Precise preimplantation genetic testing for a Chinese pedigree carrying a small segmental copy number variation

Study question How effective is the PGT-A upgrade in detecting smaller copy number variations (CNVs), uniparental disomies(UPD), and other genetic abnormalities in embryos, compared to traditional PGT-A? Summary answer The PGT-A upgrade improves detection of both large and small CNVs, enhancing embryonic genetic health understanding and potentially improving assisted reproductive technology outcomes. What is known already Preimplantation Genetic Testing for Aneuploidy (PGT-A) is a crucial tool in assisted reproductive technologies, aiming to improve implantation rates and reduce the risk of miscarriage by selecting euploid embryos for transfer. However, traditional PGT-A has limitations in detecting smaller copy number variations (CNVs) and uniparental disomies (UPD). Study design, size, duration This retrospective study evaluated 4300 embryos that underwent the PGT-A upgrade. The study used a haplotype-aware inference approach with a dataset threshold of 3M NGS reads, assessing both larger and smaller CNVs and other genetic abnormalities. Participants/materials, setting, methods A total of 4300 embryos underwent the PGT-A upgrade protocol, which included analysis for euploidy, UPD, triploidy, and CNVs in the range of 1-4Mb. The study used next-generation sequencing (NGS) with a dataset threshold of 3M reads and fluorescence in situ hybridization (FISH) to validate findings related to small CNVs. Main results and the role of chance The euploidy rate among the 4300 embryos was 49.0%. The detection rates for UPD and triploidy were 0.11% (5/4300) and 0.22% (10/4300), respectively. Among the euploid embryos, 1.09% (23/2107) were found to carry small CNVs of 1-4Mb. To validate these findings, fluorescence in situ hybridization (FISH) was used to confirm the presence of these small CNVs in 22 cases, demonstrating a high concordance with the PGT-A upgrade results. Limitations, reasons for caution This retrospective study may limit definitive conclusions about causality. While FISH validation showed high concordance, the clinical relevance of small CNVs remains uncertain. Further prospective studies and broader population validation are needed to fully understand the implications and potential impact on improving assisted reproductive technology outcomes. Wider implications of the findings The PGT-A upgrade enhances detection by identifying larger CNVs and smaller genetic anomalies affecting embryonic development, highlighting the value of comprehensive screening to improve assisted reproductive technology success rates. Trial registration number No

Noninvasive preimplantation genetic testing for aneuploidy in spent culture medium as a substitute for trophectoderm biopsy

Natural fecundity of women decreases gradually and more rapidly after age 37 years. This decrease is accompanied by rising aneuploidy rates of pregnancies and can also be observed in products of conception of spontaneous abortions [1]. These observations lead to the hypothesis that transferring only euploid embryos in association with in vitro fertilization (IVF) might decrease miscarriages and increase live birth rates (LBRs), attesting-procedure now called preimplantation genetic testing (of embryos) for aneuploidy (PGT-A), until recently generally referred to as preimplantation genetic screening (PGS). Verlinsky and Kuliev further proposed that the removal of all aneuploid embryos prior to transfer would improve implantation rates and live birth rates and suggested that the diagnosis be made via biopsy of both polar bodies [2]. Polar body biopsy, however, proved technically too difficult for general IVF practice and would have revealed only meiotic aneuploidies. The procedure was, therefore, initially performed biopsying 1–2 blastomeres of day-3 cleavage-stage embryos, often given the acronym PGS 1.0. This form of embryo testing has, since, been replaced by PGS 2.0, with the embryo biopsy being moved from day-3 cleavage stage to trophectoderm biopsy of blastocyst-stage embryos on days 5–6 after fertilization. In July 2016, another major change in PGT-A was announced, for the first time introducing the concept pf “mosaic” embryos (also called PGS 3.) (Preimplantation Genetic Diagnosis Society (PGDIS) position statement on chromosome mosaicism and preimplantation aneuploidy testing at the blastocyst stage, Chicago, IL; July 19, 2016 http://pgdis.org/docs/newsletter_071816.html). After almost two decades of PGS 1.0 through PGS 3.0, the procedure has, however, still been unable to demonstrate the promised improvements in live births and anticipated declines in miscarriage rates [3–5]. Several studies, even summarized in a meta-analysis [6], have claimed improved clinical IVF outcomes following PGT-A. They, however, reported IVF outcomes with reference point embryo transfer rather than cycle start (intent-to-treat) and, therefore, by excluding poorer prognosis patients, were severely biased [7]. The STAR study This is why the recently published STAR study [8] attracted special attention: It avoided at least some patient selection biases of earlier fresh-cycle studies by being prospectively randomized and reporting on IVF outcomes from transfers of only single frozen-thawed embryos at blastocyst stage. That qualifying patients required having at least two frozen embryos from a prior fresh cycle, however, still demonstrates a favorable patient selection bias. Importantly, however, the study at least analyzed outcomes for study and control groups with reference point initial first cycle start [7]. In doing so, the study convincingly revealed no improvements in live birth rates and no reduction in miscarriage rates when cycle outcomes were compared in singe-embryo transfers at blastocyst stage between women, randomized to either PGT-A or only morphological assessments of a single embryo prior to transfers [8]. For no declared reason, the authors then, however, performed a post hoc sub-group analysis based on age and reported, between ages 35 and 40 years, that PGT-A, still, offered significant increases in ongoing pregnancy rate (OPR). In the discussion of their manuscript, they emphasized this finding as “continuous evidence” for the clinical utility of PGT-A in at least that age group. Again, in contrast to the overall study that had been performed with reference point cycle start (intent-to-treat), their post hoc analysis was performed with reference embryo transfer and, therefore, statistically suspect. Because results of the STAR study are already impacting IVF practice worldwide, we here offer a statistically corrected analysis of the STAR study, reaffirming the study’s overall findings by refuting the results of the post hoc analysis and its interpretation by the authors, claimed benefits for PGT-A utilization for all age groups.

Aneuploidy rates and likelihood of obtaining a usable embryo for transfer among in vitro fertilization cycles using preimplantation genetic testing for monogenic disorders and aneuploidy compared with in vitro fertilization cycles using preimplantation genetic testing for aneuploidy alone

Prognostic data in fertility preservation: the role of preimplantation genetic testing for aneuploidy (PGT-A) among cancer patients undergoing embryo banking (EB)

Do donor oocyte recipients benefit from preimplantation genetic testing for aneuploidy (PGT-A)? PGT-A did not improve the likelihood of live birth for recipients of vitrified donor oocytes, but it did avoid embryo transfer in cycles with no euploid embryos. Relative to slow freeze, oocyte vitrification has led to increased live birth from cryopreserved oocytes and has led to widespread use of this technology in donor egg IVF programs. However, oocyte cryopreservation has the potential to disrupt the meiotic spindle leading to abnormal segregation of chromosome during meiosis II and ultimately increased aneuploidy in resultant embryos. Therefore, PGT-A might have benefits in vitrified donor egg cycles. In contrast, embryos derived from young donor oocytes are expected to be predominantly euploid, and trophectoderm biopsy may have a negative effect relative to transfer without biopsy. This is a paired cohort study analyzing donor oocyte-recipient cycles with or without PGT-A performed from 2012 to 2018 at 47 US IVF centers. Vitrified donor oocyte cycles were analyzed for live birth as the main outcome measure. Outcomes from donors whose oocytes were used by at least two separate recipient couples, one couple using PGT-A (study group) and one using embryos without PGT-A (control group), were compared. Generalized estimating equation models controlled for confounders and nested for individual donors contributing to both PGT-A and non-PGT-A cohorts, enabling a single donor to serve as her own control. In total, 1291 initiated recipient cycles from 223 donors were analyzed, including 262 cycles with and 1029 without PGT-A. The median aneuploidy rate per recipient was 25%. Forty-three percent of PGT-A cycles had only euploid embryos, whereas only 12.7% of cycles had no euploid embryos. On average 1.09 embryos were transferred in the PGT-A group compared to 1.38 in the group without PGT-A (P < 0.01). Live birth occurred in 53.8% of cycles with PGT-A versus 55.8% without PGT-A (P = 0.44). Similar findings persisted in cumulative live birth from per recipient cycle. Pooled clinical data from 47 IVF clinics introduced PGT-A heterogeneity as genetic testing were performed using different embryology laboratories, PGT-A companies and testing platforms. PGT-A testing in donor oocyte-recipient cycles does not improve the chance for live birth nor decrease the risk for miscarriage in the first transfer cycle but does increase cost and time for the patient. Further studies are required to test if our findings can be applied to the young infertility patient population using autologous oocytes. No external funding was used for this study. There are no conflicts of interest to declare. N/A.

The preimplantation genetic testing for aneuploidy (PGT-A) platform is not currently available for small copy-number variants (CNVs), especially those < 1 Mb. Through strategies used in PGT for monogenic disease (PGT-M), this study intended to perform PGT for families with small pathogenic CNVs. Couples who carried small pathogenic CNVs and underwent PGT at the Reproductive and Genetic Hospital of CITIC-Xiangya (Hunan, China) between November 2019 and April 2023 were included in this study. Haplotype analysis was performed through two platforms (targeted sequencing and whole-genome arrays) to identify the unaffected embryos, which were subjected to transplantation. Prenatal diagnosis using amniotic fluid was performed during 18-20 weeks of pregnancy. PGT was successfully performed for 20 small CNVs (15 microdeletions and 5 microduplications) in 20 families. These CNVs distributed on chromosomes 1, 2, 6, 7, 13, 15, 16, and X with sizes ranging from 57 to 2120 kb. Three haplotyping-based PGT-M strategies were applied. A total of 89 embryos were identified in 25 PGT cycles for the 20 families. The diagnostic yield was 98.9% (88/89). Nineteen transfers were performed for 17 women, resulting in a 78.9% (15/19) clinical pregnancy rate after each transplantation. Of the nine women who had healthy babies, eight accepted prenatal diagnosis and the results showed no related pathogenic CNVs. Our results show that the extended haplotyping-based PGT-M strategy application for small pathogenic CNVs compensated for the insufficient resolution of PGT-A. These three PGT-M strategies could be applied to couples with small pathogenic CNVs.

Reflections on preimplantation genetic testing for aneuploidy and mosaicism: how did we get here, and what does it mean clinically?

Study question Does blastocyst cohort size impact aneuploidy rates, evaluated by next generation sequencing (NGS)? Summary answer Embryo aneuploidy rates were independent of blastocyst cohort size across all patient ages. What is known already The effects of ovarian response on oocyte and embryo quality remain controversial. Several studies have proposed that a high response to ovarian stimulation may negatively impact oocyte competence. Alternatively, irrespective of maternal age, a poor ovarian response may potentially compromise embryo quality. Using blastocyst cohort size as an indirect measure of ovarian response, previous studies applying array comparative genomic hybridisation (aCGH) have demonstrated that the number of embryos available for biopsy does not impact embryo aneuploidy rates. Nevertheless, these findings remain to be confirmed in a comprehensive cohort, using current approaches for preimplantation genetic testing for aneuploidies (PGT-A). Study design, size, duration Retrospective, international, cohort study of 3998 patients from 16 clinics undergoing PGT-A from 2016–2020. We evaluated 11665 blastocysts, tested using trophectoderm (TE) biopsy and next generation sequencing (NGS). To eliminate bias of multiple treatments, we considered only the first PGT-A cycle for all patients. Both autologous and donation cycles were included in the analysis. Cycles were excluded if they utilised preimplantation genetic testing for monogenic disorders (PGT-M) or preimplantation genetic testing for structural rearrangements (PGT-SR). Participants/materials, setting, methods We evaluated aneuploidy and mosaicism rates, as well as the proportion of patients who had at least one euploid embryo suitable for transfer. Findings were stratified according to SART-defined maternal age groups, &amp;lt;35 (n = 698/2622 patients/blastocysts), 35–37 (n = 988/3141 patients/blastoycsts), 38–40 (n = 1447/3939 patients/blastocysts), 41–42 (653/1562 patients/blastocysts) and &amp;gt;42 (212/401 patients/blastocysts) and blastoycst cohort size (1–2, 3–5, 6–9 and 10 or more biopsied blastocysts). Main results and the role of chance The mean maternal age was 37.0±3.7. The overall embryo aneuploidy rate was 50.6% (5904/11665), while mosaicism was established in 4.0% (469/11665) of blastocysts. As expected, the proportion of aneuploid embryos increased steadily with advancing maternal age (31.8%, 41.5%, 58.4%, 71.2%, 87.8%; p &amp;lt; 0.0001), while mosaicism rates did not vary significantly (p = 0.2). Within each age group, we observed no association between the number of blastocysts biopsied and aneuploidy or mosaicism rates. However, as previously suggested, the chance of having at least one euploid embryo increased linearly with the number of embryos biopsied. We observed that young patients (&amp;lt;35) with 1–2 blastocysts had a 70.4% of having at least one embryo suitable for transfer, which increased to 96.4% and 99.2% with 3–5 and 6–9 blastocysts, respectively. Similar trends were observed in the 36–38 and 39–40 age groups. Patients in the 40–41 age group had a significantly lower chance of having a suitable embryo for transfer. Nevertheless, the chance increased from 27.2% with 1–2 embryos to 61.2% with 3–5 blastocysts. Patients with &amp;gt;10 embryos had at least one euploid embryo in 100% of cases, across all ages. Albeit, the numbers of patients within this category was low, and decreased significantly with advancing maternal age. Limitations, reasons for caution While blastocyst cohort size is considered to be an indirect measure of ovarian reserve, the number of oocytes retrieved was not evaluated. Our study only included the first PGT-A cycle for all patients. Subsequent, alterations in stimulation protocols may have resulted in an improved response in some patients. Wider implications of the findings: The comprehensive nature of the study, based on current PGT-A approaches and a large number of cycles across 16 centres increases clinical confidence in the notion that ovarian response is independent of embryo aneuploidy. Importantly, our findings may serve as a valuable clinical resource to guide patient counselling strategies. Trial registration number NA

What factors are associated with decision regret and anxiety following preimplantation genetic testing for aneuploidy (PGT-A)? The majority of patients viewed PGT-A favourably regardless of their outcome; although patients with negative outcomes expressed greater decision regret and anxiety. PGT-A is increasingly utilized in in vitro fertilization (IVF) cycles to aid in embryo selection. Despite the increasing use of PGT-A technology, little is known about patients' experiences and the possible unintended consequences of decision regret and anxiety related to PGT-A outcome. Anonymous surveys were distributed to 395 patients who underwent their first cycle of autologous PGT-A between January 2014 and March 2015. There were 69 respondents who underwent PGT-A at a university-affiliated fertility centre, completed the survey and met inclusion criteria. Respondents completed three validated questionnaires including the Brehaut Decision Regret (DR) Scale, short-form State-Trait Anxiety Inventory (STAI-6) and a health literacy scale. The surveys also assessed demographics, fertility history, IVF and frozen embryo transfer cycle data. The majority of respondents were Caucasian, >35years of age and educated beyond an undergraduate degree. The majority utilized PGT-A on their first IVF cycle, most commonly to 'maximize the efficiency of IVF' or reduce per-transfer miscarriage risk. The overall median DR score was low, but 39% of respondents expressed some degree of regret. Multiple regression confirmed a relationship between embryo ploidy and decision regret, with a lower number of euploid embryos associated with a greater degree of regret. Patients who conceived following euploid transfer reported less regret than those who miscarried or failed to conceive (P < 0.005). Decision regret was inversely associated with number of living children but not associated with age, education, race, insurance coverage, religion, marital status or indication for IVF/PGT-A. Anxiety was greater following a negative pregnancy test or miscarriage compared to successful conception (P < 0.0001). Anxiety was negatively associated with age, time since oocyte retrieval and number of living children, and a relationship was observed between anxiety and religious affiliation. Overall, decision regret was low, and 94% of all respondents reported satisfaction with their decision to pursue PGT-A; however, patients with a negative outcome were more likely to express decision regret and anxiety. This survey was performed at a single centre with a relatively homogenous population, and the findings may not be generalizable. Reasons for caution include the possibility of response bias and unmeasured differences among those who did and did not respond to the survey, as well as the possibility of recall bias given the retrospective nature of the survey. Few studies have examined patient perceptions of PGT-A, and our findings should be interpreted with caution. Overall decision regret was low following PGT-A, and the vast majority deemed the information gained valuable for reproductive planning regardless of outcome. However, more than one-third of the respondents expressed some degree of regret. Respondents with no euploid embryos were more likely to express regret, and those with a negative outcome following euploid embryo transfer expressed both higher regret and anxiety. These data identify unanticipated consequences of PGT-A and suggest opportunities for additional counselling and support surrounding IVF with PGT-A. No external funding was obtained for this study. D.H.M. reports personal fees, honorarium, and travel expenses from Ferring Pharmaceuticals, personal fees and travel expenses from Granata Bio, and personal fees from Biogenetics Corporation, The Sperm and Embryo Bank of New York, and ReproART: Georgian American Center for Reproductive Medicine. All conflicts are outside the submitted work.

Antimüllerian hormone is not associated with embryo ploidy in patients with and without infertility undergoing in vitro fertilization with preimplantation genetic testing

Two’s company, three’s a crowd: involvement of a gestational carrier necessitates use of best and safe practices

A review of factors influencing the implantation of euploid blastocysts after in vitro fertilization

Study question How many eggs will be required to optimize the chances of a live birth with or without PGT-A? Summary answer The number of zygotes required for live birth is higher in women with an advanced age, and the use of PGT-A does not provide improvement. What is known already Women who are undergoing PGT-A often wish to know how many eggs will be required to optimize the chances of a live birth. This important information could be provided as part of prior genetic counseling, but there are no precise data on this at present. If the number of eggs required to give the best chance of a successful live birth was known, treatment plans with or without PGT-A could be better determined. Study design, size, duration We estimated the optimal number of eggs required for IVF treatment with PGT-A to produce at least a single live birth, stratified by maternal age, on the basis of information from prior studies and in current databases. Participants/materials, setting, methods We derived our calculation parameters from three prior large-scale clinical investigations associated with PGT-A. We estimated a live birth rate using the following factors: rate of zygotes that develop a useful blastocyst, euploid rate in PGT-A, and the live birth rate after euploid embryo transfer. All of these factors were assumed to be statistically independent in this study for the purposes of our calculations and the live birth rate per single zygote was calculated. Main results and the role of chance The estimations in our present analyses however indicate a probability of less than 10% that woman over 40 years of age will have a live birth from a single zygote, regardless of whether PGT-A is performed or not. We used a negative binomial distribution approach to calculate how many zygotes are needed to obtain at least one live birth. The plot of these results is provided in Figure 2. To achieve a 50% chance of getting at least one live birth, patients required 8 zygotes at age of 40 and 21 zygotes at the age of 43. Furthermore, to achieve an 80% chance of obtaining a live birth, our calculations estimate that 18 and 47 zygotes would be required at these two ages, respectively, which would be challenging to achieve. On the other hand, by avoiding unnecessary transplants using PGT-A, women may have to wait a shorter period to accomplish a live birth or may be able to avoid wasting their limited remaining reproductive period, particularly if they are older than 42. Limitations, reasons for caution The reference data from PGT-A studies that have estimated of the live birth rate include chromosomal quantitative PCR, microarray analysis, and next generation sequencing (NGS). There is a high possibility that the embryos designated as “euploid” in those studies include mosaic embryos, which represents a limitation of our present meta-analysis. Wider implications of the findings More details on the clinical outcomes of PGT-A will be revealed as clinical studies progress in the future. It is our hope that the results of this present study will assist with future genetic counseling strategies for PGT-A in the meantime. Trial registration number not applicable

Importance of early and precise ascertainment of mosaic Turner syndrome for fertility preservation and assisted reproduction counseling