like artificial uterus also under exploration. Nonetheless, the objective of automation should be to enhance the abilities of the embryologist, not to reduce their need. Once the process has been standardized, it should pave the way for the creation of novel and more interesting tasks. This empowers the embryologist to engage in higher complexity or more high touch tasks, rather than merely replacing labor.

P-294 Intrauterine infusion of autologous platelet-rich plasma(PRP) for patients with refractory thin endometrium in euploid frozen embryo transfer cycles improve pregnancy outcomes

Abstract Study question Can intrauterine infusion of autologous platelet-rich plasma(PRP) for patients with refractory thin endometrium in euploid frozen embryo transfer(FET) cycles improve pregnancy outcomes? Summary answer Intrauterine infusion of autologous platelet-rich plasma(PRP) for patients with refractory thin endometrium in euploid FET cycles improve pregnancy outcomes. What is known already Previous studies have reported a positive correlation between endometrial thickness and pregnancy rate, which has led to the common clinical practice of canceling embryo transfer in cycles where the patient’s endometrial thickness remains below 7mm.One of the novel and promising treatments to improve pregnancy outcomes in patients with refractory thin endometrium is intrauterine infusion of autologous PRP.Recent studies demonstrated that intrauterine infusion of PRP improve pregnancy outcomes in these patients.Thus, to better assess the efficacy of intrauterine PRP treatment, further studies involving euploid embryos are necessary to rule out failed implantation or pregnancy loss due to aneuploidy of the embryos. Study design, size, duration This retrospective study analyzed all cycles(n = 131) of euploid FET in women with refractory thin endometrium at the CHA Fertility Center in Seoul Station from January 2022 to August 2023.Patients were divided into two groups according to whether they received PRP intrauterine infusion before embryo transfer in euploid FET cycles.Group A(n = 88) received intrauterine infusion of PRP at least once before transfer of euploid FET,and Group B(n = 43) underwent euploid FET without PRP infusion. Participants/materials, setting, methods Intrauterine infusion of autologous PRP was performed on patients who had more than two canceled FET cycles due to thin endometrial thickness(<7mm). Endometrial preparation was executed according to a standardized protocol for hormone replacement therapy. Group A received the last autologous PRP infusion 48 hours before euploid FET. PRP preparation was carried using an aseptic PRP centrifuge kit (PROSYS PRP; Prodizen, Korea). All embryos transferred were tested for genetic abnormalities using next-generation sequencing. Main results and the role of chance There was no significant difference in mean age and maximum endometrial thickness between the two groups. (38.1±3.1 vs. 38.3±3.2, p = 0.986 and 6.6±1.1 vs. 6.0±0.8, p = 0.320, respectively) Group A had significantly higher clinical pregnancy rates and ongoing pregnancy rates than Group B. (51.1% vs. 30.2%, p = 0.024 and 44.3 vs. 16.3%, p = 0.0017, respectively) Implantation rate and beta HCG positivity were also higher in group A, but there was no statistical difference. (63.6% vs. 46.5%, p = 0.063 and 37.2 vs. 23.2%, p = 0.064, respectively) Limitations, reasons for caution This is a retrospective study and results must be confirmed on a well-designed randomized controlled study. Also, follow-up study consisting of larger number of patients is necessary. Wider implications of the findings Autologous PRP intrauterine infusion has shown positive effects in assisting implantation in patients with refractory thin endometrium. In particular, euploid frozen embryo transfer in these patients excludes failed implantation and pregnancy loss due to embryo aneuploidy, suggesting that intrauterine PRP treatment contributes to successful implantation and pregnancy. Trial registration number not applicable

P-551 Application of simultaneous PGT-M/PGT-A using whole genome sequencing and haplotyping for monogenetic disorders, multiple variants, low penetrance, and de novo disorders

Abstract Study question Is the concurrent use of high-resolution PGT-A and genome-wide haplotyping with disease-specific assays for PGT-M effective in an unselected patient population at-risk for monogenetic disorders? Summary answer PGT-A/PGT-M is an effective treatment for couples/patients burdened with genetic diseases, that provides optimal family planning, prevents vertical transmission, and reduces personal and societal encumbrance What is known already Societal awareness of genetic testing expanded the use of carrier screening, diagnostic genetic tests, and higher in vitro fertilization (IVF) utilization has increased demand for preimplantation genetic testing for monogenetic disorders (PGT-M), as an alternative to invasive prenatal tests. The scope of PGT-M testing expands beyond common severe childhood disorders, to include serious and mild late onset disorders, cancer predisposition, variants of unknown significance, de novo mutations, and variants in multiple genes. This highlights novel ethical dilemmas, necessitating adjustment of generic genome wide approaches to complement, or eventually replace, targeted disease-specific PGT-M assays. Study design, size, duration This is a retrospective study of 359 couples at-risk for monogenetic disorders referred to the CReATe Fertility Centre, Toronto, Canada. Our objective was to evaluate the efficiency of concurrent use of genome wide haplotyping and disease specific assays for PGT-M and high resolution PGT-A in an unselected patient population at risk for monogenetic disorders, multiple and de novo variants, low penetrance and variants of unknown significance. Participants/materials, setting, methods All patients received comprehensive genetic counselling. For each couple/patient, DNA from gamete providers, related family member/s and whole-genome amplified (WGA)DNA from a single trophectoderm(TE) biopsy were analyzed. Whole-genome SNP-array (Karyomapping, Illumina, CA) and STR-analysis were used to obtain parental haplotypes and Sanger-sequencing was used for direct variant analysis. PGT-A was performed using Illumina platform(NextSeq 550). NxClinical (BioDiscovery) and BlueFuse Multi(Illumina) software were used for data analysis. Main results and the role of chance 1826 embryos, from 235 IVF cycles were analyzed for 88 unique genes (14 cancer predisposing, 42 autosomal recessive and 32 dominant genes) and a total of 282 unique variants. The most frequent genes tested were BRCA1/2 -21% of all cases, followed by HBB-8.7%, CFTR-8.7% and CYP21A2-8.7%. PGT-M for 11 cases was done for a de novo variant detected in a sibling and in 2 cases for a parental mosaic variant. Multiple genes and VUS were analyzed in 12 and 6 cases, respectively. PGT-A analysis showed 31.6% aneuploidy, 9.6% mosaicism and 58.8% euploidy. Genome wide haplotyping was used for euploid and mosaic embryos with an average case call rate of 95%, and average case Allele Drop Out (ADO) rate of 2.7%. Recombination events in 3% of cases prevented assignment of haplotype. Direct mutation testing was obtained for all samples and was 100% concordant with the predicted haplotypes. In cases with no predicted haplotype, direct testing established the PGT-M diagnosis. The distribution for both recessive and dominant disorders was in Mendelian order. To date 66 live births confirmed the results from PGT-M diagnosis. Limitations, reasons for caution Ethical considerations and extensive genetic counselling are prerequisites for PGT-A/M testing implementation. Simultaneous screening for embryo aneuploidy and monogenetic disorders optimizes patient treatment and family planning, but may not be available in all jurisdictions. Although the data was collected prospectively, retrospective design is a limitation. Wider implications of the findings Simultaneous PGT-A and PGT-M with direct variant testing provides optimal embryo prioritization for transfer and significantly reduces time to a healthy pregnancy in couples at risk for inherited disorders. Karyomapping is effective for generic haplotyping, with a low risk of undetected recombination events in the genomic region of interest. Trial registration number not applicable

P-588 Serum Luteinizing Hormone levels before progesterone supplementation are significantly associated with live birth rate in single euploid frozen embryo transfers performed in hormonal replacement cycles

Abstract Study question Do luteinizing hormone (LH) levels measured prior to initiating progesterone supplementation influence live birth rate (LBR) in artificial frozen-thawed single euploid blastocyst transfer cycles? Summary answer Serum LH levels, measured prior to progesterone initiation in artificial frozen-thawed single euploid blastocyst transfer cycles, are significantly associated with LBR. What is known already In the absence of a dominant follicle, estrogen supplementation in artificial endometrial preparation for frozen embryo transfer (FRET) can lead to a rise in endogenous LH levels, as observed in a natural cycle. Since the LH rise is a key feature in natural conception and the embryo, the endometrium, and the myometrium display receptors for LH, the rise observed during FRET preparation may have an impact on the clinical outcomes of the treatment. Previous studies found contradictory results hence none of them excluded embryo aneuploidy by implementation of preimplantation genetic testing for aneuploidies and the transfer of euploid embryos. Study design, size, duration Retrospective analysis of 639 frozen-thawed single euploid blastocyst transfer cycles performed in a tertiary referral IVF center from January 2018 to August 2023. Cycles with hormonal pre-treatment or pituitary suppression were excluded. Participants/materials, setting, methods Single euploid FRET cycles were included. Oral estradiol administration commenced on cycle-day 2/3. Estradiol, progesterone, and LH were measured at the start of endometrial preparation and up to 3 days before progesterone initiation, and the slope of LH was calculated. Patients were stratified in groups according to their LH values before progesterone administration, and the impact of LH on LBR and implantation rate was assessed. Logistic regression analysis was performed to adjust for potential confounders. Main results and the role of chance In total 639 cycles were included. Patients’ characteristics (mean+/-SD) were: age 33.2±5.9 years; BMI 27.5±4.9 kg/m2. The median duration of estrogen administration was 15 days (range: 11-27 days). The overall implantation rate and LBR were 65.9% (421/639) and 46.9% (300/639), respectively. Patients were stratified into six groups according to their LH levels prior to progesterone supplementation: <10th percentile (≤6mIU/ml, n = 57), p10th-p25th (>6 to ≤ 8.5mIU/ml, n = 81), p25th-p50th (>8.5 to ≤ 12.4mIU/ml, n = 176), p50th-p75th (>12.4 to ≤ 18.1mIU/ml, n = 180), p75th-p90th (>18.1 to ≤ 25.0mIU/ml, n = 91) and >p90th (>25.0mIU/ml, n = 54). A significant trend for higher LBR with increasing serum LH levels was observed (Cochran-Armitage Test, P = 0.041) but not for implantation rate (P = 0.158). Those with higher basal serum estradiol levels before estrogen supplementation had a steeper LH increase from basal to progesterone-supplementation day (0.40 mIU/mL higher per 10pmol/L increase in basal estradiol, P = 0.027). Regression analysis identified as independent predictors of LBR BMI (OR 0.96, 95%CI: 0.93-0.99) and embryo quality (CC vs. AA p < 0.001, CB vs. AA p = 0.027, BC vs. AA p = 0.013). Patients with steeper LH increases from estrogen to progesterone-supplementation day had higher LBR in both univariable (OR:1.27, 95%CI: 1.00–1.63, P = 0.052) and multivariable analyses (OR:1.26, 95%CI: 0.97–1.64, P = 0.085) but the difference didn’t reach statistical significance. Limitations, reasons for caution The retrospective design of the study is a limitation. Results should be interpreted with caution due to the small number of patients in the lowest and highest LH percentile groups. Wider implications of the findings The results of this study suggest that serum LH levels measured before progesterone supplementation may have an impact on the clinical outcome of artificial frozen-thawed blastocyst transfer cycles. Further studies with a higher sample size are necessary to confirm or refute our findings. Trial registration number not applicable

Excessive Exogenous Gonadotropins and Genetic and Pregnancy Outcomes After Euploidy Embryo Transfer

The safety of exogenous gonadotropin treatment, based on its effect on embryos and pregnancy outcomes, remains inconclusive. To evaluate the associations of different doses and durations of gonadotropins with embryonic genetic status and pregnancy outcomes after euploid embryo transfer in couples with infertility. This study was a post hoc analysis of a multicenter randomized clinical trial (RCT) conducted at 14 reproductive centers throughout China from July 2017 to June 2018 that evaluated the cumulative live birth rate with or without preimplantation genetic testing for aneuploidy (PGT-A) among couples with infertility and good prognosis. The PGT-A group from the original RCT was selected for secondary analysis. Patients were divided into 4 groups according to the total dosage of exogenous gonadotropins and treatment duration: group 1 (≤1500 IU and <10 days), group 2 (≤1500 IU and ≥10 days), group 3 (>1500 IU and <10 days), and group 4 (>1 500 IU and ≥10 days). Group 1 served as the control group. Data were analyzed from June through August 2023. Blastocyst biopsy and PGT-A. The primary outcomes were embryonic aneuploidy, embryonic mosaicism, and cumulative live birth rates after euploid embryo transfer. A total of 603 couples (mean [SD] age of prospective mothers, 29.13 [3.61] years) who underwent PGT-A were included, and 1809 embryos were screened using next-generation sequencing. The embryo mosaicism rate was significantly higher in groups 2 (44 of 339 embryos [13.0%]; adjusted odds ratio [aOR], 1.69 [95% CI, 1.09-2.64]), 3 (27 of 186 embryos [14.5%]; aOR, 1.98 [95% CI, 1.15-3.40]), and 4 (82 of 651 embryos [12.6%]; aOR, 1.60 [95% CI, 1.07-2.38]) than in group 1 (56 of 633 embryos [8.8%]). There were no associations between gonadotropin dosage or duration and the embryo aneuploidy rate. The cumulative live birth rate was significantly lower in groups 2 (83 of 113 couples [73.5%]; aOR, 0.49 [95% CI, 0.27-0.88]), 3 (42 of 62 couples [67.7%]; aOR, 0.41 [95% CI, 0.21-0.82]), and 4 (161 of 217 couples [74.2%]; aOR, 0.53 [95% CI, 0.31-0.89]) than in group 1 (180 of 211 couples [85.3%]). In this study, excessive exogenous gonadotropin administration was associated with increased embryonic mosaicism and decreased cumulative live birth rate after euploid embryo transfer in couples with a good prognosis. These findings suggest that consideration should be given to minimizing exogenous gonadotropin dosage and limiting treatment duration to improve embryo outcomes and increase the live birth rate. ClinicalTrials.gov Identifier: NCT03118141.

Single-cell sequencing shows mosaic aneuploidy in most human embryos.

Mammalian preimplantation embryos often contain chromosomal defects that arose in the first divisions after fertilization and affect a subpopulation of cells - an event known as mosaic aneuploidy. In this issue of the JCI, Chavli et al. report single-cell genomic sequencing data for rigorous evaluation of the incidence and degree of mosaic aneuploidy in healthy human in vitro fertilization (IVF) embryos. Remarkably, mosaic aneuploidy occurred in at least 80% of human blastocyst-stage embryos, with often less than 20% of cells showing defects. These findings confirm that mosaic aneuploidy is prevalent in human embryos, indicating that the process is a widespread event that rarely has clinical consequences. There are major implications for preimplantation genetic testing of aneuploidy (PGT-A), a test commonly used to screen and select IVF embryos for transfer. The application and benefit of this technology is controversial, and the findings provide more cause for caution on its use.

Early onset of APC/C activity renders SAC inefficient in mouse embryos.

Control mechanisms of spindle assembly and chromosome segregation are vital for preventing aneuploidy during cell division. The mammalian germ cells and embryos are prone to chromosome segregation errors, and the resulting aneuploidy is a major cause of termination of development or severe developmental disorders. Here we focused on early mouse embryos, and using combination of methods involving microinjection, immunodetection and confocal live cell imaging, we concentrated on the Spindle Assembly Checkpoint (SAC) and Anaphase Promoting Complex/Cyclosome (APC/C). These are two important mechanisms cooperating during mitosis to ensure accurate chromosome segregation, and assessed their activity during the first two mitoses after fertilization. Our results showed, that in zygotes and 2-cell embryos, the SAC core protein Mad1 shows very low levels on kinetochores in comparison to oocytes and its interaction with chromosomes is restricted to a short time interval after nuclear membrane disassembly (NEBD). Exposure of 2-cell embryos to low levels of spindle poison does not prevent anaphase, despite the spindle damage induced by the drug. Lastly, the APC/C is activated coincidentally with NEBD before the spindle assembly completion. This early onset of APC/C activity, together with precocious relocalization of Mad1 from chromosomes, prevents proper surveillance of spindle assembly by SAC. The results contribute to the understanding of the origin of aneuploidy in early embryos.

Novel MEI1 mutations cause chromosomal and DNA methylation abnormalities leading to embryonicarrest and implantation failure.

This study presents a case of a female infertile patient suffering from embryonic arrest and recurrent implantation failure. The primary objective was to assess the copy number variations (CNVs) and DNA methylation of her embryos. Genetic diagnosis was conducted by whole-exome sequencing and validated through Sanger sequencing. CNV evaluation of two cleavage stage embryos was performed using whole-genome sequencing, while DNA methylation and CNV assessment of two blastocysts were carried out using whole-genome bisulfite sequencing. We identified two novel pathogenic frameshift variants in the MEI1 gene (NM_152513.3, c.3002delC, c.2264_2268 + 11delGTGAGGTATGGACCAC) in the proband. These two variants were inherited from her heterozygous parents, consistent with autosomal recessive genetic transmission. Notably, two Day 3 embryos and two Day 6 blastocysts were all aneuploid, with numerous monosomy and trisomy events. Moreover, global methylation levels greatly deviated from the optimized window of 0.25-0.27, measuring 0.344 and 0.168 for the respective blastocysts. This study expands the mutational spectrum of MEI1 and is the first to document both aneuploidy and abnormal methylation levels in embryos from a MEI1-affected female patient presenting with embryonic arrest. Given that females affected by MEI1 mutations might experience either embryonic arrest or monospermic androgenetic hydatidiform moles due to the extrusion of all maternal chromosomes, the genetic makeup of the arrested embryos of MEI1 patients provides important clues for understanding the different disease mechanisms of the two phenotypes.

Blastocyst telomere length predicts successful implantation after frozen-thawed embryo transfer.

Do embryos with longer telomere length (TL) at the blastocyst stage have a higher capacity to survive after frozen-thawed embryo transfer (FET)? Digitally estimated TL using low-pass whole genome sequencing (WGS) data from the preimplantation genetic testing for aneuploidy (PGT-A) process demonstrates that blastocyst TL is the most essential factor associated with likelihood of implantation. The lifetime TL is established in the early cleavage cycles following fertilization through a recombination-based lengthening mechanism and starts erosion beyond the blastocyst stage. In addition, a telomerase-mediated slow erosion of TL in human fetuses has been observed from a gestational age of 6-11 weeks. Finally, an abnormal shortening of telomeres is likely involved in embryo loss during early development. Blastocyst samples were obtained from patients who underwent PGT-A and FET in an IVF center from March 2015 to May 2018. Digitally estimated mitochondrial copy number (mtCN) and TL were used to study associations with the implantation potential of each embryo. In total, 965 blastocysts from 232 cycles (164 patients) were available to investigate the biological and clinical relevance of TL. A WGS-based workflow was applied to determine the ploidy of each embryo. Data from low-pass WGS-PGT-A were used to estimate the mtCN and TL for each embryo. Single-variant and multi-variant logistic regression, decision tree, and random forest models were applied to study various factors in association with the implantation potential of each embryo. Of the 965 blastocysts originally available, only 216 underwent FET. While mtCN from the transferred embryos is significantly associated with the ploidy call of each embryo, mtCN has no role in impacting IVF outcomes after an embryo transfer in these women. The results indicate that mtCN is a marker of embryo aneuploidy. On the other hand, digitally estimated TL is the most prominent univariant factor and showed a significant positive association with pregnancy outcomes (P < 0.01, odds ratio 79.1). We combined several maternal and embryo parameters to study the joint effects on successful implantation. The machine learning models, namely decision tree and random forest, were trained and yielded classification accuracy of 0.82 and 0.91, respectively. Taken together, these results support the vital role of TL in governing implantation potential, perhaps through the ability to control embryo survival after transfer. The small sample size limits our study as only 216 blastocysts were transferred. The number was further reduced to 153 blastocysts, where pregnancy outcomes could be accurately traced. The other limitation of this study is that all data were collected from a single IVF center. The uniform and controlled operation of IVF cycles in a single center may cause selection bias. We present novel findings to show that digitally estimated TL at the blastocyst stage is a predictor of pregnancy capacity after a FET cycle. As elective single-embryo transfer has become the mainstream direction in reproductive medicine, prioritizing embryos based on their implantation potential is crucial for clinical infertility treatment in order to reduce twin pregnancy rate and the time to pregnancy in an IVF center. The AI-powered, random forest prediction model established in this study thus provides a way to improve clinical practice and optimize the chances for people with fertility problems to achieve parenthood. This study was supported by a grant from the National Science and Technology Council, Taiwan (MOST 108-2321-B-006-013 -). There were no competing interests. N/A.

Preimplantation genetic testing in the current era, a review.

Preimplantation genetic testing (PGT), also referred to as preimplantation genetic diagnosis (PGD), is an advanced reproductive technology used during in vitro fertilization (IVF) cycles to identify genetic abnormalities in embryos prior to their implantation. PGT is used to screen embryos for chromosomal abnormalities, monogenic disorders, and structural rearrangements. Over the past few decades, PGT has undergone tremendous development, resulting in three primary forms: PGT-A, PGT-M, and PGT-SR. PGT-A is utilized for screening embryos for aneuploidies, PGT-M is used to detect disorders caused by a single gene, and PGT-SR is used to detect chromosomal abnormalities caused by structural rearrangements in the genome. In this review, we thoroughly summarized and reviewed PGT and discussed its pros and cons down to the minutest aspects. Additionally, recent studies that highlight the advancements of PGT in the current era, including their future perspectives, were reviewed. This comprehensive review aims to provide new insights into the understanding of techniques used in PGT, thereby contributing to the field of reproductive genetics.

The reproductive potential of vitrified-warmed euploid embryos declines following repeated uterine transfers

BackgroundRecurrent implantation failure (RIF) represents a vague clinical condition with an unclear diagnostic challenge that lacks solid scientific underpinning. Although euploid embryos have demonstrated consistent implantation capabilities across various age groups, a unanimous agreement regarding the advantages of preimplantation genetic testing for aneuploidy (PGT-A) in managing RIF is absent. The ongoing discussion about whether chromosomal aneuploidy in embryos significantly contributes to recurrent implantation failure remains unsettled. Despite active discussions in recent times, a universally accepted characterization of recurrent implantation failure remains elusive. We aimed in this study to measure the reproductive performance of vitrified-warmed euploid embryos transferred to the uterus in successive cycles.MethodsThis observational cohort study included women (n = 387) with an anatomically normal uterus who underwent oocyte retrieval for PGT-A treatment with at least one biopsied blastocyst, between January 2017 and December 2021 at a university-affiliated public fertility center. The procedures involved in this study included ICSI, blastocyst culture, trophectoderm biopsy and comprehensive 24-chromosome analysis of preimplantation embryos using Next Generation Sequencing (NGS). Women, who failed a vitrified-warmed euploid embryo transfer, had successive blastocyst transfer cycles (FET) for a total of three using remaining cryopreserved euploid blastocysts from the same oocyte retrieval cycle. The primary endpoints were sustained implantation rate (SIR) and live birth rate (LBR) per vitrified-warmed single euploid embryo. The secondary endpoints were mean euploidy rate (m-ER) per cohort of biopsied blastocysts from each patient, as well as pregnancy and miscarriage rates.ResultsThe mean age of the patient population was 33.4 years (95% CI 32.8–33.9). A total of 1,641 embryos derived from the first oocyte retrieval cycle were biopsied and screened. We found no associations between the m-ER and the number of previous failed IVF cycles among different ranges of maternal age at oocyte retrieval (P = 0.45). Pairwise comparisons showed a significant decrease in the sustained implantation rate (44.7% vs. 30%; P = 0.01) and the livebirth rate per single euploid blastocyst (37.1% vs. 25%; P = 0.02) between the 1st and 3rd FET. The cumulative SIR and LBR after up to three successive single embryo transfers were 77.1% and 68.8%, respectively. We found that the live birth rate of the first vitrified-warmed euploid blastocyst transferred decreased significantly with the increasing number of previously failed IVF attempts by categories (45.3% vs. 35.8% vs. 27.6%; P = 0.04). A comparable decrease in sustained implantation rate was also observed but did not reach statistical significance (50% vs. 44.2 vs. 37.9%; P = NS). Using a logistic regression model, we confirmed the presence of a negative association between the number of previous IVF failed attempts and the live birth rate per embryo transfer cycle (OR = 0.76; 95% CI 0.62–0.94; P = 0.01).ConclusionsThese findings are vital for enhancing patient counseling and refining management strategies for individuals facing recurrent implantation failure. By tailoring interventions based on age and ovarian reserve, healthcare professionals can offer more personalized guidance, potentially improving the overall success rates and patient experiences in fertility treatments.Trial registration numberN/A.

Association Between Anti-Müllerian Hormone and Early Spontaneous Abortion in Assisted Reproduction Treatment: A Case-Control Study Integrated with Biological Evidence.

Early spontaneous abortion (ESA) is a common adverse pregnancy outcome mainly attributed to embryo chromosomal abnormalities. However, as a quantitative marker, whether the anti-Müllerian hormone (AMH) can reflect oocyte quality is still controversial. By integrating biological evidence and adjusting many cofounders, this study aimed to clarify the controversies about the association between AMH and ESA caused by embryo aneuploidy during assisted reproductive technology (ART) treatment. We strictly preselected 988 patients receiving first ART treatment for analyzing clinical data, while 55 of them acquired chorionic villi karyotype results. In addition, 373 biopsied embryos from 126 patients receiving preimplantation genetic diagnosis (PGT) were tracked to compare embryo karyotypes. Univariate and multiple factor regressions were applied to analyze the risk factors leading to ESA. As covariates unadjusted, AMH (odds ratio 0.87, 95% CI 0.82-0.93) was the significant variable contributing to ESA. However, AMH played no significant role in the following regression models after age was adjusted. Also, AMH had no significant association with ESA in most age-adjusted subgroups, except in the male factors engaged subgroup. Additionally, compared to the patients with euploid chorionic villi karyotypes, those with aneuploid karyotypes were older and acquired fewer oocytes, yet their AMH levels were not significantly different. Furthermore, the embryo aneuploidy was independent of AMH while associated with maternal age, retrieved oocyte number, and embryo quality. This study suggested that AMH was unassociated with the ESA caused by embryo aneuploidy in ART therapy. As a critical cofounder, age remains the variable closely related to ESA.

Healthy Live Births after the Transfer of Mosaic Embryos: Self-Correction or PGT-A Overestimation?

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.

Y chromosome AZFc microdeletion may have negative effect on embryo euploidy: a retrospective cohort study

BackgroundEmbryo aneuploidy is a main of principal reason of pregnancy loss, in vitro fertilization (IVF) failure and birth defects in offspring. Previous researchs have demonstrated that Y chromosome AZFc microdeletion was associated with reproduction outcomes, however, the relationship between Y chromosome AZFc microdeletion and embryo aneuploidy remains unexplored.MethodsThis retrospective cohort study enrolled 513 patients with 603 cycles in the reproductive center of Nanjing Maternity and Child Health Care Hospital from January 1, 2016 to June 30, 2022. The study cohort was divided into two groups: the AZFc microdeletion group, comprising 53 patients and 58 cycles, and the control group, comprising 460 patients and 545 cycles. Statistical methods including restricted cubic spline and generalized estimating equation (GEE) were employed to evaluate the relationship between Y chromosome AZFc microdeletion and embryo euploidy.Results294 and 2833 blastocysts were selected as AZFc microdeletion group and control group, respectively. Patients with Y chromosome AZFc microdeletion had significantly higher embryo aneuploid rate (33.0% vs. 27.3%, P < 0.05), lower rate of normal fertilization rate (81.5% vs. 90.3%, P < 0.05) and lower blastocysts formation rate (47.0% vs. 57.8%, P < 0.05) compared with the control group. However, no significant differences in pregnancy outcomes after euploid embryos transfer were observed between these two groups.ConclusionsOur study underscored the association between Y chromosome AZFc microdeletion and an elevated risk of embryo aneuploidy. Before the conventional intracytoplasmic sperm injection (ICSI) treatment, couples with Y chromosome AZFc microdeletion should be apprised of the heightened susceptibility to embryo aneuploidy. Preimplantation genetic testing for aneuploidy (PGT-A) should be introduced for selection.

#88 : Correlation Between AMH Levels and Aneuploidy Rates in Patients Over 40 Years of Age: A Novel Finding

Background and Aims: AMH is a crucial biomarker of ovarian reserve and predictor of ovarian response to stimulation. Ovarian reserve is inversely proportional to women’s age, consequently lower AMH levels are seen in older women particularly at [Formula: see text]40 yrs of age. A correlation between age and embryo aneuploidy was established but not with AMH level. We hypothesize that older women with good ovarian reserve produce less aneuploid embryos and produce the same blastocyst rate. Method: A retrospective study was undertaken involving 507 IVF cycles in patients aged [Formula: see text]40 years who had ICSI with PGT-A using NGS. The cycles were categorized into three main cohorts based on the patient’s AMH level: low (&lt; 1.1 ng/mL), moderate (&gt;1.1 to [Formula: see text] 3.0 ng/mL) and high (&gt; 3.0 ng/mL). Each cohort was subdivided into 3 age sub-cohorts as follow: (40 to &lt;42), (42 to &lt;44), and (&gt;44). Our primary outcome was aneuploidy rate whereas the secondary outcome was blastulation rate. Results: The results will be mentioned in sequence: low AMH, moderate AMH and, high AMH levels. In the [Formula: see text]40 to &lt; 42 age group, the aneuploidy rates were 78.2%, 72.3% and, 67.2%, respectively (n = 232; p = 0.027). The blastulation rates were 53.8%, 47.7% and, 48.5% respectively (p&gt; 0.05). In the [Formula: see text]42 and &lt;44 age group, the aneuploidy rates were 89.2%, 83.2% and, 81.5% respectively (n = 136 p=0.039). The blastulation rates were 45.2%, 46.2% and, 43.8% respectively p&gt; 0.05. In the [Formula: see text]44 age group, the aneuploidy rates were 93.9%, 91.2% and, 89.4% respectively (n = 139; p = 0.043). The blastulation rates were 41.2%, 39.2% and, 38.8% respectively (p&gt;0.05). Conclusion: The aneuploidy rates rises when AMH levels decreases; whereas the blastulation rates was not associated with AMH levels in the population studied.

#176 : The Relationship Between Blastocyst Morphology and PGD Result

Background and Aims: Blastocyst morphology has been clinically assessed during the selection of blastocyst for transfer and cryopreservation, and the morphological grading scheme has gained wide acceptance. Blastocyst grading according to Gardner et al. (20) early blastocyst, blastocoel less than half of the blastocyst (1AA), blastocyst with blastocoel more than half of the blastocyst (2AA), blastocyst with blastocoel fill the blastocyst (3AA), and Expanded Blastocyst (4AA/4BB/4CC). Morphology blastocyst with good quality determining the success of PGD. Preimplantation genetic diagnosis (PGD) are routine procedures performed in many in vitro fertilization (IVF) clinics. In patients with genetic or chromosomal abnormalities, PGD is an integral part of the IVF program. This study aims to determine whether embryos with normal blastocyst morphology have euploidy result on the PGD test. Method: Observational research study. Results: Dari 22 embryo blastocyst yang di PGD, 59% embryo euploidy 4AA/4BB, 22,7% embryo low level mosaicism 4AA/4BB, 13% embryo aneuploidy 2AA/3AA and 4,5% embryo multiple aneuploidy 3AA. The result suggested that the euploidy rate of blastocyst grade 4AA was the highest. Conclusion: Based on data, embryo morphology with quality 4AA/4BB show PGD result Euploidy was the highest. Euploidy of embryo is one of the important factors affecting embryo implantation and development. Euploidy of blastocyst during PGD treatment was found to be correlated with morphological assessment (Gardner grade)

Aberrant landscapes of maternal meiotic crossovers contribute to aneuploidies in human embryos.

Meiotic recombination is crucial for human genetic diversity and chromosome segregation accuracy. Understanding its variation across individuals and the processes by which it goes awry are long-standing goals in human genetics. Current approaches for inferring recombination landscapes rely either on population genetic patterns of linkage disequilibrium (LD)-capturing a time-averaged view-or on direct detection of crossovers in gametes or multigeneration pedigrees, which limits data set scale and availability. Here, we introduce an approach for inferring sex-specific recombination landscapes using data from preimplantation genetic testing for aneuploidy (PGT-A). This method relies on low-coverage (<0.05×) whole-genome sequencing of in vitro fertilized (IVF) embryo biopsies. To overcome the data sparsity, our method exploits its inherent relatedness structure, knowledge of haplotypes from external population reference panels, and the frequent occurrence of monosomies in embryos, whereby the remaining chromosome is phased by default. Extensive simulations show our method's high accuracy, even at coverages as low as 0.02×. Applying this method to PGT-A data from 18,967 embryos, we mapped 70,660 recombination events with ∼150 kbp resolution, replicating established sex-specific recombination patterns. We observed a reduced total length of the female genetic map in trisomies compared with disomies, as well as chromosome-specific alterations in crossover distributions. Based on haplotype configurations in pericentromeric regions, our data indicate chromosome-specific propensities for different mechanisms of meiotic error. Our results provide a comprehensive view of the role of aberrant meiotic recombination in the origins of human aneuploidies and offer a versatile tool for mapping crossovers in low-coverage sequencing data from multiple siblings.

#27 : A Sibling Study: How Individual Culture Strategy Affects Embryo Ploidy Status in IVF/PGT-A Cycles?

Background and Aims: The result of individual and group culture embryo studies remained conflicting. Several studies assumed that the blastulation rate was lower when culturing embryos separately. Meanwhile, there has been little information on whether individual culture can affect embryo ploidy status. This study was designed to find a relationship between culture strategies and the embryo’s ploidy status in IVF/PGT-A cycles. Method: 91 IVF/PGT-A cycles with at least four zygotes enrolled in a randomized control trial from September 2021 to June 2022 in Tam Anh general hospital. Zygotes from each cycle were randomly assigned to individual culture(10 ul/drop) or group cultured of 3-5 zygotes (50 ul/drop). Among those, 280 blastocysts derived from biopsy-all cycles (137 from individual culture and 143 from group culture) and already determined for ploidy status were to be analyzed. Information of the embryo, including maternal age, paternal age, day of the biopsy, embryo quality (excellent, good, fair, poor), blastocoele expansion (2-6), ICM score (A, B, C), TE score (A, B, C), and PGT-A result (euploid, aneuploidy or else) was collected and analyzed along with the type of culture. Simple and multiple regression analyses using generalized linear mixed model were applied to evaluate the relation between embryo ploidy status as the dependent variable and independent factors. Results: The rates of euploid, aneuploidy and other results of embryos cultured individually were 39.4%, 37.2%, and 23.4%, respectively. These results did not yield a significant difference compared with the cultured group. There was no relationship between culture strategies and the aneuploidy rate of the embryos (OR = 1.16, CI 0.64-2.13). However, biopsy days 6-7 and decreasing grade of TE might be related factors for a higher rate of aneuploidy. Conclusion: Culture embryos individually or in groups hadnot yet shown any effect on embryo’s ploidy status.

PGT-A: What Is Its Value in Treating Recurrent Pregnancy Loss?

PGT has been widely applied in the world, but its effectiveness for the treatments of recurrent pregnancy loss is still inconclusive. There is currently no consensus over the precise definition of RPL, according to the ESHRE guideline in 2022, a diagnosis of Recurrent Pregnancy Loss (RPL) could be considered after the loss of two or more pregnancies. The etiology of RPL is complex and highly heterogeneous, the higher proportion of embryo aneuploidy is an important reason, which can be affected significantly by maternal age. RPL has been listed as one of the common indications for PGT-A worldwide. However, the new RPL guideline by EHSRE in 2022, indicated that limited evidence for PGT in couples with RPL shows no clear benefit of treatment with very low quality. We reviewed the published studies, three retrospective studies showed that PGT-A may improve live birth rate for RPL patients, while two studies showed negative results. Most importantly, there were no randomized studies published on the application of PGT-A among RPL couples. Our data also showed similar cumulative live birth rates between PGT-A group and the control, regardless of maternal age, but PGT-A may favour in reducing miscarriage recurrence risk for RPL couples over 37 years old who obtained transferrable embryos. The presence of mosaic embryos and false positive/negative results, which can cause a high rate of cycle cancellation, may be the main reasons that compromise the efficacy of PGT-A. The cost-effectiveness of PGT-A is another important concern. In conclusion, there is still no sufficient evidence to support whether PGT-A can effectively improve the pregnancy outcomes for RPL couples, and proper-designed RCT is required in the future.