A Broadly Neutralizing Antibody Targets the Dynamic HIV Envelope Trimer Apex via a Long, Rigidified, and Anionic ß-Hairpin Structure.

Broadly neutralizing antibodies (bnAbs) to HIV delineate vaccine targets and are prophylactic and therapeutic agents. Some of the most potent bnAbs target a quaternary epitope at the apex of the surface HIV envelope (Env) trimer. Using cryo-electron microscopy, we solved the atomic structure of an apex bnAb, PGT145, in complex with Env. We showed that the long anionic HCDR3 of PGT145 penetrated between glycans at the trimer 3-fold axis, to contact peptide residues from all three Env protomers, and thus explains its highly trimer-specific nature. Somatic hypermutation in the other CDRs of PGT145 were crucially involved in stabilizing the structure of the HCDR3, similar to bovine antibodies, to aid in recognition of a cluster of conserved basic residues hypothesized to facilitate trimer disassembly during viral entry. Overall, the findings exemplify the creative solutions that the human immune system can evolve to recognize a conserved motif buried under a canopy of glycans.

Development of pre-implantation genetic testing protocol for monogenic disorders (PGT-M) of Hb H disease.

Hb H disease is the most severe form of α-thalassemia compatible with post-natal life. Compound heterozygous α0-thalassemia- SEA deletion/α+-thalassemia- 3.7kb deletion is the commonest cause of Hb H disease in Thailand. Preimplantation genetics testing for monogenic disorders (PGT-M) is an alternative for couples at risk of the disorder to begin a pregnancy with a healthy baby. This study aims to develop a novel PCR protocol for PGT-M of Hb H disease- SEA/-3.7kb using multiplex fluorescent PCR. A novel set of primers for α+-thalassemia- 3.7kb deletion was developed and tested. The PCR protocol for α0-thalassemia- SEA deletion was combined for Hb H disease- SEA/-3.7kb genotyping. The PCR protocols were applied to genomic DNA extracted from subjects with different thalassemia genotypes and on whole genome amplification (WGA) products from clinical PGT-M cycles of the families at risk of Hb Bart's. The results were compared and discussed. The results showed three PCR products from α+-thalassemia- 3.7kb primer set, and three from α0thalassemiaSEA primer set. The results were consistent with the known thalassemia genotypes. The novel -α3.7 primers protocol was also tested on 37 WGA products from clinical PGT-M cycles giving accurate genotyping results and a satisfying amplification efficiency with the ADO rates of 2.7%, 0%, and 0% for HBA2, HBA1, and internal control fragments, respectively. This novel PCR protocol can precisely distinguish Hb H disease- SEA/-3.7kb from other genotypes. Additionally, this is the first PCR protocol for Hb H disease- SEA/-3.7kb which is optimal for PGT-M.

Implicit bias in diagnosing mosaicism amongst preimplantation genetic testing providers: results from a multicenter study of 36 395 blastocysts.

STUDY QUESTION: Does the diagnosis of mosaicism affect ploidy rates across different providers offering preimplantation genetic testing for aneuploidies (PGT-A)? SUMMARY ANSWER: Our analysis of 36 395 blastocyst biopsies across eight genetic testing laboratories revealed that euploidy rates were significantly higher in providers reporting low rates of mosaicism. WHAT IS KNOWN ALREADY: Diagnoses consistent with chromosomal mosaicism have emerged as a third category of possible embryo ploidy outcomes following PGT-A. However, in the era of mosaicism, embryo selection has become increasingly complex. Biological, technical, analytical, and clinical complexities in interpreting such results have led to substantial variability in mosaicism rates across PGT-A providers and clinics. Critically, it remains unknown whether these differences impact the number of euploid embryos available for transfer. Ultimately, this may significantly affect clinical outcomes, with important implications for PGT-A patients. STUDY DESIGN, SIZE, DURATION: In this international, multicenter cohort study, we reviewed 36 395 consecutive PGT-A results, obtained from 10 035 patients across 11 867 treatment cycles, conducted between October 2015 and October 2021. A total of 17 IVF centers, across eight PGT-A providers, five countries and three continents participated in the study. All blastocysts were tested using trophectoderm biopsy and next-generation sequencing. Both autologous and donation cycles were assessed. Cycles using preimplantation genetic testing for structural rearrangements were excluded from the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The PGT-A providers were randomly categorized (A to H). Providers B, C, D, E, F, G, and H all reported mosaicism, whereas Provider A reported embryos as either euploid or aneuploid. Ploidy rates were analyzed using multilevel mixed linear regression. Analyses were adjusted for maternal age, paternal age, oocyte source, number of embryos biopsied, day of biopsy, and PGT-A provider, as appropriate. We compared associations between genetic testing providers and PGT-A outcomes, including the number of chromosomally normal (euploid) embryos determined to be suitable for transfer. MAIN RESULTS AND THE ROLE OF CHANCE: The mean maternal age (±SD) across all providers was 36.2 (±5.2). Our findings reveal a strong association between PGT-A provider and the diagnosis of euploidy and mosaicism. Amongst the seven providers that reported mosaicism, the rates varied from 3.1% to 25.0%. After adjusting for confounders, we observed a significant difference in the likelihood of diagnosing mosaicism across providers (P < 0.001), ranging from 6.5% (95% CI: 5.2-7.4%) for Provider B to 35.6% (95% CI: 32.6-38.7%) for Provider E. Notably, adjusted euploidy rates were highest for providers that reported the lowest rates of mosaicism (Provider B: euploidy, 55.7% (95% CI: 54.1-57.4%), mosaicism, 6.5% (95% CI: 5.2-7.4%); Provider H: euploidy, 44.5% (95% CI: 43.6-45.4%), mosaicism, 9.9% (95% CI: 9.2-10.6%)); and Provider D: euploidy, 43.8% (95% CI: 39.2-48.4%), mosaicism, 11.0% (95% CI: 7.5-14.5%)). Moreover, the overall chance of having at least one euploid blastocyst available for transfer was significantly higher when mosaicism was not reported, when we compared Provider A to all other providers (OR = 1.30, 95% CI: 1.13-1.50). Differences in diagnosing and interpreting mosaic results across PGT-A laboratories raise further concerns regarding the accuracy and relevance of mosaicism predictions. While we confirmed equivalent clinical outcomes following the transfer of mosaic and euploid blastocysts, we found that a significant proportion of mosaic embryos are not used for IVF treatment. LIMITATIONS, REASONS FOR CAUTION: Due to the retrospective nature of the study, associations can be ascertained, however, causality cannot be established. Certain parameters such as blastocyst grade were not available in the dataset. Furthermore, certain platform-related and clinic-specific factors may not be readily quantifiable or explicitly captured in our dataset. As such, a full elucidation of all potential confounders accounting for variability may not be possible. WIDER IMPLICATIONS OF THE FINDINGS: Our findings highlight the strong need for standardization and quality assurance in the industry. The decision not to transfer mosaic embryos may ultimately reduce the chance of success of a PGT-A cycle by limiting the pool of available embryos. Until we can be certain that mosaic diagnoses accurately reflect biological variability, reporting mosaicism warrants utmost caution. A prudent approach is imperative, as it may determine the difference between success or failure for some patients. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Torres Quevedo Grant, awarded to M.P. (PTQ2019-010494) by the Spanish State Research Agency, Ministry of Science and Innovation, Spain. M.P., L.B., A.R.L., A.L.R.d.C.L., N.P.P., M.P., D.S., F.A., A.P., B.M., L.D., F.V.M., D.S., M.R., E.P.d.l.B., A.R., and R.V. have no competing interests to declare. B.L., R.M., and J.A.O. are full time employees of IB Biotech, the genetics company of the Instituto Bernabeu group, which performs preimplantation genetic testing. M.G. is a full time employee of Novagen, the genetics company of Cegyr, which performs preimplantation genetic testing. TRIAL REGISTRATION NUMBER: N/A.

Association between the length of in vitro embryo culture, mode of ART, and the initial endogenous hCG rise in ongoing singleton pregnancies.

STUDY QUESTION: Is there an association between the length of in vitro culture, mode of ART and the initial endogenous hCG rise, in cycles with a foetal heartbeat after single embryo transfer (ET) and implantation? SUMMARY ANSWER: Both the length of in vitro culture and the mode of ART have an impact on the initial endogenous rise in hCG in singleton pregnancies. WHAT IS KNOWN ALREADY: Different factors have been identified to alter the kinetics of hCG in pregnancies. Current studies show conflicting results regarding the kinetics of hCG after different types of ART (fresh vs frozen ET (FET)), the inclusion or not of preimplantation genetic testing (PGT), and the length of time in in vitro culture. STUDY DESIGN, SIZE, DURATION: This was a multicentre cohort study, using prospectively collected data derived from 4938 women (5524 treatment cycles) undergoing IUI (cycles, n = 608) or ART (cycles, n = 4916) treatments, resulting a in singleton ongoing pregnancy verified by first-trimester ultrasound scan. Data were collected from the Danish Medical Data Centre, used by the three participating Danish public fertility clinics at Copenhagen University hospitals: Herlev Hospital, Hvidovre Hospital, and Rigshospitalet, from January 2014 to December 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: The fresh ET cycles included cleavage-stage (2 or 3 days in vitro) and blastocyst (5 days in vitro) transfers. FET cycles included cleavage-stage (3 days in vitro before cryopreservation) or blastocyst (5 or 6 days in vitro before cryopreservation) transfers. The IUI cycles represented no time in vitro. To attain a comparable interval for serum-hCG (s-hCG), the ovulation induction time was identical: 35-37 h before oocyte retrieval or IUI. The conception day was considered as: the insemination day for pregnancies conceived after IUI, the oocyte retrieval day for fresh ET, or the transfer day minus 3 or 5 as appropriate for FET of Day 3 or 5 embryos. Multiple linear regression analysis was used, including days post-conception for the hCG measurement as a covariate, and was adjusted for the women's age, the cause of infertility, and the centre. For FET, a sensitivity analysis was used to adjust for endometrial preparation. MAIN RESULTS AND THE ROLE OF CHANCE: The study totally includes 5524 cycles: 2395 FET cycles, 2521 fresh ET cycles, and 608 IUI cycles. Regarding the length of in vitro culture, with IUI as reference (for no time in in vitro culture), we found a significantly lower s-hCG in pregnancies achieved after fresh ET (cleavage-stage ET or blastocyst transfer). S-hCG was 18% (95% CI: 13-23%, P < 0.001) lower after fresh cleavage-stage ET, and 23% (95% CI: 18-28%, P < 0.001) lower after fresh blastocyst transfer compared to IUI. In FET cycles, s-hCG was significantly higher after blastocyst transfers compared to cleavage-stage FET, respectively, 26% (95% CI: 13-40%, P < 0.001) higher when cryopreserved on in vitro Day 5, and 14% (95% CI: 2-26%, P = 0.02) higher when cryopreserved on in vitro Day 6 as compared to Day 3. Regarding the ART treatment type, s-hCG after FET blastocyst transfer (Day 5 blastocysts) cycles was significantly higher, 33% (95% CI: 27-45%, P < 0.001), compared to fresh ET (Day 5 blastocyst), while there was no difference between cleavage-stage FET (Days 2 + 3) and fresh ET (Days 2 + 3). S-hCG was 12% (95% CI: 4-19%, 0.005) lower in PGT FET (Day 5 blastocysts) cycles as compared to FET cycles without PGT (Day 5 blastocysts). LIMITATIONS, REASONS FOR CAUTION: The retrospective design is a limitation which introduces the risk of possible bias and confounders such as embryo score, parity, and ovarian stimulation. WIDER IMPLICATIONS OF THE FINDINGS: This study elucidates how practices in medically assisted reproduction treatment are associated with the hCG kinetics, underlining a potential impact of in vitro culture length and mode of ART on the very early embryo development and implantation. The study provides clinicians knowledge that the type of ART used may be relevant to take into account when evaluating s-hCG for the prognosis of the pregnancy. STUDY FUNDING/COMPETING INTEREST(S): No funding was received for this study. AP has received consulting fees, research grants, or honoraria from the following companies: Preglem, Novo Nordisk, Ferring Pharmaceuticals, Gedeon Richter, Cryos, Merck A/S, and Organon. AZ has received grants and honoraria from Gedeon Richter. NLF has received grants from Gedeon Richter, Merck A/S, and Cryos. MLG has received honoraria fees or research grants from Gedeon Richter, Merck A/S, and Cooper Surgical. CB has received honoraria from Merck A/S. MB has received research grants and honoraria from IBSA. MPR, KM, and PVS all report no conflicts of interest. TRIAL REGISTRATION NUMBER: The study was registered and approved by the Danish Protection Agency, Capital Region, Denmark (Journal-nr.: 21019857). No approval was required from the regional ethics committee according to Danish law.

The impact of implementing a non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) embryo culture protocol on embryo viability and clinical outcomes.

STUDY QUESTION: Are modifications in the embryo culture protocol needed to perform non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) affecting clinical reproductive outcomes, including blastocyst development and pregnancy outcomes? SUMMARY ANSWER: The implementation of an embryo culture protocol to accommodate niPGT-A has no impact on blastocyst viability or pregnancy outcomes. WHAT IS KNOWN ALREADY: The recent identification of embryo cell-free (cf) DNA in spent blastocyst media has created the possibility of simplifying PGT-A. Concerns, however, have arisen at two levels. First, the representativeness of that cfDNA to the real ploidy status of the embryo. Second, the logistical changes that need to be implemented by the IVF laboratory when performing niPGT-A and their effect on reproductive outcomes. Concordance rates of niPGT-A to invasive PGT-A have gradually improved; however, the impact of culture protocol changes is not as well understood. STUDY DESIGN, SIZE, DURATION: As part of a trial examining concordance rates of niPGT-A versus invasive PGT-A, the IVF clinics implemented a specific niPGT-A embryo culture protocol. Briefly, this involved initial culture of fertilized oocytes following each laboratory standard routine up to Day 4. On Day 4, embryos were washed and cultured individually in 10 µl of fresh media. On Day 6 or 7, blastocysts were then biopsied, vitrified, and media collected for the niPGT-A analysis. Six IVF clinics from the previously mentioned trial were enrolled in this analysis. In the concordance trial, Clinic A cultured all embryos (97 cycles and 355 embryos) up to Day 6 or 7, whereas in the remaining clinics (B-F) (379 cycles), nearly a quarter of all the blastocysts (231/985: 23.5%) were biopsied on Day 5, with the remaining blastocysts following the niPGT-A protocol (754/985: 76.5%). During the same period (April 2018-December 2020), the IVF clinics also performed standard invasive PGT-A, which involved culture of embryos up to Days 5, 6, or 7 when blastocysts were biopsied and vitrified. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 428 (476 cycles) patients were in the niPGT-A study group. Embryos from 1392 patients underwent the standard PGT-A culture protocol and formed the control group. Clinical information was obtained and analyzed from all the patients. Statistical comparisons were performed between the study and the control groups according to the day of biopsy. MAIN RESULTS AND THE ROLE OF CHANCE: The mean age, number of oocytes, fertilization rates, and number of blastocysts biopsied were not significantly different for the study and the control group. Regarding the overall pregnancy outcomes, no significant effect was observed on clinical pregnancy rate, miscarriage rate, or ongoing pregnancy rate (≥12 weeks) in the study group compared to the control group when stratified by day of biopsy. LIMITATIONS, REASONS FOR CAUTION: The limitations are intrinsic to the retrospective nature of the study, and to the fact that the study was conducted in invasive PGT-A patients and not specifically using niPGT-A cases. WIDER IMPLICATIONS OF THE FINDINGS: This study shows that modifying current IVF laboratory protocols to adopt niPGT-A has no impact on the number of blastocysts available for transfer and overall clinical outcomes of transferred embryos. Whether removal of the invasive biopsy step leads to further improvements in pregnancy rates awaits further studies. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Igenomix. C.R., L.N.-S., and D.V. are employees of Igenomix. D.S. was on the Scientific Advisory Board of Igenomix during the study. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (NCT03520933).

Ready for polygenic risk scores? An analysis of regulation of preimplantation genetic testing in European countries.

STUDY QUESTION: Would the different regulatory approaches for preimplantation genetic testing (PGT) in Europe permit the implementation of preimplantation genetic testing using polygenic risk scores (PGT-P)? SUMMARY ANSWER: While the regulatory approaches for PGT differ between countries, the space provided for potential implementation of PGT-P seems limited in all three regulatory models. WHAT IS KNOWN ALREADY: PGT is a reproductive genetic technology that allows the testing for hereditary genetic disorders and chromosome abnormalities in embryos before implantation. Throughout its history, PGT has largely been regarded as an ethically sensitive technology. For example, ethical questions have been raised regarding the use of PGT for adult-onset conditions, non-medical sex selection, and human leukocyte antigen typing for the benefit of existing siblings. Countries in which PGT is offered each have their own approach of regulating the clinical application of PGT, and a clear overview of legal and practical regulation of PGT in Europe is lacking. An emerging development within the field of PGT, namely PGT-P, is currently bringing new ethical tensions to the forefront. It is unclear whether PGT-P may be applied within the current regulatory frameworks in Europe. Therefore, it is important to investigate current regulatory frameworks in Europe and determine whether PGT-P fits within these frameworks. STUDY DESIGN, SIZE, DURATION: The aim of this study was to provide an overview of the legal and practical regulation of the use of PGT in seven selected European countries (Belgium, France, Germany, Italy, the Netherlands, Spain, and the UK) and critically analyse the different approaches with regards to regulatory possibilities for PGT-P. Between July and September 2023, we performed a thorough and extensive search of websites of governments and governmental agencies, websites of scientific and professional organizations, and academic articles in which laws and regulations are described. PARTICIPANTS/MATERIALS, SETTING, METHODS: We investigated the legal and regulatory aspects of PGT by analysing legal documents, regulatory frameworks, scientific articles, and guidelines from scientific organizations and regulatory bodies to gather relevant information about each included country. The main sources of information were national laws relating to PGT. MAIN RESULTS AND THE ROLE OF CHANCE: We divided the PGT regulation approaches into three models. The regulation of PGT differs per country, with some countries requiring central approval of PGT for each new indication (the medical indication model: the UK, the Netherlands), other countries evaluating each individual PGT request at the local level (the individual requests model: France, Germany), and countries largely leaving decision-making about clinical application of PGT to healthcare professionals (the clinical assessment model: Belgium, Italy, Spain). In the countries surveyed that use the medical indication model and the individual requests model, current legal frameworks and PGT criteria seem to exclude PGT-P. In countries using the clinical assessment model, the fact that healthcare professionals and scientific organizations in Europe are generally negative about implementation of PGT-P due to scientific and socio-ethical concerns, implies that, even if it were legally possible, the chance that PGT-P would be offered in the near future might be low. LIMITATIONS, REASONS FOR CAUTION: The results are based on our interpretation of publicly available written information and documents, therefore not all potential discrepancies between law and practice might have been identified. In addition, our analysis focuses on seven-and not all-European countries. However, since these countries are relevant players within PGT in Europe and since they have distinct PGT regulations, the insights gathered give relevant insights into diverse ways of PGT regulation. WIDER IMPLICATIONS OF THE FINDINGS: To the best of our knowledge, this is the first paper that provides a thorough overview of the legal and practical regulation of PGT in Europe. Our analysis of how PGT-P fits within current regulation models provides guidance for healthcare professionals and policymakers in navigating the possible future implementation of PGT-P within Europe. STUDY FUNDING/COMPETING INTEREST(S): This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no. 813707. The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Prenatal screening after preimplantation genetic testing for aneuploidy: time to evaluate old strategies.

RESEARCH QUESTION: How does first-trimester aneuploidy screening perform in pregnancies achieved through IVF with preimplantation genetic testing for aneuploidy (PGT-A) in a medical setting? DESIGN: This retrospective cohort study was undertaken in a single tertiary care centre between January 2013 and June 2022. In total, 20,237 women had prenatal follow-up at the study centre and were included in the study. The women were divided into three groups: singleton pregnancies conceived through the transfer of a PGT-A-screened euploid embryo (n = 510); singleton pregnancies conceived through IVF without PGT-A (n = 3291); and singleton pregnancies conceived naturally (n = 16,436). RESULTS: The conventional combined screening test for pregnancies conceived through IVF with PGT-A had specificity of 91%; sensitivity could not be calculated as there were no cases of fetal aneuploidy in this group. In 89.1% of pregnancies conceived through IVF with PGT-A with high risk for trisomy 21, 18 or 13, the result was related to advanced maternal age (>35 years at time of screening). CONCLUSIONS: The current screening strategy for trisomies 21, 18 and 13 can generate unnecessary tests in pregnancies achieved through IVF with PGT-A. A new protocol is needed for these patients, with greater weight given to ultrasound markers.

Ongoing pregnancy rates in single euploid frozen embryo transfers remain unaffected by female age: a retrospective study.

RESEARCH QUESTION: Is female age a significant factor in the likelihood of an ongoing pregnancy in single euploid frozen embryo transfers (FET)? DESIGN: Retrospective study of 1923 single euploid FET cycles in 1464 women, either in a natural cycle or a hormone replacement therapy cycle. The primary outcome was the ongoing pregnancy rate (OPR). RESULTS: There were 990 (51.48%) ongoing pregnancies among 1923 included transfers. The OPR were 51.4%, 49.1%, 53.3% and 52.3% for women aged ≤35, >35-≤37, >37-≤40 and >40 years at oocyte retrieval (OCR), without a significant trend for decreasing OPR (P = 0.679). No significant differences in female age at embryo transfer (P = 0.609) and female age at OCR (P = 0.816) were found between the groups (ongoing pregnancy versus no pregnancy or miscarriage). Women who received good-quality embryos (P < 0.001), had a lower body mass index (BMI) (P < 0.001), had achieved at least one pregnancy previously (P < 0.001), and underwent natural cycle endometrial preparation (P < 0.001) were more likely to achieve an ongoing pregnancy. Multivariable regression analysis (adjusted for BMI, embryo quality and endometrial preparation) did not show a significant effect of female age at OCR on achieving an ongoing pregnancy. Compared with women aged ≤35 years, none of the age groups had significantly higher or lower OPR. A multinomial regression analysis showed that BMI, embryo quality and endometrial preparation were associated with miscarriage/no pregnancy versus ongoing pregnancy (P = 0.001, 0.001 and 0.001, respectively). Female age had no significant association with either outcome. CONCLUSIONS: Female age in itself does not have a substantial impact on the OPR in single euploid FET cycles, but the OPR is impacted significantly by embryo quality, BMI, previous parity, and a natural cycle endometrial preparation protocol.

Multiple embryo manipulations in PGT-A cycles may result in inferior clinical outcomes.

RESEARCH QUESTION: Do embryos that undergo a thaw, biopsy and re-vitrification (TBR) for pre-implantation genetic testing for aneuploidy (PGT-A) have different ploidy and transfer outcomes compared with fresh biopsied embryos? DESIGN: Retrospective cohort study of all embryos that underwent the following procedures: fresh biopsy for PGT-A (fresh biopsy); embryos that were warmed, biopsied for PGT-A and re-vitrified (single biopsy TBR); embryos with a no signal result after initial biopsy that were subsequently warmed, biopsied and re-vitrified (double biopsy TBR). The patients who underwent transfers of those embryos at a single academic institution between March 2013 and December 2021 were also studied. RESULTS: About 30% of embryos planned for TBR underwent attrition. Euploidy rates were similar after biopsy: fresh biopsy (42.7%); single biopsy TBR (47.5%) (adjusted RR: 0.99, 0.88 to 1.12); and double biopsy TBR 50.3% (adjusted RR: 0.99, 0.80 to 1.21). Ongoing pregnancy over 8 weeks was not statistically significant (double biopsy TBR: 6/19 [31.6%] versus fresh biopsy: 650/1062 [61.2%]) (adjusted RR 0.52, 95% CI 0.26 to 1.03). The miscarriage rate increased (double biopsy TBR: 4/19 [21.1%] versus fresh biopsy: 66/1062 [6.2%])(RR 3.39, 95% CI 1.38 to 8.31). Live birth rate was also lower per transfer for the double biopsy TBR group (double biopsy TBR [18.75%] versus fresh biopsy [53.75%]) (RR 0.35, 95% CI 0.12 to 0.98), though not after adjustment (adjusted RR 0.37, 95% CI 0.13 to 1.09). These differences were not seen when single biopsy TBR embryos were transferred. CONCLUSIONS: Embryos that undergo TBR have an equivalent euploidy rate to fresh biopsied embryos. Despite that, double biopsy TBR embryos may have impaired transfer outcomes.

Double vitrification and warming of blastocysts does not affect pregnancy, miscarriage or live birth rates.

RESEARCH QUESTION: Does double blastocyst vitrification and warming affect pregnancy, miscarriage or live birth rates, or birth outcomes, from embryos that have undergone preimplantation genetic testing for aneuploidies (PGT-A) testing? DESIGN: This retrospective observational analysis of embryo transfers was performed at a single centre between January 2017 and August 2022. The double-vitrification group included frozen blastocysts that were vitrified after 5-7 days of culture, warmed, biopsied (either once or twice) and re-vitrified. The single vitrification (SV) group included fresh blastocysts that were biopsied at 5-7 days and then vitrified. RESULTS: A comparison of the 84 double-vitrification blastocysts and 729 control single-vitrification blastocysts indicated that the double-vitrification embryos were frozen later in development and had expanded more than the single-vitrification embryos. Of the 813 embryo transfer procedures reported, 452 resulted in the successful delivery of healthy infants (56%). There were no significant differences between double-vitrification and single-vitrification embryos in the pregnancy, miscarriage or live birth rates achieved after single-embryo transfer (55% versus 56%). Logistic regression indicated that while reduced live birth rates were associated with increasing maternal age at oocyte collection, longer culture prior to freezing and lower embryo quality, double vitrification was not a significant predictor of live birth rate. CONCLUSIONS: Blastocyst double vitrification was not shown to impact pregnancy, miscarriage or live birth rates. Although caution is necessary due to the study size, no effects of double vitrification on miscarriage rates, birthweight or gestation period were noted. These data offer reassurance given the absence of the influence of double vitrification on all outcomes after PGT-A.

Variant analysis and PGT-M of OTC gene in a Chinese family with ornithine carbamoyltransferase deficiency.

BACKGROUND: Ornithine carbamoyltransferase deficiency (OTCD) is a kind of X-linked metabolic disease caused by a deficiency in ornithine transcarbamylase leading to urea cycle disorders. The main reason is that the OTC gene variants lead to the loss or decrease of OTC enzyme function, which hinders the ammonia conversion to urea, resulting in hyperammonemia and severe neurological dysfunction. Here, we studied one Chinese family of three generations who consecutively gave birth to two babies with OTCD. This study aims to explore the pathogenicity of two missense variants in the OTC gene and investigate the application of preimplantation genetic testing for monogenic (PGT-M) for a family troubled by Ornithine carbamoyltransferase deficiency (OTCD). METHODS: The retrospective method was used to classify the pathogenicity of two missense variants in the OTC gene in a family tortured by OTCD. Sanger sequencing was used to validate the variants in the OTC gene, and then the pathogenicity of variants was confirmed through family analysis and bioinformatics software. We used PGT-M to target the OTC gene and select a suitable embryo for transplantation. Prenatal diagnosis was recommended to confirm previous results using Sanger sequencing and karyotyping at an appropriate gestational stage. Tandem mass spectrometry (MS-MS) and gas chromatography-mass spectrometry (GC-MS) were used to detect fetal metabolism after birth. The number of the study cohort is ChiCTR2100053616. RESULTS: Two missense variants, c.959G > C (p.Arg320Pro) and c.634G > A (p.Gly212Arg), were validated in the OTC gene in this family. According to the ACMG genetic variation classification criteria, the missense variant c.959G > C can be considered as "pathogenic", and the missense variant c.634G > A can be regarded as "likely benign." PGT-M identified a female embryo carrying the heterozygous variant c.959G > C (p.Arg320Pro), which was selected for transplantation. Prenatal diagnosis revealed the same variant in the fetus, and continued pregnancy was recommended. A female baby was born, and her blood amino acid testing and urine organic acid testing were regular. Follow-up was conducted after six months and indicated the girl was healthy. CONCLUSION: Our research first validated the segregation of both c.959G > C and c.634G > A variants in the OTC gene in a Chinese OTCD family. Then, we classified variant c.959G > C as "pathogenic" and variant c.634G > A as "likely benign", providing corresponding theoretical support for genetic counseling and fertility guidance in this family. PGT-M and prenatal diagnosis were recommended to help the couple receive a female baby successfully with a six-month follow-up.

Female obesity increases the risk of preterm birth of single frozen-thawed euploid embryos: a retrospective cohort study.

INTRODUCTION: Obesity has been associated with an increased risk of reproductive failure, especially preterm birth. As preimplantation genetic testing for aneuploidies (PGT-A) is increasingly used worldwide, however, it is still unclear whether body mass index (BMI) has an effect on the preterm birth rate in patients undergoing in vitro fertilization (IVF) with PGT-A when transferring a single euploid blastocyst. MATERIALS AND METHODS: This retrospective, single-center cohort study included 851 women who underwent the first cycle of frozen-thawed single euploid blastocyst transfer with PGT-A between 2015 and 2020. The primary outcome was the preterm birth rate. Secondary outcomes were clinical pregnancy, miscarriage, ectopic pregnancy, pregnancy complications, and live birth. RESULTS: Patients were grouped by World Health Organization (WHO) BMI class: underweight (<18.5, n = 81), normal weight (18.5-24.9, n = 637), overweight (25-30, n = 108), and obese (≥30, n = 25). There was no difference in the clinical pregnancy, miscarriage, ectopic pregnancy, pregnancy complication, and live birth by BMI category. In multivariate logistic regression analysis, preterm birth rates were significantly higher in women with overweight (adjusted odds ratio [aOR] 3.18; 95% confidence interval [CI], 1.29-7.80, p = .012) and obese (aOR 1.49; 95% CI, 1.03-12.78, p = .027) compared with the normal weight reference group. CONCLUSION: Women with obesity experience a higher rate of preterm birth after euploid embryo transfer than women with a normal weight, suggesting that the negative impact of obesity on IVF and clinical outcomes may be related to other mechanisms than aneuploidy.

The Blastulation Rate Is Negatively Associated With Euploid Rate.

OBJECTIVES: To study the association between the blastulation rate, the presence of 1 pronucleus (1PN) zygotes, and the ploidy of the cohort of blastocysts. METHODS: A cross-sectional study using the existing databases of 2 university fertility centres in Canada. We included 345 cycles from 235 couples who underwent next-generation sequencing preimplantation genetic testing for the detection of aneuploidy in the study. RESULTS: A total of 1456 blastocysts were biopsied. In multivariate analysis, only female age and the number of 1PN/2PN embryos showed a negative association with euploid ratio. Surprisingly, when the analysis was limited to cycles with no delayed blastulation, the blastulation rate was also negatively associated with the euploid ratio. CONCLUSIONS: This study sheds some light on the stages of early embryo development. Further study on the mechanisms governing embryo development and the different cell cycle checkpoints in embryo development is warranted.

Best quality vs. sex selection - an analysis of embryo selection preferences for patients undergoing preimplantation genetic testing for aneuploidy over a 10-year period.

PURPOSE: Investigate patient preferences in embryo selection for transfer regarding quality versus sex in IVF/ICSI cycles with PGT-A and assess associated clinical implications. METHODS: Retrospective cohort study at a university fertility practice from January 2012 to December 2021. Included were patients undergoing single frozen euploid transfers with at least one embryo of each sex available. Primary outcomes were preference for embryo selection (quality vs. sex) and sex preference (male vs. female). Trends over 10 years were evaluated and clinical outcomes, including clinical pregnancy rate (CPR), sustained implantation rate (SIR), and live birth rate (LBR), were compared. RESULTS: A total of 5,145 embryo transfer cycles were included; 54.5% chose the best-quality embryo, while 45.5% selected based on sex. Among those choosing based on sex, 56.5% chose male embryos and 43.5% chose female. Preference for quality remained consistent over the decade (p = 0.30), while male embryos were consistently favored (p = 0.64). Best-quality embryos had higher grades (p < 0.001). Clinical outcomes were similar between groups (CPR: 74.4% vs. 71.9%, p = 0.05; SIR: 64.9% vs. 63.4%, p = 0.26; LBR: 58.8% vs. 56.7%, p = 0.13), and between male and female embryo selections. CONCLUSIONS: Sex selection remains common, with 45.5% selecting embryos based on sex, predominantly favoring males. This trend persisted over 10 years, with comparable clinical outcomes regardless of selection criteria.

Basal FSH values are positively associated with aneuploidy incidence in pre-advanced maternal age (AMA) but not in AMA patients.

PURPOSE: To assess the impact of maternal age on the association between maternal basal FSH and aneuploidy. METHODS: A retrospective study including data from 1749 blastocysts diagnosed as euploid or aneuploid by PGT-A (preimplantation genetic testing for aneuploidy). Aneuploidy incidence was compared between embryos from mothers with high vs. low basal FSH levels (above and below the group median, respectively) in total, pre-AMA (advanced maternal age; < 35 years, 198 embryos) and AMA (≥ 35 years, 1551 embryos) patient groups, separately. To control for the interference of potentially confounding variables, the association between aneuploidy and high basal FSH levels was assessed by multivariate logistic analysis in overall, pre-AMA and AMA patient groups. RESULTS: Overall, aneuploidy rate was 9% higher (p = 0.02) in embryos from patients with high basal FSH (63.7%) compared to those with low basal FSH (58.4%). In the pre-AMA subgroup, aneuploidy incidence was 35% higher (p = 0.04) in embryos from patients with high basal FSH (53.5%) compared to those with low basal FSH (39.4%). Differently, aneuploidy occurrence did not vary between embryos from AMA patients with low (61.0%) and high (64.8%) basal FSH (p = 0.12). The multivariate analysis revealed that, in pre-AMA embryos, the association between aneuploidy occurrence and high basal FSH is independent of potential confounding variables (p = 0.04). CONCLUSION: Maternal basal FSH values are associated with embryo aneuploidy in pre-AMA but not in AMA patients. The present findings suggest that basal FSH is a useful parameter to assess aneuploidy risk in pre-AMA patients and reinforce the hypothesis that excessive FSH signalling can predispose to oocyte meiotic errors.

Mosaic embryo transfer versus additional IVF with PGT-A Cycle: a decision model comparing live birth rate and cost.

PURPOSE: To evaluate the relative live birth rate and net cost difference between mosaic embryo transfer and an additional cycle of IVF with PGT-A for patients whose only remaining embryos are non-euploid. METHODS: A decision analytic model was designed with model parameters varying based on discrete age cutoffs (<35, 35-37, 38-39, 40-42, 43-44, >44). Model inputs included probabilities of successful IVF, clinical pregnancy, and live birth as well as costs of IVF with PGT-A, embryo transfer, live birth, amniocentesis, and dilation and curettage. All costs were modeled from the healthcare system perspective and adjusted for inflation to 2023 $USD. Model outcomes were sub-stratified by degree and type of mosaicism. RESULTS: For patients younger than 43, an additional cycle of IVF with PGT-A resulted in a higher relative live birth rate (<35, +20%; 35-37, +15%; 38-39, +17%; 40-42, +6%; average, +14.5%) compared to mosaic embryo transfer with an average additional cost of $16,633. For patients older than 42, mosaic embryo transfer resulted in a higher live birth rate (43-44, +5%; >44, +3%; average, +4%) while on average costing $9572 less than an additional cycle of IVF with PGT-A. CONCLUSION: Mosaic embryo transfers are a superior alternative to an additional cycle of IVF with PGT-A for patients older than 42 whose only remaining embryos are non-euploid. Mosaic embryo transfers also should be considered for patients younger than 42 who are unable to pursue additional autologous IVF cycles. Counseling and care should be personalized to individual patients and embryos.

Mechanism of chromosomal mosaicism in preimplantation embryos and its effect on embryo development.

Aneuploidy is one of the main causes of miscarriage and in vitro fertilization failure. Mitotic abnormalities in preimplantation embryos are the main cause of mosaicism, which may be influenced by several endogenous factors such as relaxation of cell cycle control mechanisms, defects in chromosome cohesion, centrosome aberrations and abnormal spindle assembly, and DNA replication stress. In addition, incomplete trisomy rescue is a rare cause of mosaicism. However, there may be a self-correcting mechanism in mosaic embryos, which allows some mosaicisms to potentially develop into normal embryos. At present, it is difficult to accurately diagnose mosaicism using preimplantation genetic testing for aneuploidy. Therefore, in clinical practice, embryos diagnosed as mosaic should be considered comprehensively based on the specific situation of the patient.

Primary sex ratio in euploid embryos of consanguine couples after IVF/ICSI.

PURPOSE: To assess the primary sex ratio (males-to-females at time of conception) in blastocysts from consanguine couples undergoing IVF/ICSI treatments and its correlation with chromosomal constitution. METHOD: A total of 5135 blastocysts were analyzed by preimplantation-genetic testing for aneuploidy (PGT-A) with next-generation sequencing (NGS) from November 2016 to December 2020. From those, a total of 1138 blastocysts were from consanguine couples (CS) and 3997 from non-consanguine couples (NCS). Only blastocysts presenting normal sex chromosome constitution with or without autosomal aneuploidies were included. Primary sex ratio (PSR) of biopsied blastocysts was compared between CS and NCS couples. RESULTS: Expanded blastocysts derived from CS had 47.7% XY versus 52.3% XX constitutions, presenting a PSR of 0.91. In NCS, 48.9% of expanded blastocysts were XY and 51.2% XX, with a less pronounced PSR of 0.95. When stratifying embryos by ploidy, euploid embryos from CS had the lowest PSR (0.87) with 46.6% XY versus 53.4% XX blastocysts (OR 0.89, 95% CI 0.70-1.14; NS), but it did not achieve statistical significance. The lower PSR seemed rather related to euploid embryos from first-degree cousins (PSR = 0.80 versus 0.98 in second-degree cousins, NS). Euploid embryos from NCS presented a PSR of 0.96, with 49.1% XY versus 50.9% XX blastocysts (OR 0.98, 95% CI 0.79-1.22; NS). Significant differences in prevalence of euploidy of specific chromosomes were encountered between CS and NCS. CONCLUSIONS: The primary sex ratio was generally similar in expanded blastocysts from consanguine and non-consanguine couples, with a slight decrease in primary sex ratio of euploid blastocysts from consanguine couples.

A heatmap for expected cumulative live birth rate in preimplantation genetic testing for monogenic disorders and chromosomal structural rearrangements.

PURPOSE: Our objective is to predict the cumulative live birth rate (CLBR) and identify the specific subset within the population undergoing preimplantation genetic testing for monogenic disorders (PGT-M) and chromosomal structural rearrangements (PGT-SR) which is likely to exhibit a diminished expected CLBR based on various patient demographics. METHODS: We performed a single-centre retrospective cohort study including 1522 women undergoing 3130 PGT cycles at a referral centre for PGT. A logistic regression analysis was performed to predict the CLBR per ovarian stimulation in women undergoing PGT-M by polymerase chain reaction (PCR) or single-nucleotide polymorphism (SNP) array, and in women undergoing PGT-SR by SNP array, array comparative genomic hybridization (CGH) or next-generation sequencing (NGS). RESULTS: The mean age of women was 32.6 years, with a mean AMH of 2.75 µg/L. Female age and AMH significantly affected the expected CLBR irrespective of the inheritance mode or PGT technology. An expected CLBR < 10% was reached above the age of 42 years and AMH ≤ 1.25 µg/L. We found no significant difference in outcome per ovarian stimulation between the different PGT technologies, i.e. PCR, SNP array, array CGH and NGS. Whereas per embryo transfer, we noticed a significantly higher probability of live birth when SNP array, array CGH and NGS were used as compared to PCR. CONCLUSION: In a PGT-setting, couples with an unfavourable female age and AMH should be informed of the prognosis to allow other reproductive choices. The heatmap produced in this study can be used as a visual tool for PGT couples.

Preimplantation genetic testing for aneuploidy: predictive embryonic factors.

PURPOSE: In a preimplantation genetic testing for aneuploidy (PGT-A) cycle, does the blastocyst quality before biopsy, or the day of biopsy, or the embryo hatching status have an impact on either euploidy or the rate of embryo survival after freezing? METHODS: This was a retrospective study including 6130 biopsied blastocysts coming from 1849 PGT-A cycles performed in our center (2016-2022). Embryos were categorized according to the inner cell mass and trophectoderm quality, using Gardner's scoring (excellent: AA; good: AB, BA, BB; poor: AC, CA, BC, CB, CC); the day of biopsy (5 or 6); and their hatching status (fully hatched blastocysts [FHB] or non-fully hatched blastocysts [nFHB]). The independent relationship between each group and both euploidy and survival rate was assessed. RESULTS: Excellent-quality embryos were more euploid than both good- and poor-quality embryos (52.69%, 39.69%, and 26.21%; p < 0.001), and day 5-biopsied embryos were more euploid than day 6-biopsied embryos (39.98% and 34.80%; p < 0.001). Survival rates of excellent-quality (92.26%) and good-quality (92.47%) embryos were higher than survival rates in the poor-quality group (84.61%) (p = 0.011 and p = 0.002). Day 5-biopsied embryos survived better than day 6-biopsied embryos (93.71% vs. 83.69%; p < 0.001) and FHB had poorer survival than nFHB (78.61% vs. 93.52%; p < 0.001). CONCLUSIONS: Excellent-quality and day 5-biopsied embryos are more prone to be euploid than good and poor or day 6-biopsied embryos, respectively. Poor-quality, day 6-biopsied embryos, and FHB have significantly lower survival after biopsy and vitrification.