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.

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).

Factors affecting biochemical pregnancy loss (BPL) in preimplantation genetic testing for aneuploidy (PGT-A) cycles: machine learning-assisted identification.

PURPOSE: To determine the factors influencing the likelihood of biochemical pregnancy loss (BPL) after transfer of a euploid embryo from preimplantation genetic testing for aneuploidy (PGT-A) cycles. METHODS: The study employed an observational, retrospective cohort design, encompassing 6020 embryos from 2879 PGT-A cycles conducted between February 2013 and September 2021. Trophectoderm biopsies in day 5 (D5) or day 6 (D6) blastocysts were analyzed by next generation sequencing (NGS). Only single embryo transfers (SET) were considered, totaling 1161 transfers. Of these, 49.9% resulted in positive pregnancy tests, with 18.3% experiencing BPL. To establish a predictive model for BPL, both classical statistical methods and five different supervised classification machine learning algorithms were used. A total of forty-seven factors were incorporated as predictor variables in the machine learning models. RESULTS: Throughout the optimization process for each model, various performance metrics were computed. Random Forest model emerged as the best model, boasting the highest area under the ROC curve (AUC) value of 0.913, alongside an accuracy of 0.830, positive predictive value of 0.857, and negative predictive value of 0.807. For the selected model, SHAP (SHapley Additive exPlanations) values were determined for each of the variables to establish which had the best predictive ability. Notably, variables pertaining to embryo biopsy demonstrated the greatest predictive capacity, followed by factors associated with ovarian stimulation (COS), maternal age, and paternal age. CONCLUSIONS: The Random Forest model had a higher predictive power for identifying BPL occurrences in PGT-A cycles. Specifically, variables associated with the embryo biopsy procedure (biopsy day, number of biopsied embryos, and number of biopsied cells) and ovarian stimulation (number of oocytes retrieved and duration of stimulation), exhibited the strongest predictive power.

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.

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.

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.

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.

Comparison of Clinical Outcomes in the Slow-Developing Blastocysts With or Without Preimplantation Genetic Testing-Aneuploidy on Day 6 in the Frozen-Thawed Cycle.

OBJECTIVE: This study investigated the potential of the slow-developing blastocysts using preimplantation genetic testing-aneuploidy (PGT-A) in patients undergoing frozen-thawed embryo transfer, stratified by age. STUDY DESIGN: A retrospective analysis was performed including a total of 743, the first frozen embryo transfer (FET) cycle with single embryo transfer (SET), who underwent treatment between January 2020 and July 2023 in a single fertility center, XXXX Fertility Center. A total of 743 cycles, in which we performed intracellular sperm injection and freeze all strategy, from 743 patients were included. The patient group was divided into 4 groups as follows: Group 1 (G1), 208 FET on day 5; Group 2 (G2), 177 FET with PGT-A on day 5; Group 3 (G3), 220 FET on day 6; Group 4 (G4), 138 FET with PGT-A on day 6. We also divided into two groups-under 35 years of age and over 35 years of age-and performed the analysis separately for each group. RESULTS: In under 35 years of age groups, there were no significant differences in clinical pregnancy and miscarriage rates in G1 and G2 (67.2% vs 63.8%, NS). Also, G4 had a higher clinical pregnancy rate than G3, but it was not significant (51.8% vs 54.7%, NS). In 35 years or older group, G2 had higher pregnancy rates than G1 and lower miscarriage rates (CPR: 43.3% vs 67.7%, P = 0.001, MR: 22.5% vs 3.4%, P = 0.001). In addition, G4 had a higher pregnancy rate than G3 and lower miscarriage rate (CPR: 31.8% vs. 46.9%, P = 0.003, MR: 22.9% vs 2.2%, P = 0.023). CONCLUSIONS: In ≥35 years group, PGT-A on day 5 and day 6 showed a high pregnancy rate and a low miscarriage rate. Therefore, using PGT-A seems advantageous for patients of an advanced maternal age.

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.

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.

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.

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.

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.

Optimizing non-invasive preimplantation genetic testing: investigating culture conditions, sample collection, and IVF treatment for improved non-invasive PGT-A results.

PURPOSE: This study aimed to optimize the non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) in the laboratory by comparing two collection timing of the spent culture medium (SCM), two embryo rinsing protocols, and the use of conventional insemination instead of intracytoplasmic sperm injection (ICSI). METHODS: Results of two embryo rinsing methods (one-step vs sequential) and SCM collected on day 5 vs day 6 after retrieval were compared against trophectoderm (TE) biopsies as reference. Results from day 6 SCM in cycles fertilized by conventional insemination were compared with PGT-A using ICSI. RESULTS: The rate of concordance was higher in day 6 samples than in day 5 samples when the sequential method was used, in terms of total concordance (TC; day 6 vs day 5: 85.0% vs 60.0%, p = 0.0228), total concordance with same sex (TCS, 82.5% vs 28,0%, p < 0.0001), and full concordance with same sex (FCS, 62.5% vs 24.0%, p = 0.0025). The sequential method significantly out-performed the one-step method when SCM were collected on day 6 (sequential vs one-step, TC: 85.0% vs 64.5%, p = 0.0449; TCS: 82.5% vs 54.8%, p = 0.0113; FCS: 62.5% vs 25.8%, p = 0.0021). There was no significant difference in niPGT-A results between cycles fertilized by the conventional insemination and ICSI. CONCLUSION: We have shown a higher concordance rate when SCM was collected on day 6 and the embryos were rinsed in a sequential manner. Comparable results of niPGT-A when oocytes were fertilized by conventional insemination or ICSI. These optimization steps are important prior to commencement of a randomized trial in niPGT-A.

Evaluating the developmental potential of 2.1PN-derived embryos and associated chromosomal analysis.

PURPOSE: Zygotes with 2.1 pronuclei (2.1PN) present with two normal-sized pronuclei, and an additional smaller pronucleus, that is approximately smaller than two thirds the size of a normal pronucleus. It remains unclear whether the additional pronucleus causes embryonic chromosome abnormalities. In the majority of cases, in vitro fertilization (IVF) clinics discarded 2.1PN zygotes. Thus, the present study aimed to evaluate the developmental potential and value of 2.1PN zygotes. METHODS: 2.1PN-derived embryos from 164 patients who underwent IVF or intracytoplasmic sperm injection (ICSI) treatment between January 2021 and December 2022 were included in the present study. All embryos were monitored using a time-lapse system, and blastocyst formation was used to assess 2.1PN-derived embryo developmental potential. The blastocyst formation was quantified using generalized estimating equations, and chromosome euploidy was analyzed using next-generation sequencing (NGS). In addition, the potential association between age and occurrence of 2.1PN zygotes was determined. RESULTS: The present study demonstrated that numerous 2.1PN zygotes developed into blastocysts. Early cleavage patterns and embryo quality on Day 3 were the independent predictors for the blastocyst formation of 2.1PN-derived embryos. The 2.1PN zygotes displayed a comparable developmental potential compared to 2PN zygotes in advanced age patients (≥ 38). Moreover, there was a tendency that 2.1PN-derived blastocysts showed a similar euploidy rate compared to 2PN-derived blastocysts. CONCLUSION: Clinicians should consider using 2.1PN-derived euploid embryos for transfer after preimplantation genetic testing in the absence of available 2PN embryo cycles. 2.1PN-derived embryos could be a candidate, particularly beneficial for patients at advanced age.

Reassessing the impact of letrozole co-administration in controlled ovarian hyperstimulation: findings from a single-center repeated measures study.

PURPOSE: To explore whether letrozole improved outcomes in subsequent controlled ovarian hyperstimulation (COH) cycles. METHODS: This was a retrospective repeated measures cohort study examining COH cycles. Patients were included if they underwent two cycles for unexplained infertility, male factor infertility, or planned oocyte/embryo cryopreservation. The first cycles for all patients implemented a non-letrozole, conventional gonadotropin protocol. Second cycles for the study group included letrozole (2.5-7.5 mg for 5 days) with no medication change to second cycles amongst controls. Our primary objective was to compare oocyte yield. Cohorts were then subdivided by pursuit of oocyte (OC) or embryo (IVF) cryopreservation. Secondary outcome amongst the OC subgroup was oocyte maturation index (metaphase II (MII)/total oocytes). Secondary outcomes amongst the IVF subgroup were normal fertilization rate (2-pronuclear zygotes (2PN)/oocytes exposed to sperm), blastocyst formation rate (blastocysts/2PNs), and embryo ploidy (%euploid and aneuploid). RESULTS: Fifty-four cycles (n = 27) were included in letrozole and 108 cycles (n = 54) were included in control. Oocyte yield was higher in second cycles (p < 0.008) in the letrozole group but similar in second cycles (p = 0.26) amongst controls. Addition of letrozole did not impact MII index (p = 0.90); however, MII index improved in second cycles amongst controls (p < 0.001). Both groups had similar rates of normal fertilization (letrozole: p = 0.52; control: p = 0.61), blast formation (letrozole: p = 0.61; control: p = 0.84), euploid (letrozole: p = 0.29; control: p = 0.47), and aneuploid embryos (letrozole: p = 0.17; control: p = 0.78) between cycles. CONCLUSIONS: Despite improved oocyte yield, letrozole did not yield any difference in oocyte maturation or embryo outcomes.

Leukocytospermia does not negatively impact outcomes in in vitro fertilization cycles with intracytoplasmic sperm injection and preimplantation genetic testing for aneuploidy: findings from 5435 cycles.

PURPOSE: To investigate whether leukocytospermia (defined as the presence of ≥ 1 × 106 white blood cells/mL) affects clinical and embryologic outcomes in in vitro fertilization (IVF) cycles with intracytoplasmic sperm injection (ICSI) and preimplantation genetic testing for aneuploidy (PGT-A). METHODS: This was a retrospective cohort study including 5425 cycles between January 2012 to December 2021 at a single large university-affiliated fertility clinic. The primary outcome was live birth rate (LBR). RESULTS: The prevalence of leukocytospermia was 33.9% (n = 1843). Baseline characteristics including female age, BMI, AMH, Day 3 FSH, and male partner's age were similar in cycles with and without leukocytospermia. The LBR after the first euploid embryo transfer was similar in those with and without leukocytospermia (62.3% vs. 63% p = 0.625). Secondary outcomes including clinical pregnancy rate (CPR), sustained implantation rate (SIR), fertilization (2PN) rate, blastulation rate, and aneuploidy rate were also evaluated. The CPR (73.3% vs 74.9%, p = 0.213) and SIR (64.6% vs. 66%, p = 0.305) were similar in both groups. The 2PN rate was also similar in both groups (85.7% vs. 85.8%, p = 0.791), as was the blastulation rate per 2PN (56.7% vs. 57.5%, p = 0.116). The aneuploidy rate was not significantly different between groups (25.7% vs 24.4%, p = 0.053). A generalized estimation equation with logistic regression demonstrated that the presence leukocytospermia did not influence the LBR (adjusted OR 0.878; 95% CI, 0.680-1.138). CONCLUSION: Leukocytospermia diagnosed just prior to an IVF cycle with PGT-A does not negatively impact clinical or embryologic outcomes.

Embryo drop-out rates in preimplantation genetic testing for aneuploidy (PGT-A): a retrospective data analysis from the DoLoRes study.

PURPOSE: To evaluate the decline in transferable embryos in preimplantation genetic testing for aneuploidy (PGT-A) cycles due to (a) non-biopsable blastocyst quality, (b) failure of genetic analysis, (c) diagnosis of uniform numerical or structural chromosomal aberrations, and/or (d) chromosomal aberrations in mosaic constitution. METHODS: This retrospective multicenter study comprised outcomes of 1562 blastocysts originating from 363 controlled ovarian stimulation cycles, respectively, 226 IVF couples in the period between January 2016 and December 2018. Inclusion criteria were PGT-A cycles with trophectoderm biopsy (TB) and next generation sequencing (NGS). RESULTS: Out of 1562 blastocysts, 25.8% were lost due to non-biopsable and/or non-freezable embryo quality. In 10.3% of all biopsied blastocysts, genetic analysis failed. After exclusion of embryos with uniform or chromosomal aberrations in mosaic, only 18.1% of those originally yielded remained as diagnosed euploid embryos suitable for transfer. This translates into 50.4% of patients and 57.6% of stimulated cycles with no euploid embryo left for transfer. The risk that no transfer can take place rose significantly with a lower number of oocytes and with increasing maternal age. The chance for at least one euploid blastocyst/cycle in advanced maternal age (AMA)-patients was 33.3% compared to 52.1% in recurrent miscarriage (RM), 59.8% in recurrent implantation failure (RIF), and 60.0% in severe male factor (SMF). CONCLUSIONS: The present study demonstrates that PGT-A is accompanied by high embryo drop-out rates. IVF-practitioners should be aware that their patients run a high risk of ending up without any embryo suitable for transfer after (several) stimulation cycles, especially in AMA patients. Patients should be informed in detail about the frequency of inconclusive or mosaic results, with the associated risk of not having an euploid embryo available for transfer after PGT-A, as well as the high cost involved in this type of testing.

PGT-M for spinocerebellar ataxia type 1: development of a STR panel and a report of two clinical cases.

PURPOSE: To present the developed preimplantation genetic testing (PGT) for spinocerebellar ataxia type 1 (SCA1) and the outcomes of IVF with PGT. METHODS: PGT was performed for two unrelated couples from the Republic of Sakha (Yakutia) with the risk of SCA1 in one spouse. We have developed a system for PGT of a monogenic disease (PGT-M) for SCA1, which includes the analysis of a panel of 11 polymorphic STR markers linked to the ATXN1 gene and a pathogenic variant of the ATXN1 gene using nested PCR and fragment analysis. IVF/ICSI programs were performed according to standard protocols. Multiple displacement amplification (MDA) was used for whole genome amplification (WGA) and array comparative genomic hybridization (aCGH) for aneuploidy testing (PGT-A). RESULTS: Eight STRs were informative for the first couple and ten for the second. Similarity of the haplotypes carrying pathogenic variants of the ATXN1 gene was noted. In the first case, during IVF/ICSI-PGT, three embryos reached the blastocyst stage and were biopsied. One embryo was diagnosed as normal by maternal STR haplotype and the ATXN1 allele. PGT-A revealed euploidy. The embryo transfer resulted in a singleton pregnancy, and a healthy boy was born. Postnatal diagnosis confirmed normal ATXN1. In the second case, two blastocysts were biopsied. Both were diagnosed as normal by PGT-M, but PGT-A revealed aneuploidy. CONCLUSION: Birth of a healthy child after PGT for SCA1 was the first case of successful preimplantation prevention of SCA1 for the Yakut couple and the first case of successful PGT for SCA1 in Russia.

Preimplantation genetic testing in the current era, a review.

BACKGROUND: 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. DEVELOPMENT OF PGT: 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. PURPOSE OF REVIEW: 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. CONCLUSIONS: This comprehensive review aims to provide new insights into the understanding of techniques used in PGT, thereby contributing to the field of reproductive genetics.