Looking past the appearance: a comprehensive description of the clinical contribution of poor-quality blastocysts to increase live birth rates during cycles with aneuploidy testing.

Which are the clinical benefits and risks of including poor-quality blastocysts (PQBs) in the cohort of biopsied embryos during a cycle with preimplantation genetic testing for aneuploidies (PGT-A)? PQBs show a worse prognosis with respect to sibling non-PQBs, but their clinical use allows an overall 2.6% increase in the number of live births (LBs) achievable after PGT-A. PQBs (<BB according to Gardner and Schoolcraft's classification) are generally disregarded for clinical use and/or research purposes. Therefore, limited data exist in literature to estimate the benefits and risks deriving from the transfer of a PQB. In Italy, the law imposes the transfer or cryopreservation of all embryos, unless proven not viable. This regulation has allowed the production of a large amount of data regarding poor-quality embryos. Previous reports outlined a lower chance of euploidy and implantation for PQBs. Yet, a comprehensive picture of their real clinical contribution is missing. This observational cohort study including 2757 oocyte retrievals for PGT-A (mean maternal age, 39.6 ± 3.3 years) conducted at a private IVF centre between April 2013 and May 2018. A total of 1497 PQBs were obtained and their embryological, chromosomal and clinical features were compared to 5250 non-PQBs (≥BB according to Gardner and Schoolcraft's classification) and adjusted for all significant confounders. After defining the overall increase in LBs due to PQBs, we outlined the population of patients who might benefit the most from their clinical use. ICSI cycles, involving ovarian stimulation, blastocyst culture, trophectoderm biopsy, vitrification, comprehensive chromosome testing and vitrified-warmed euploid single embryo transfers (SETs), were conducted. Overall analyses and sub-analyses in populations of patients clustered according to maternal age at retrieval and size of the cohort of sibling non-PQBs were performed. Finally, the risk of miscarriage and the chance of LB per biopsied PQB and non-PQB were estimated. PQBs allowed a 12.4% increase in the cycles where ≥1 blastocyst was biopsied. To date, we report a concurrent 2.6% increase in the cycles resulting in ≥1 LB. On average 0.7 ± 0.9 (range, 0-9) PQBs were obtained per cycle for biopsy, including 0.2 ± 0.4 (range, 0-5) euploid PQBs. Maternal age solely correlates with the prevalence of PQBs from both overall and cycle-based analyses. Indeed, the patients who benefit the most from these embryos (i.e. 18 women achieving their only LBs thanks to PQBs) cluster among women older than 42 years and/or those with no or few sibling non-PQBs (1.1 ± 1.1; range, 0-3). The 1497 PQBs compared to the 5250 non-PQBs showed slower development (Day 5, 10.1% versus 43.9%; Day 6, 60.5% versus 50.8%; Day 7, 29.4% versus 5.2%) and lower euploidy rates (23.5% versus 51%; adjusted OR, 0.36). Among the 195 and 1697 transferred euploid PQBs and non-PQBs, the former involved a lower implantation rate (16.9% versus 52.3%) and a higher miscarriage rate per clinical pregnancy (36.4% versus 13.9%), therefore resulting in a lower LB rate (LBR, 10.8% versus 44.6%; adjusted OR, 0.22). Based on these rates, we estimated an overall 1.5% risk of miscarriage and 2.6% chance of LB after euploid vitrified-warmed SET per each biopsied PQB. The same estimates for non-PQBs were 3.7% and 22.8%. The clinical benefit of PQBs is underestimated since they are the last option for transfer and this analysis entailed only the first LB. The higher miscarriage rate per clinical pregnancy here reported might be the consequence of a population of patients of poorer prognosis undergoing the SET of euploid PQBs, an option that requires further investigation. Finally, a cost-benefit analysis is needed in a prospective non-selection fashion. PQBs show higher aneuploidy rates. If to be included, PGT-A is recommended. When selected against aneuploid-PQBs, euploid ones could still involve a worse prognosis, yet, their LBR is not negligible. Women should be informed that a poor morphology does not define a non-viable embryo per se, although PQBs show a reduced chance of resulting in an LB. No external funding was used for this study. The authors have no conflict of interest related to this study. N/A.

Cimadomo D ，Soscia D ，Vaiarelli A ，Maggiulli R ，Capalbo A ，Ubaldi FM ，Rienzi L ... -  《-》

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 ... -  《-》

Associations of blastocyst features, trophectoderm biopsy and other laboratory practice with post-warming behavior and implantation.

Cimadomo D ，Capalbo A ，Levi-Setti PE ，Soscia D ，Orlando G ，Albani E ，Parini V ，Stoppa M ，Dovere L ，Tacconi L ，Ievoli E ，Maggiulli R ，Ubaldi FM ，Rienzi L ... -  《-》

The euploid blastocysts obtained after luteal phase stimulation show the same clinical, obstetric and perinatal outcomes as follicular phase stimulation-derived ones: a multicenter study.

Are the reproductive outcomes (clinical, obstetric and perinatal) different between follicular phase stimulation (FPS)- and luteal phase stimulation (LPS)-derived euploid blastocysts? No difference was observed between FPS- and LPS-derived euploid blastocysts after vitrified-warmed single embryo transfer (SET). Technical improvements in IVF allow the implementation non-conventional controlled ovarian stimulation (COS) protocols for oncologic and poor prognosis patients. One of these protocols begins LPS 5 days after FPS is ended (DuoStim). Although, several studies have reported similar embryological outcomes (e.g. fertilization, blastulation, euploidy) between FPS- and LPS-derived cohort of oocytes, information on the reproductive (clinical, obstetric and perinatal) outcomes of LPS-derived blastocysts is limited to small and retrospective studies. Multicenter study conducted between October 2015 and March 2019 including all vitrified-warmed euploid single blastocyst transfers after DuoStim. Only first transfers of good quality blastocysts (≥BB according to Gardner and Schoolcraft's classification) were included. If euploid blastocysts obtained after both FPS and LPS were available the embryo to transfer was chosen blindly. The primary outcome was the live birth rate (LBR) per vitrified-warmed single euploid blastocyst transfer in the two groups. To achieve 80% power (α = 0.05) to rule-out a 15% difference in the LBR, a total of 366 first transfers were required. Every other clinical, as well as obstetric and perinatal outcomes, were recorded. Throughout the study period, 827 patients concluded a DuoStim cycle and among them, 339 did not identify any transferable blastocyst, 145 had an euploid blastocyst after FPS, 186 after LPS and 157 after both FPS and LPS. Fifty transfers of poor quality euploid blastocysts were excluded and 49 patients did not undergo an embryo transfer during the study period. Thus, 389 patients had a vitrified-warmed SET of a good quality euploid blastocyst (182 after FPS and 207 after LPS). For 126 cases (32%) where both FPS- and LPS-derived good quality blastocysts were available, the embryo transferred was chosen blindly with a 'True Random Number Generator' function where '0' stood for FPS-derived euploid blastocysts and '1' for LPS-derived ones (n = 70 and 56, respectively) on the website random.org. All embryos were obtained with the same ovarian stimulation protocol in FPS and LPS (GnRH antagonist protocol with fixed dose of rec-FSH plus rec-LH and GnRH-agonist trigger), culture conditions (continuous culture in a humidified atmosphere with 37°C, 6% CO2 and 5% O2) and laboratory protocols (ICSI, trophectoderm biopsy in Day 5-7 without assisted hatching in Day 3, vitrification and comprehensive chromosome testing). The women whose embryos were included had similar age (FPS: 38.5 ± 3.1 and LPS: 38.5 ± 3.2 years), prevalence of male factor, antral follicle count, basal hormonal characteristics, main cause of infertility and previous reproductive history (i.e. previous live births, miscarriages and implantation failures) whether the embryo came from FPS or LPS. All transfers were conducted after warming in an artificial cycle. The blastocysts transferred after FPS and LPS were similar in terms of day of full-development and morphological quality. The positive pregnancy test rates for FPS- and LPS-derived euploid blastocysts were 57% and 62%, biochemical pregnancy loss rates were 10% and 8%, miscarriage rates were 15% and 14% and LBRs were 44% (n = 80/182, 95% CI 37-51%) and 49% (n = 102/207, 95% CI 42-56%; P = 0.3), respectively. The overall odds ratio for live birth (LPS vs FPS (reference)) adjusted for day of blastocyst development and quality, was 1.3, 95% CI 0.8-2.0, P = 0.2. Among patients with euploid blastocysts obtained following both FPS and LPS, the LBRs were also similar (53% (n = 37/70, 95% CI 41-65%) and 48% (n = 27/56, 95% CI 35-62%) respectively; P = 0.7). Gestational issues were experienced by 7.5% of pregnant women after FPS- and 10% of women following LPS-derived euploid single blastocyst transfer. Perinatal issues were reported in 5% and 0% of the FPS- and LPS-derived newborns, respectively. The gestational weeks and birthweight were similar in the two groups. A 5% pre-term delivery rate was reported in both groups. A low birthweight was registered in 2.5% and 5% of the newborns, while 4% and 7% showed high birthweight, in FPS- and LPS-derived euploid blastocyst, respectively. Encompassing the 81 FPS-derived newborns, a total of 9% were small and 11% large for gestational age. Among the 102 LPS-derived newborns, 8% were small and 6% large for gestational age. No significant difference was reported for all these comparisons. The LPS-derived blastocysts were all obtained after FPS in a DuoStim protocol. Therefore, studies are required with LPS-only, late-FPS and random start approaches. The study is powered to assess differences in the LBR per embryo transfer, therefore obstetric and perinatal outcomes should be considered observational. Although prospective, the study was not registered. This study represents a further backing of the safety of non-conventional COS protocols. Therefore, LPS after FPS (DuoStim protocol) is confirmed a feasible and efficient approach also from clinical, obstetric and perinatal perspectives, targeted at patients who need to reach the transfer of an euploid blastocyst in the shortest timeframe possible due to reasons such as cancer, advanced maternal age and/or reduced ovarian reserve and poor ovarian response. None. N/A.

Vaiarelli A ，Cimadomo D ，Alviggi E ，Sansone A ，Trabucco E ，Dusi L ，Buffo L ，Barnocchi N ，Fiorini F ，Colamaria S ，Giuliani M ，Argento C ，Rienzi L ，Ubaldi FM ... -  《-》

How slow is too slow? A comprehensive portrait of Day 7 blastocysts and their clinical value standardized through artificial intelligence.

What is the clinical value of Day 7 blastocysts? Ending embryo culture at 144 hours post-insemination (h.p.i.; i.e. 6 days) would involve 7.3% and 4.4% relative reductions in the number of patients obtaining euploid blastocysts and live birth(s) (LBs), respectively. Many studies showed that Day 7 blastocysts are clinically valuable, although less euploid and less competent than faster-growing embryos. Nevertheless, a large variability exists in: (i) the definition of 'Day 7'; (ii) the criteria to culture embryos to Day 7; (iii) the clinical setting; (iv) the local regulation; and/or (v) the culture strategies and incubators. Here, we aimed to iron out these differences and portray Day 7 blastocysts with the lowest possible risk of bias. To this end, we have also adopted an artificial intelligence (AI)-powered software to automatize developmental timings annotations and standardize embryo morphological assessment. Observational study including 1966 blastocysts obtained from 681 patients cultured in a time-lapse incubator between January 2013 and December 2020 at a private Italian IVF center. According to Italian Law 40/2004, embryos were not selected based on their morphology and culture to ≥168 h.p.i. is standard care at our center. ICSI, continuous culture with Day 5 media refresh, trophectoderm biopsy without assisted hatching and comprehensive chromosome testing (CCT) to diagnose full-chromosome non-mosaic aneuploidies, were all performed. Blastocysts were clustered in six groups based on the time of biopsy in h.p.i. at 12 hr intervals starting from <120 h.p.i. (set as control) up to >168 h.p.i. Blastocyst quality was assessed using Gardner's scheme and confirmed with AI-powered software. AI was also used to automatically annotate the time of expanding blastocyst (tEB) and the hours elapsing between this moment and the achievement of full expansion when blastocysts were biopsied and vitrified. Also, blastocyst area at tEB and at the time of biopsy was automatically assessed, as well as the hour of the working day when the procedure was performed. The main outcomes were the euploidy rate and the LB rate (LBR) per vitrified-warmed euploid single blastocyst transfer. The results were adjusted for confounders through multivariate logistic regressions. To increase their generalizability, the main outcomes were reported also based on a 144-h.p.i. cutoff (i.e. 6 exact days from ICSI). Based on this cutoff, all the main patient outcomes (i.e. number of patients obtaining blastocysts, euploid blastocysts, LBs, with supernumerary blastocysts without a LB and with surplus blastocysts after an LB) were also reported versus the standard care (>168 h.p.i.). All hypothetical relative reductions were calculated. A total of 14.6% of the blastocysts reached full expansion beyond 144 h.p.i. (5.9% in the range 144-156 h.p.i., 7.9% in the range 156-168 h.p.i. and 0.8% beyond 168 h.p.i.). Slower blastocysts were of a worse quality based on the evaluation of both embryologists and AI. Both later tEB and longer time between tEB and full blastocyst expansion concurred to Day 7 development, quite independently of blastocyst quality. Slower growing blastocysts were slightly larger than faster-growing ones at the time of biopsy, but no difference was reported in the risk of hatching, mainly because two dedicated slots have been set along the working day for these procedures. The lower euploidy rate among Day 7 blastocysts is due to their worse morphology and more advanced oocyte age, rather than to a slower development per se. Conversely, the lower LBR was significant even after adjusting for confounders, with a first relevant decrease for blastocysts biopsied in the range 132-144 h.p.i. (N = 76/208, 36.5% versus N = 114/215, 53.0% in the control, multivariate odds ratio 0.61, 95% CI 0.40-0.92, adjusted-P = 0.02), and a second step for blastocysts biopsied in the range 156-168 h.p.i. (N = 3/21, 14.3%, multivariate odds ratio: 0.24, 95% CI 0.07-0.88, adjusted-P = 0.03). Nevertheless, when the cutoff was set at 144 h.p.i., no significant difference was reported. In this patient population, ending embryo culture at 144 h.p.i. would have caused 10.6%, 7.3%, 4.4%, 13.7% and 5.2% relative reductions in the number of patients obtaining blastocysts, euploid blastocysts, LBs, supernumerary blastocysts without an LB and surplus blastocysts after an LB, respectively. Gestational and perinatal outcomes were not assessed, and a cost-effectiveness analysis is missing. Moreover, we encourage other groups to investigate this topic with different culture and biopsy protocols, as well as in different clinical settings and regulatory contexts. In view of the increasing personalization and patient-centeredness of IVF, whenever allowed from the local regulations, the choice to culture embryos to Day 7 should be grounded on the careful evaluation of couples' reproductive history. Patients should be aware that Day 7 blastocysts are less competent than faster-growing ones; still, poor prognosis couples, couples less compliant toward other attempts in case of a failure and couples wishing for more than one child, may benefit from them. AI tools can help improving the generalizability of the evidence worldwide. This study did not receive any funding. I.E., A.B.M. and I.H.-V. are employees of Fairtility Ltd. N/A.

Cimadomo D ，Soscia D ，Casciani V ，Innocenti F ，Trio S ，Chiappetta V ，Albricci L ，Maggiulli R ，Erlich I ，Ben-Meir A ，Har-Vardi I ，Vaiarelli A ，Ubaldi FM ，Rienzi L ... -  《-》