Abstract Study question Does the limited exposure of embryos outside the incubator, during evaluation and changeover, have an impact on the blastocyst development, blastocyst quality and euploid outcomes? Summary answer Exposure of embryos outside the incubator, negatively impacts the number, quality and euploidy rate of day 5 blastocysts. What is known already The laboratory environment with its culture conditions is one of the crucial elements of the delicate equation to a successful ART outcome. It has been shown that increased fluctuations in the culture conditions have a considerable impact on the number of blastocysts obtained and cycle outcomes. Compared to conventional benchtop incubators, Time Lapse Technology (TLT) incubators capture images of the embryo and allow morphologic and morphokinetic assessment without disturbance during incubation. Several studies have been published comparing the efficiency, safety and outcome performance between conventional and TLT incubators, however, none of them explored the euploid outcomes. Study design, size, duration An observational sibling oocyte study was performed at ART Fertility Clinics, Abu Dhabi between March 2018 and April 2020 and included data of 796 mature oocytes injected from 42 stimulation cycles. Sibling oocytes were randomly split between 2 different incubators: 12 oocytes were assigned to the twelve wells of the EmbryoscopeTM (ES) and the remaining oocytes were cultured in a conventional benchtop incubator, G185 K-System (KS). Participants/materials, setting, methods Embryos from patients with primary or secondary infertility, who underwent ovarian stimulation for ICSI and PGT-A through NGS on trophectoderm biopsies, were eligible. All patients had at least 16 fresh mature oocytes, randomly allocated to two different incubators after ICSI: 503 (63.2%) oocytes were cultured in ES and 293 (36.8%) in KS. The fertilization, cleavage, useable blastocyst and euploid rates, as well as embryo/blastocyst qualities were assessed to evaluate each incubator’s performance. Main results and the role of chance The fertilization and cleavage rates were similar between incubators. Total useable blastocyst rate (64.8% vs 49.6%, p < 0.001) was significantly higher for embryos cultured in ES, mainly due a higher percentage of blastocysts biopsied on day 5 in ES (67.8% vs 57.0%, p = 0.037), with improved quality (p = 0.008). There was no difference in the total euploid rate between ES and KS (59.9% vs 50.4%, p = 0.314), but a significantly higher euploid rate was seen for blastocysts cultured in ES and biopsied on day 5 (63.5% vs 37.4%, p = 0.001). Day 3 embryo quality and total biopsied blastocyst quality was not different between incubators. No difference was observed in the total useable blastocyst development from good (p = 0.0832) and poor (p = 0.112) quality day 3 cleavage stage embryos. However, when stratifying according to the day of blastocyst development, poor quality embryos on day 3 showed superior blastocyst formation on day 5 when cultured in ES (64.1% vs 39.1% for day 5 and 35.9% vs 60.9% for day 6, p = 0.005). Accordingly, blastocyst formation from poor quality embryos on day 3, was shifted to day 6 for embryos cultured in KS. This difference in the day of blastocyst development was not observed for good quality cleavage stage embryos (p = 0.917). Limitations, reasons for caution The current observational study needs confirmation in a prospective trial and should also include the implantation potential of the euploid blastocysts, which was not followed in the current study. A good prognosis population (≥16 mature oocytes) was studied and may not reflect the outcomes in patients with lower oocyte numbers. Wider implications of the findings: This work builds evidence to the solid introduction of the TLT incubators to the clinical routine, as the reduced exposure of embryos outside the incubator – and hence decreased stress - improves the blastocyst development. Trial registration number NA
Abstract Study question Are there differences in post-insemination events between fresh and frozen-derived gametes when testicular sperm extraction (TESE) is used? Summary answer Frozen-TESE leads to decreased fertilization, but not blastocyst euploidy rates; 2PN-lag time is significantly longer when frozen gametes are utilized compared to their fresh counterparts What is known already Studies indicate that the origin of testicular sperm influences the development potential of cleavage-stage embryos into blastocysts. Recent morphokinetic analysis suggest that embryos from frozen-TESE exhibit prolonged pronuclear stages and more frequent uneven cleavage compared to those from ejaculated sperm. Notably, no comparison with fresh-TESE was performed. To date, no studies have explored analysis in embryo morphokinetic parameters comparing fresh and frozen-TESE, especially when derived from the same patient.Such comparisons could provide a more accurate understanding of potential differences. Given that TESE occasionally involves the use of vitrified oocytes, it is essential to thoroughly examine outcomes across all combinations. Study design, size, duration Retrospective cohort study in a tertiary referral IVF centre including 72 cycles of 30 couples between January 2017 to September 2023. Following insemination by TESE-sperm, embryos were cultured in time-lapse (TL) incubator. Blastocysts ≥BL3CC (Gardner’s) underwent trophectoderm (TE) biopsy for Preimplantation Genetic Testing for aneuploidy (PGT-A) by next generation sequency (NGS). Participants/materials, setting, methods Gamete sources were fresh (fresh-TESE) or frozen-thawed-TESE (frozen-TESE) from the same patients with non-obstructive azoospermia (NOA), and fresh or vitrified (frozen) oocytes from the same female partner. Fertilization, usable blastocysts, and euploidy rates were recorded. Morphokinetics were annotated for time (t) of PB2 (tPB2), tPNa (PN-appearance), tPNf (PN-fading), t2-t9, CP (compaction), tM (Morula), tSB (start blastulation), tB (blastocyst) and timings between each developmental event. Main results and the role of chance Among included 980 oocyte/TESE dyads, 253 (25.8%) used fresh oocyte/sperm, 141 (14.4%) frozen oocyte/fresh sperm, 558 (56.9%) fresh oocyte/frozen sperm and 28 (2.9%) frozen oocyte/sperm. In oocyte/sperm dyads using fresh-TESE, normal fertilization (2PN) (52.0% vs. 44.5%) were higher compared to dyads using frozen-TESE (P < 0.01). Mixed-effects logistic-regression accounting for dependency structure between oocytes/sperm dyads of the same couple, showed an association of frozen-TESE with lower fertilization (OR: 0.64, 95%CI: 0.45-0.89, P = 0.008) and lower blastulation (³BL3CC)/MII (OR: 0.53, 95%CI: 0.37-0.77, P = 0.001), but similar blastulation (³BL3CC)/2PN (OR: 0.69, 95%CI: 0.45-1.07, P = 0.09). After accounting for female age, euploidy per tested blastocyst was similar whether fresh or frozen sperm was used (OR: 0.91, 95% CI: 0.46-1.79, P = 0.788). Frozen oocyte use on the other hand was not significantly associated with these outcomes (P > 0.05 for all). Analysis of morphokinetic parameters revealed that use of frozen dyads was associated with longer duration of 2PN after tPNa to tPNf: in frozen-TESE, 2.10h longer (95%CI: 0.55-3.64, P = 0.008); and in frozen oocytes, 2.34h longer (95%CI: 0.31-4.36, P = 0.024). Among embryos reaching blastulation, frozen-TESE embryos reached 6-cell stage earlier (tPNf to t6, 2.4h earlier, P = 0.009) but they were somewhat delayed in reaching expansion from there on (t6 to tEB, 4.4 hours later, P = 0.059). Limitations, reasons for caution In certain oocytes, insemination was conducted using either fresh or frozen immotile sperm that showed unresponsiveness to activation methods. However, the samples size hinders a comprehensive comparison. Wider implications of the findings Use of frozen-thawed TESE from NOA-patients may decrease fertilization and consequently lower blastocyst yield, but not euploidy rates. Synchronizing fresh-TESE with oocyte retrieval appears safer to maximize fertilization. Regardless of the gamete type, frozen-sourced sperm or oocyte impact 2PN-lag time, possibly indicating nuclear repair mechanisms post-freezing, requiring further investigation. Trial registration number 2310-ABU-020-VF
Abstract Study question Is the live birth rate (LBR) in euploid frozen blastocyst transfer (FET) affected by the quality of ICM (Inner cell mass) and TE (Trophectoderm)? Summary answer ICM and TE significantly impacts the LBR with a decline of LB from 57.3% (ICM-A) to 48.5% (ICM-B) to 22.1% (ICM-C) (p < 0.001) What is known already The morphological blastocyst grading system proposed by Gardner-Schoolcraft remains the most accepted system to identify blastocysts with higher implantation potential. It relies on morphological features within the blastocyst, including ICM and TE. Several studies tried to identify the individual contribution of each. However, the conclusions remain contradictory and no clear consensus has yet been achieved. Due to heterogeneity of parameters evaluated between different publications, where embryos with unknown ploidy status were transferred in conjunction with a variability of stimulation protocols and in the number of transferred embryos, the real effect of the ICM and TE is difficult to infer. Study design, size, duration This two-center retrospective observational study includes a total of 977 euploid single FET cycles between March 2017 and March 2020 at ART Fertility Clinics Muscat, Oman and Abu Dhabi, UAE. Participants/materials, setting, methods Trophectoderm biopsies were analyzed with Next Generation Sequencing (NGS). All blastocysts available on D5 or D6 with a quality ≥ BL3CC were subjected to TE biopsy for PGT-A analysis and LBR was recorded. Vitrification/warming of blastocysts was performed using Cryotop method (Kitazato). Bivariate and multivariate analysis were performed between LB outcomes and ICM and TE grade while controlling for confounding factors. Main results and the role of chance A total of 977 single FET cycles were analyzed: 651 in hormone replacement therapy (HRT) and 326 in natural cycle regimen (NC) resulting in a 46.88% LBR. The mean patients’ age was 33.80 years with a mean Body Mass Index (BMI) of 26.80 kg/m2. Though all qualities of ICM and TE were associated with LB, blastocyst ICM-A LBR was statistically significantly higher (57.3%) than ICM-B (48.4%) and ICM-C (22.1%) (p < 0.001). Similarly, blastocyst TE-A LBR was statistically significantly higher (59.2%) than TE- B (48.6%) and TE- C (30.3%) (p < 0.001). Miscarriage rate was similar in all groups. The grade of ICM and TE were significantly associated with Anti-Mullerian-Hormone (AMH) and day of blastocyst biopsy. Mean AMH (ng/ml) was higher in ICM groups (A: 3.78, B: 3.24, p < 0.001) and TE group (A: 3.63, B: 3.38, p < 0.05) compared to lower grade (ICM-C: 2.86, TE-C: 2.82). In multivariate analysis, endometrial preparation for FET, BMI and AMH were the parameters influencing LBR: OR:1.45, [CI:1.07-1.96], p < 0.015) for NC; OR 0.96 [CI:0.93-0.99], p = 0.004 for BMI; OR 0.95 [CI:0.90-1.00], p = 0.033 for AMH; Both, ICM-C and TE-C, resulted in a significantly lower chance of LB [ICM: OR 0.32, CI:0.17-0.61, p < 0.001; TE: OR 0.44, CI:0.27-0.73, p = 0.002), compared to grade A. Limitations, reasons for caution The retrospective nature of the study and inter-observer variability in blastocyst scoring is a limitation. The physician/embryologist performing the embryo transfer could not been standardized due to the multicenter design. Randomized controlled studies are needed to determine whether ICM or TE should be prioritized in the selection of the blastocyst. Wider implications of the findings The ICM and TE scoring in FET may influence the LBR and should be considered as an important factor for the success of embryo transfer cycles. Whether these results can be extrapolated to fresh embryo transfer and to blastocysts with unknown ploidy status, needs further investigation. Trial registration number not applicable
Abstract Study question Does the transfer of a poor-quality blastocyst along with a good-quality blastocyst improve clinical outcomes compared to single good-quality blastocyst transfer in euploid-only transfer cycles? Summary answer Transferring a poor-quality with a good-quality blastocyst improved clinical pregnancy rates but live birth rates were not significantly increased compared to single good-quality blastocyst transfer. What is known already Single embryo transfers (SET) are often preferred when using euploid blastocysts with good morphological characteristics. However, co-transfer of a euploid blastocyst with poor morphological characteristics along with a good-quality euploid blastocyst is still practiced. Studies on embryos with unknown ploidy status suggested that poor-quality blastocysts may hinder the outcomes of good-quality blastocysts when transferred together. Controversial results imply that co-transfer of a poor-quality along with a good-quality blastocyst is associated with similar clinical pregnancy rates and significantly increases multiple pregnancy rates. Outcomes of such double embryo transfers (DET) compared to SETs are yet to be determined in euploid-only blastocyst transfers. Study design, size, duration Retrospective cohort of two centers including 500 SETs and 718 DETs using only euploid blastocysts in FET autologous cycles between March 2017 to January 2021. Transferred blastocysts were graded ≥BL3CC (Gardner criteria) and underwent trophectoderm biopsy on days 5-7 for aneuploidy testing with Next Generation Sequencing. Classification of blastocyst quality (good vs poor) was based on inner cell mass, trophectoderm grades, and the day of blastocyst biopsy. Participants/materials, setting, methods Patients underwent SET of good-quality or DET of good and poor-quality euploid-blastocysts. Endometrial preparation of frozen embryo transfer cycles was achieved with hormonal replacement or within a natural cycle. Reported outcomes included clinical pregnancy (CP), live birth (LB), and high-order pregnancy rates. For comparison of expected and observed LB rates, theoretical rates were estimated with Monte-Carlo simulations using binomial density function and SET data, under the assumption of independency for success rates of individual embryos. Main results and the role of chance After adjusting for Age, AMH, BMI, and cycle preparation method (artificial vs natural), DET of good-quality and poor-quality blastocysts was associated with higher CP rates compared to SET of a good-quality blastocyst (73.1% vs 63.8%, OR: 1.67, 95% CI: 1.12-2.55, P = 0.014). After adjusting for confounders, DET of good-quality and poor-quality blastocysts was not associated with significantly higher LB rates compared to SET of a good-quality blastocyst (56.8% vs 52.8%, OR: 1.25, 95% CI: 0.87-1.83, P = 0.234). Moreover, high-order pregnancy rates were significantly higher in the DET group compared to SET (40.4% vs 1.1%, P < 0.001). Using a model based on SET data, we estimated theoretical LB rates that should be achieved if the LB chance of co-transferred embryos were independent. The model showed that the observed LB rates of DET good and poor-quality blastocysts were significantly lower than the expected average (observed 56.8% vs expected 66.4% [95% CI: 59.0 to 73.8%], P = 0.0154). Limitations, reasons for caution This study has a retrospective design, and estimates are susceptible to residual confounding from unobserved or unaccounted variables. Theoretical rates were estimated under the assumption of independency of transferred embryos. Wider implications of the findings Despite higher CP, DET of poor and good-quality blastocysts did not increase LB compared to SET of good-quality blastocyst and was associated with higher multiple-pregnancy rates. Lower than expected LB rates imply DET to be detrimental to cumulative-LB, and sequential-SET may be preferable, regardless of morphological quality of euploid blastocysts. Trial registration number Not applicable
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