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).

Fresh or frozen day 6 blastocyst transfer: is there still a question?

BACKGROUND: The Live Birth Rate (LBR) after day 5 (D5) blastocyst transfer is significantly higher than that with D6 embryos in both fresh and frozen-vitrified embryo transfer cycles, according to the most recently published meta-analyses. Therefore, for women obtaining only D6 blastocysts, the chances of pregnancy may be lower but nonetheless sufficient to warrant transferring such embryos. The best strategy for transfer (i.e., in fresh versus frozen cycles) remains unclear and there is a paucity of data on this subject. METHODS: A total of 896 couples with D6 single blastocyst transfers were retrospectively analyzed: patients receiving a fresh D6 embryo transfer (Fresh D6 transfer group, n = 109) versus those receiving a frozen-thawed D6 embryo transfer (Frozen D6 transfer group, n = 787). A subgroup comprising a freeze-all cycle without any previous fresh or frozen D5 embryo transfers (Elective frozen D6, n = 77) was considered and also compared with the Fresh D6 transfer group. We compared LBR between these two groups. Correlation between D6 blastocyst morphology according to Gardner's classification and live birth occurrence was also evaluated. Statistical analysis was carried out using univariate and multivariate logistic regression models. RESULTS: The LBR was significantly lower after a fresh D6 blastocyst transfer compared to the LBR with a frozen-thawed D6 blastocyst transfer [5.5% (6/109) vs. 12.5% (98/787), p = 0.034]. Comparison between LBR after Elective frozen D6 group to the Fresh D6 blastocyst transfers confirmed the superiority of frozen D6 blastocyst transfers. Statistical analysis of the blastocyst morphology parameters showed that both trophectoderm (TE) and inner cell mass (ICM) grades were significantly associated with the LBR after D6 embryo transfer (p < 0.001, p = 0.037). Multiple logistic regression revealed that frozen D6 thawed transfer was independently associated with a higher LBR compared with fresh D6 transfer (OR = 2.54; 95% CI: [1.05-6.17]; p = 0.038). Our results also show that transferring a good or top-quality D6 blastocyst increased the chances of a live birth by more than threefold. CONCLUSIONS: Our results indicate that transferring D6 blastocysts in frozen cycles improves the LBR, making it the best embryo transfer strategy for these slow-growing embryos. CLINICAL TRIAL NUMBER: Not applicable.

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

OBJECTIVES: 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. METHODS: A retrospective analysis was performed including a total of 743 cycles, the first frozen embryo transfer (FET) cycle with single embryo transfer, who underwent treatment between January 2020 and July 2023 in a single fertility centre, Gangnam CHA 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 2 groups-under 35 years of age and over 35 years of age-and performed the analysis separately for each group. RESULTS: In the under 35 years of age group, there were no significant differences in clinical pregnancy and miscarriage rates in G1 and G2 (67.2% vs. 63.8%, not statistically significantly different). Also, G4 had a higher clinical pregnancy rate than G3, but it was not significant (51.8% vs. 54.7%, not statistically significantly different). In the 35 years or older group, G2 had higher pregnancy rates than G1 and lower miscarriage rates (clinical pregnancy rate: 43.3% vs. 67.7%, P = 0.001, miscarriage rate: 22.5% vs. 3.4%, P = 0.001). In addition, G4 had a higher pregnancy rate than G3 and a lower miscarriage rate (clinical pregnancy rate: 31.8% vs. 46.9%, P = 0.003, miscarriage rate: 22.9% vs. 2.2%, P = 0.023). CONCLUSIONS: In the under-35-year-old 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.

Is a day 7 blastocyst predictive of the reproductive potential of sibling day 5 and day 6 blastocysts?

PURPOSE: To explore if a day 7 blastocyst is predictive of the reproductive potential of sibling day 5 or day 6 blastocysts? METHODS: Retrospective cohort of autologous frozen embryo transfers (FET), February 2019 to April 2022. Cycles divided into groups 1 to 5, according to the day of embryo cryopreservation and the presence of a day 7 blastocyst sibling within the cohort: group 1/group 2-day 5 blastocyst without/with a day 7 sibling, group 3/group 4-day 6 blastocyst without/with a day 7 sibling, group 5-day 7 blastocyst. Clinical, ongoing pregnancy and miscarriage rates, cycle, and patient characteristics are reported. Multivariable generalized estimating equations (GEE) logistic regression analysis accounts for confounders and assesses the effect of a sibling day 7 blastocyst on ongoing pregnancy rates of day 5 or day 6 blastocyst FETs. RESULTS: Ongoing pregnancy rates are 38.4%, 59.5%, 30.8%, 32.7%, and 4.4% in groups 1-5, respectively. When correcting for maternal age, number of oocytes retrieved and discarded per cohort, and ploidy, embryos cryopreserved on either day 6 or day 7 have reduced odds of ongoing pregnancy after FET compared to day 5 blastocysts (OR = 0.76, IQR [0.61-0.95], p-value = 0.01). However, the presence of a day 7 sibling does not significantly affect odds of ongoing pregnancy of day 5 or day 6 blastocysts compared to the same-day blastocyst without a day 7 sibling (p-value = 0.20 and 0.46, respectively). This finding is consistent within both the Preimplantation Genetic Testing for Aneuploidy (PGT-A) unscreened and screened (euploid) embryo subgroups. CONCLUSIONS: Day of embryo cryopreservation significantly affects ongoing pregnancy rates. However, day 7 embryos within a cohort do not affect the reproductive potential of sibling day 5 and day 6 blastocysts, suggesting that slow embryo development is an embryo-specific trait.

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.

Do clinical outcomes differ for day-5 versus day-6 single embryo transfers controlled for endometrial factor?

RESEARCH QUESTION: Is there is a difference in clinical outcomes between day-5 versus day-6 blastocysts when transferred in a personalized embryo transfer (PET) cycle guided by Endometrial Receptivity Analysis (ERA)? DESIGN: Multicentre, retrospective study; 260 patients who underwent a single embryo transfer with either a day-5 or day-6 blastocyst in a PET cycle guided by ERA between January 2017 and December 2019. RESULTS: A total of 260 blastocysts were transferred in a single embryo PET cycle guided by ERA. Of those, 183 (70.4%) were day-5 blastocysts and 77 (29.6%) were day-6 blastocysts. Clinical outcomes were similar when transferring day-5 blastocysts versus day-6 blastocysts: pregnancy rate was 75.4% (138/183) and 70.1% (54/77) (P = 0.465); implantation rate was 67.8% (124/183) and 63.6% (49/77) (P = 0.476); and ongoing pregnancy rate was 57.9% (106/183) and 58.4% (45/77) (P = 0.728), respectively. CONCLUSIONS: The data suggest that the clinical potential of day-5 and day-6 blastocysts are similar, as no difference in clinical outcomes are observed when transferring at the time of optimal endometrial receptivity as determined by ERA.

Day 5 vs day 6 single euploid blastocyst frozen embryo transfers: which variables do have an impact on the clinical pregnancy rates?

OBJECTIVE: To determine which variables affect most the clinical pregnancy rate with positive fetal heartbeat (CPR FHB+) when frozen embryo transfer (FET) cycles are performed with day 5 (D5) or day 6 (D6) euploid blastocysts. Design and method A single center retrospective study was performed from March 2017 till February 2021 including all single FET cycles with euploid D5 or D6 blastocysts and transferred in natural cycles (NC) or hormone replacement therapy (HRT) cycles. Trophectoderm (TE) and inner cell mass (ICM) qualities were recorded before biopsy. RESULTS: A total of 1102 FET cycles were included, 678 with D5 and 424 with D6 blastocysts. Pregnancy rate (PR), clinical PR (CPR), and CPR FHB+ were significantly higher with D5 blastocysts (PR: 70.7% vs 62.0%, OR = 0.68 [0.53-0.89], p = 0.004; CPR: 63.7% vs 54.2%, OR = 0.68 [0.52-0.96], p = 0.002 and CPR FHB+: 57.8% vs 49.8%, OR = 0.72 [0.53-0.96], p = 0.011). However, miscarriage rate (12.5% vs 11.4%, OR = 0.78 [0.48-1.26], p = 0.311) did not differ. From a multivariate logistic regression model, endometrial thickness (OR = 1.11 [1.01-1.22], p = 0.028), patient's age (OR = 1.03 [1.00-1.05], p = 0.021), BMI (OR = 0.97 [0.94-0.99], p = 0.023), and ICM grade C (OR = 0.23 [0.13-0.43], p < 0.001) were significant in predicting CPR FHB+. CONCLUSION: Although clinical outcomes are higher with D5 blastocysts, CPR FHB+ is more affected by endometrial thickness, patient age, BMI, and ICM grade C rather than biopsy day or endometrial preparation protocol.

Comparison of pregnancy and live birth rates between fresh day 5 morula transfer and fresh day 6 blastocyst transfer following extended culture for slow growing embryos.

PURPOSE: The aim of this study is to evaluate and compare the outcomes of fresh day 5 morula transfers and fresh day 6 blastocyst transfers after extended culture, in women with a whole cohort of slow growing embryos. METHODS: Fresh embryo transfer cycles of patients that underwent intracytoplasmic sperm injection between 2013 and 2020 with a whole cohort of slow developing embryos on 5th day of fertilization were evaluated for this retrospective cohort study. Outcomes of day 5 morula transfers and day 6 blastocyst transfers after extended culture were compared. RESULTS: Out of 479 patients, day 5 morula transfers were performed to 194 and embryo culturing was extended to day 6 in 285 women. Blastocyst formation was observed in 129 of 285 patients in extended culture group. Implantation rate of day 6 blastocyst transfer group was significantly higher than day 5 morula transfer group (27% vs. 12%, p < 0.001). However, clinical pregnancy (17% vs 15.4%) and live birth rates (14.4% vs 13%) were found similar in day 5 morula transfer and extended culture group per initiated cycle. CONCLUSION: Although implantation capacity of day 6 blastocysts seems higher comparing to day 5 morulas, clinical pregnancy and live birth rates are similar among intention to treat population in fresh day 5 morula transfers and day 6 blastocyst transfers after extended culture for women that cohorts consist of only slow developing embryos.

Suboptimal trophectoderm mitochondrial DNA level is associated with delayed blastocyst development.

PURPOSE: To provide a comprehensive analysis of mtDNA quantity in D5 and D6 blastocysts, as well as a further insight to the origin of delayed blastocyst development. METHODS: A retrospective cohort analysis of 829 D5 and 472 D6 blastocysts from 460 patients who underwent in vitro fertilization (IVF) with next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A). The quantity of trophectoderm mtDNA was extrapolated from the NGS data, followed by the analysis of mean mtDNA levels between D5 and D6 blastocysts of the same ploidy (aneuploid/euploid) and transfer outcomes (positive/negative clinical pregnancy). RESULTS: D5 blastocysts had significantly higher euploidy rate and clinical pregnancy rate when compared with D6 blastocysts. The proportion of blastocysts derived from patients ≧ 40 years old were similar between the D5 and D6 cohorts. When blastocysts with identical ploidy were analyzed, the D5 cohorts all had significantly higher mean mtDNA levels than their D6 counterparts. Similarly, when embryo transfers with identical outcome were analyzed, the D5 cohorts also had significantly higher mean mtDNA levels than the D6 cohorts. Trophectoderm mtDNA level was independent of maternal age and blastocyst morphology grades. CONCLUSIONS: Our data provided further evidence D5 blastocysts contained significantly greater mtDNA quantity than D6 blastocysts, and mtDNA quantity could be a key factor that affects the development rate of blastocysts. Furthermore, one must avoid using an arbitrary threshold when incorporating mtDNA quantity into the embryo selection criteria, as the observed value may have vastly different clinical implication when blastulation rate is also considered.

Pregnancy outcomes after day 5 versus day 6 blastocyst-stage embryo transfer: A systematic review and meta-analysis.

AIM: To compare the pregnancy outcomes after day 5 blastocyst-stage embryo transfers (BET) versus day 6 BET following vitrified-warmed cycle and to evaluate whether the number of embryos transferred and the chromosomal status of embryo influence effect estimates. METHODS: A literature search (PubMed, Embase and MEDLINE) up to January 2019 was conducted to identify studies where women with day 6 BET were compared to women with day 5 BET. Only studies published in English language, on peer-reviewed journal were considered eligible. The following subgroup analyses were performed: (i) number of embryos transferred and (ii) chromosomal status of embryo. RESULTS: From a total of 1956 articles identified, 23 observational studies were included in the meta-analysis. We observed that day 6 BET were associated with lower implantation rate (risk ratio, RR: 1.17, 95% confidence interval, CI: 1.10-1.24), clinical pregnancy rate (RR: 1.17, 95% CI: 1.10-1.24), ongoing pregnancy rate (RR: 1.15, 95% CI: 1.07-1.24) and live birth rate (RR: 1.22, 95% CI: 1.11-1.33) than day 5 BET following vitrified-warmed cycle. The subgroup analysis found that the superiority of day 5 BET compared with day 6 BET is influenced by the number of embryos transferred and chromosomal status of embryos. CONCLUSION: Current evidence shows that day 5 BET is superior to day 6 BET following vitrified-warmed cycle in clinical practice. Due to the overall low quality of available evidence, more larger and well-conducted studies are needed to compare the pregnancy outcomes between day 5 and day 6 BET before drawing a clear conclusion.