Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts.

Capalbo A ，Rienzi L ，Cimadomo D ，Maggiulli R ，Elliott T ，Wright G ，Nagy ZP ，Ubaldi FM ... -  《-》

Correlation between aneuploidy, standard morphology evaluation and morphokinetic development in 1730 biopsied blastocysts: a consecutive case series study.

Are there correlations among human blastocyst ploidy status, standard morphology evaluation and time-lapse kinetics? Correlations were observed, in that euploid human blastocysts showed a higher percentage with top quality inner cell mass (ICM) and trophectoderm (TE), higher expansion grades and shorter time to start of blastulation, expansion and hatching, compared to aneuploid ones. Embryo quality has always been considered an important predictor of successful implantation and pregnancy. Nevertheless, knowledge of the relative impact of each morphological parameter at the blastocyst stage needs to be increased. Recently, with the introduction of time-lapse technology, morphokinetic parameters can also be evaluated. However, a large number of studies has reported conflicting outcomes. This was a consecutive case series study. The morphology of 1730 blastocysts obtained in 530 PGS cycles performed from September 2012 to April 2014 that underwent TE biopsy and array comparative genomic hybridization was analyzed retrospectively. A total of 928 blastocysts were cultured in a time-lapse incubator allowing morphokinetic parameters to be analyzed. Mean female age was 36.8 ± 4.24 years. Four hunderd fifty-four couples were enrolled in the study: 384, 64 and 6 of them performed single, double or triple PGS cycles, respectively. In standard morphology evaluation, the expansion grade, and quality of the ICM and TE were analyzed. The morphokinetic parameters observed were second polar body extrusion, appearance of two pronuclei, pronuclear fading, onset of two- to eight-cell divisions, time between the two- and three-cell (cc2) and three- and four-cell (s2) stages, morulae formation time, starting blastulation, full blastocyst stage, expansion and hatching timing. Of the 1730 biopsied blastocysts, 603 were euploid and 1127 aneuploid. We observed that 47.2% of euploid and 32.8% of aneuploid blastocysts showed top quality ICM (P < 0.001), and 17.1% of euploid and 28.5% of aneuploid blastocysts showed poor quality ICM (P < 0.001). Top quality TE was present in 46.5% of euploid and 31.1% of aneuploid blastocysts (P < 0.001), while 26.6% of euploid and 38.1% of aneuploid blastocysts showed poor quality TE (P < 0.001). Regarding expansion grade, 81.1% of euploid and 72.4% of aneuploid blastocysts were fully expanded (Grade 5-6; P < 0.001). The timing of cleavage from the three- to four-cell stage, of reaching four-cell stage, of starting blastulation, reaching full blastocyst stage, blastocyst expansion and hatching were 2.6 (95% confidence interval (CI): 1.7-3.5), 40.0 (95% CI: 39.3-40.6), 103.4 (95% CI: 102.2-104.6), 110.2 (95% CI: 108.8-111.5), 118.7 (95% CI: 117.0-120.5) and 133.2 (95% CI: 131.2-135.2) hours in euploid blastocysts, and 4.2 (95% CI: 3.6-4.8), 41.1 (95% CI: 40.6-41.6), 105.0 (95% CI: 104.0-106.0), 112.8 (95% CI: 111.7-113.9), 122.1 (95% CI: 120.7-123.4) and 137.4 (95% CI: 135.7-139.1) hours in aneuploid blastocysts (P < 0.05 for early and P < 0.0001 for later stages of development), respectively. No statistically significant differences were found between euploid and aneuploid blastocysts for the remaining morphokinetic parameters.A total of 407 embryo transfers were performed (155 fresh, 252 frozen-thawed blastocysts). Higher clinical pregnancy, implantation and live birth rates were obtained in frozen-thawed compared to fresh embryo transfers (P = 0.0104, 0.0091 and 0.0148, respectively). The miscarriage rate was 16.1% and 19.6% in cryopreserved and fresh embryo transfer, respectively. The mean female age was lower in the euploid compared to aneuploid groups (35.0 ± 3.78 versus 36.7 ± 4.13 years, respectively), We found an increasing probability for aneuploidy with female age of 10% per year (odds ratio (OR) = 1.1, 95% CI: 1.1-1.2, P < 0.001). The main limitation of morphology assessment is that it is a static system and can be operator-dependent. In this study, eight embryologists performed morphology assessments. The main limitation of the time-lapse technology is that it is impossible to rotate the embryos making it very difficult to observe them in case of blastomere overlapping or increased cytoplasmic fragmentation. Although there seems to be a relationship between the ploidy status and blastocyst morphology/development dynamics, the evaluation of morphological and morphokinetic parameters cannot currently be improved upon, and therefore replace, PGS. Our results on ongoing pregnancy and miscarriage rates suggest that embryo evaluation by PGS or time-lapse imaging may not improve IVF outcome. However, time-lapse monitoring could be used in conjunction with PGS to choose, within a cohort, the blastocysts to analyze or, when more than one euploid blastocyst is available, to select which one should be transferred. No specific funding was obtained for this study. None of the authors have any competing interests to declare.

Minasi MG ，Colasante A ，Riccio T ，Ruberti A ，Casciani V ，Scarselli F ，Spinella F ，Fiorentino F ，Varricchio MT ，Greco E ... -  《-》

Leave the past behind: women's reproductive history shows no association with blastocysts' euploidy and limited association with live birth rates after euploid embryo transfers.

Is there an association between patients' reproductive history and the mean euploidy rates per biopsied blastocysts (m-ER) or the live birth rates (LBRs) per first single vitrified-warmed euploid blastocyst transfers? Patients' reproductive history (as annotated during counselling) showed no association with the m-ER, but a lower LBR was reported after euploid blastocyst transfer in women with a history of repeated implantation failure (RIF). Several studies have investigated the association between the m-ER and (i) patients' basal characteristics, (ii) ovarian stimulation strategy and dosage, (iii) culture media and conditions, and (iv) embryo morphology and day of full blastocyst development. Conversely, the expected m-ER due to women's reproductive history (previous live births (LBs), miscarriages, failed IVF cycles and transfers, and lack of euploid blastocysts among prior cohorts of biopsied embryos) still needs investigations. Yet, this information is critical to counsel new patients about a first cycle with preimplantation genetic testing for aneuploidy (PGT-A), but even more so after former adverse outcomes to prevent treatment drop-out. This observational study included all patients undergoing a comprehensive chromosome testing (CCT)-based PGT-A cycle with at least one biopsied blastocyst in the period April 2013-December 2019 at a private IVF clinic (n = 2676 patients undergoing 2676 treatments and producing and 8151 blastocysts). m-ER were investigated according to women's reproductive history of LBs: no/≥1, miscarriages: no/1/>1; failed IVF cycles: no/1/2/>2, and implantation failures after previous transfers: no/1/2/>2. Among the 2676 patients included in this study, 440 (16%) had already undergone PGT-A before the study period; the data from these patients were further clustered according to the presence or absence of euploid embryo(s) in their previous cohort of biopsied blastocysts. The clinical outcomes per first single vitrified-warmed euploid blastocyst transfers (n =1580) were investigated according to the number of patients' previous miscarriages and implantation failures. The procedures involved in this study included ICSI, blastocyst culture, trophectoderm biopsy without hatching in Day 3, CCT-based PGT-A without reporting segmental and/or putative mitotic (or mosaic) aneuploidies and single vitrified-warmed euploid blastocyst transfer. For statistical analysis, Mann-Whitney U or Kruskal-Wallis tests, as well as linear regressions and generalised linear models among ranges of maternal age at oocyte retrieval were performed to identify significant differences for continuous variables. Fisher's exact tests and multivariate logistic regression analyses were instead used for categorical variables. Maternal age at oocyte retrieval was the only variable significantly associated with the m-ER. We defined five clusters (<35 years: 66 ± 31%; 35-37 years: 58 ± 33%; 38-40 years: 43 ± 35%; 40-42 years: 28 ± 34%; and >42 years: 17 ± 31%) and all analyses were conducted among them. The m-ER did not show any association with the number of previous LBs, miscarriages, failed IVF cycles or implantation failures. Among patients who had already undergone PGT-A before the study period, the m-ER did not associate with the absence (or presence) of euploid blastocysts in their former cohort of biopsied embryos. Regarding clinical outcomes of the first single vitrified-warmed euploid blastocyst transfer, the implantation rate was 51%, the miscarriage rate was 14% and the LBR was 44%. This LBR was independent of the number of previous miscarriages, but showed a decreasing trend depending on the number of previous implantation failures, reaching statistical significance when comparing patients with >2 failures and patients with no prior failure (36% versus 47%, P < 0.01; multivariate-OR adjusted for embryo quality and day of full blastocyst development: 0.64, 95% CI 0.48-0.86, P < 0.01). No such differences were shown for previous miscarriage rates. The sample size for treatments following a former completed PGT-A cycle should be larger in future studies. The data should be confirmed from a multicentre perspective. The analysis should be performed also in non-PGT cycles and/or including patients who did not produce blastocysts, in order to investigate a putative association between women's reproductive history with outcomes other than euploidy and LBRs. These data are critical to counsel infertile couples before, during and after a PGT-A cycle, especially to prevent treatment discontinuation due to previous adverse reproductive events. Beyond the 'maternal age effect', the causes of idiopathic recurrent pregnancy loss (RPL) and RIF are likely to be endometrial receptivity and selectivity issues; transferring euploid blastocysts might reduce the risk of a further miscarriage, but more information beyond euploidy are required to improve the prognosis in case of RIF. No funding was received and there are no competing interests. N/A.

Cimadomo D ，Capalbo A ，Dovere L ，Tacconi L ，Soscia D ，Giancani A ，Scepi E ，Maggiulli R ，Vaiarelli A ，Rienzi L ，Ubaldi FM ... -  《-》

Consistent and reproducible outcomes of blastocyst biopsy and aneuploidy screening across different biopsy practitioners: a multicentre study involving 2586 embryo biopsies.

Is blastocyst biopsy and quantitative real-time PCR based comprehensive chromosome screening a consistent and reproducible approach across different biopsy practitioners? The blastocyst biopsy approach provides highly consistent and reproducible laboratory and clinical outcomes across multiple practitioners from different IVF centres when all of the embryologists received identical training and use similar equipment. Recently there has been a trend towards trophectoderm (TE) biopsy in preimplantation genetic screening (PGS)/preimplantation genetic diagnosis (PGD) programmes. However, there is still a lack of knowledge about the reproducibility that can be obtained from multiple biopsy practitioners in different IVF centres in relation also to blastocysts of different morphology. Although it has been demonstrated that biopsy at the blastocyst stage has no impact on embryo viability, it remains a possibility that less experienced individual biopsy practitioners or laboratories performing TE biopsy may affect certain outcomes. We investigated whether TE biopsy practitioners can have an impact on the quality of the genetic test and the subsequent clinical outcomes. This longitudinal cohort study, between April 2013 and December 2014, involved 2586 consecutive blastocyst biopsies performed at three different IVF centres and the analysis of 494 single frozen euploid embryo transfer cycles (FEET). Seven biopsy practitioners performed the blastocyst biopsies in the study period and quantitative PCR was used for comprehensive chromosome screening (CCS). The same practitioner performed both the biopsy and tubing procedures for each blastocyst they biopsied. To investigate the quality of the biopsied samples, the diagnostic rate, sample-specific concurrence and the cell number retrieved in the biopsy were evaluated for each biopsy operator. Clinical outcomes following FEET cycles were stratified by biopsy operator and compared. Cellularity of the biopsy sample was also correlated with clinical outcomes. The seven practitioners performed 2586 biopsies, five in centre IVF-1 and one in each of the other two IVF centres (IVF-2 and IVF-3). Overall, 2437 out of 2586 (94.2%) blastocyst biopsies resulted in a conclusive diagnosis, 119 (4.6%) showed a nonconcurrent result and 30 (1.2%) failed to amplify, suggesting the absence of TE cells in the test tube or presence of degenerated/lysed cells only. Among the samples producing a conclusive diagnosis, a mean concurrence value of 0.253 (95% CI = 0.250-0.257) was observed. Logistic regression analysis adjusted for confounding factors showed no differences in the diagnosis rate and in the concurrence of the genetic analysis between different biopsy practitioners. An overall mean number of 7.32 cells (95% CI = 6.82-7.81; range 2-15) were predicted from all biopsies. Higher cellularity was significantly associated with a better quality of the CCS diagnosis (P < 0.01) and with the conclusive diagnosis rate, with nonconcurrent samples showing significantly lower numbers of cells (2.1; 95% CI=1.5-2.7) compared with samples resulting in a conclusive diagnosis (mean cells number 7.5; 95% CI = 7.1-7.9, P < 0.01). However, no differences were recorded between different biopsy practitioners regarding cellularity of the biopsy. Finally, logistic analysis showed no impact of the biopsy practitioners on the observed ongoing rates of implantation, biochemical pregnancy loss and miscarriage after the FEET cycles. These data come from a restricted set of laboratories where all of the embryologists received identical training and use identical equipment. A single TE biopsy method and CCS technology was used and these data particularly apply to PGS programmes using blastocyst biopsy without zona opening at the cleavage stage and using qPCR-based CCS. To make firm conclusions on the potential impact of biopsy on biochemical pregnancy loss and miscarriages according to practitioner and biopsy cellularity, a larger sample size is needed. We reported a very high consistency and reproducibility of the blastocyst biopsy approach coupled with qPCR-based CSS for both genetic and clinical outcomes across different practitioners working in different IVF centres when appropriate training is provided and when the same laboratory setting is used. These data are important considering the trend towards the use of blastocyst biopsy worldwide for PGD/PGS applications. None.

Capalbo A ，Ubaldi FM ，Cimadomo D ，Maggiulli R ，Patassini C ，Dusi L ，Sanges F ，Buffo L ，Venturella R ，Rienzi L ... -  《-》

What is the reproductive potential of day 7 euploid embryos?

What is the rate of euploidy and the reproductive potential of embryos biopsied after 6 days of development? Embryos biopsied after 6 days of development have higher rates of aneuploidy; however, day 7 euploid embryos selected at transfer can achieve acceptable pregnancy rates and live birth (LB) outcomes. Recent publications have shown promising treatment results after euploid day 7 embryo transfers (ETs), albeit these studies were limited by small sample sizes. Whereas the current clinical standard has been to discard embryos that do not reach expansion by day 6 of development, the lack of robust data surrounding the clinical utility of day 7 embryos warrants further evaluation. Retrospective cohort analysis in a single, academic in vitro fertilization (IVF) center from January 2012 to March 2018. A total of 25 775 embryos underwent trophectoderm (TE) biopsy and preimplantation genetic testing for aneuploidy (PGT-A). Additionally, the clinical IVF outcomes of 3824 single, euploid frozen embryo transfer (FET) cycles were evaluated. Cohorts were segregated by day of TE biopsy following oocyte retrieval (day 5, day 6 or day 7). PGT-A was performed to identify embryonic ploidy rates. Secondly, IVF and LB outcomes after single, euploid FET were evaluated for each cohort. A total of day 5 (n = 12 535), day 6 (n = 11 939) and day 7 (n = 1298) embryos were included in the study analysis. The rate of embryo euploidy was significantly lower in day 7 blastocysts compared to day 5 or day 6 cohorts (day 7 = 40.5%; day 5 = 54.7%; day 6 = 52.9%; (P < 0.0001)). After adjusting for age, anti-Müllerian hormone, BMI, embryo quality and number of embryos biopsied, there was a significant association between aneuploidy and embryos biopsied on day 7 when compared to day 5 biopsied embryos (OR = 1.34, CI 95% 1.09-1.45, P = 0.001) and day 6 biopsied embryos (OR = 1.26, CI95% 1.07-1.16, P < 0.001).A sub-analysis of subsequent 3824 single, euploid FET cycles (day 5: n = 2321 cycles; day 6: n = 1381 cycles; and day 7: n = 116 cycles) showed significant differences among cohorts in implantation, clinical pregnancy, LB and clinical loss rates. There was a significant decrease in the odds of implantation, clinical pregnancy and LB, but no association with clinical loss or multiple pregnancy rates in patients who utilized day 7-biopsied embryos during treatment. The retrospective nature of the study and potential variability in the study center's laboratory protocol(s) compared to other reproductive treatment centers may limit the external validity of our findings. Additionally, patients who transferred euploid embryos, biopsied on day 7 of development due to an absence of day 5 or day 6 counterparts, may have introduced selection bias in this study. Embryonic developmental stage, morphological grade and ploidy status are paramount factors affecting ET selection and implantation potential. This study reveals that embryos ineligible for TE biopsy on day 5 or day 6 of development may benefit from extended culture to day 7. Our study demonstrates patient benefit when extended culture to day 7 of development is routinely performed for embryos failing to meet biopsy criteria by day 5 or 6. No funding was received for the realization of this manuscript. Dr Alan Copperman is Advisor or Board Member of Sema 4 (Stake holder in Data), Progyny and Celmatix. This retrospective analysis was approved by an Institutional Review Board (WIRB PRO NUM: 20161791; Study Number: 1167398).

Hernandez-Nieto C ，Lee JA ，Slifkin R ，Sandler B ，Copperman AB ，Flisser E ... -  《-》