Association between oocyte donors' or recipients' body mass index and clinical outcomes after first single blastocyst transfers-the uterus is the most affected.

OBJECTIVE: To assess whether high body mass index (BMI) in either oocyte donors or recipients is associated with poorer outcomes after the first single blastocyst transfer. DESIGN: Retrospective study including 1,394 first blastocyst single embryo transfers (SETs) conducted by 1,394 recipients during oocyte donation cycles with the gametes retrieved from 1,394 women (January 2019-July 2021). Four BMI clusters were defined for both donors and recipients (underweight: <18.5 kg; normal weight: 18.5-24.9 kg; overweight: 25-29.9 kg; and obese: ≥30 kg). SETTING: Network of private IVF centers. PATIENTS: A total of 1,394 recipients aged 42.4 ± 4.0 and with a BMI of 23.2 ± 3.8 kg/m2, and 1,394 donors aged 26.1 ± 4.2 and with a BMI of 21.9 ± 2.5 kg/m2. INTERVENTION: All oocytes were vitrified at 2 egg banks and warmed at 8 in vitro fertilization clinics that were part of the same network. Intracytoplasmic sperm injection, blastocyst culture, and either fresh or vitrified-warmed SETs were conducted. Putative confounders were investigated, and the data were adjusted through regression analyses. MAIN OUTCOME MEASURES: The primary outcome was the live birth rate (LBR) per SET according to donors' and/or recipients' BMI. The main secondary outcome was the miscarriage rate (<22 gestational weeks) per clinical pregnancy. RESULTS: The LBR per blastocyst SET showed no significant association with donors' BMI. Regarding recipients' BMI, instead, the multivariate odds ratio was significant in obese vs. normal-weight recipients (0.58, 95% confidence interval, 0.37-0.91). The miscarriage rate per clinical pregnancy was also significantly associated with recipients' obesity, with a multivariate odds ratio of 2.31 (95% confidence interval, 1.18-4.51) vs. normal-weight patients. A generalized additive model method was used to represent the relationship between predicted LBR or miscarriage rates and donors' or recipients' BMI; it pictured a scenario where the former outcome moderately but continuously decreases with increasing recipients' BMI to then sharply decline in the BMI range of 25-35 kg/m2. The miscarriage rate, instead, increases almost linearly with respect to both donors' and recipients' increasing BMI. CONCLUSION: Obesity mostly affects the uterus, especially because of higher miscarriage rates. Yet, poorer outcomes can be appreciated already with a BMI of 25 kg/m2 in both oocyte donors and recipients. Finer markers of nutritional homeostasis are therefore desirable; recipients should be counseled about poorer expected outcomes in cases of overweight and obesity; and oocyte banks should avoid assigning oocytes from overweight donors to overweight and obese recipients.

Discard or not discard, that is the question: an international survey across 117 embryologists on the clinical management of borderline quality blastocysts.

STUDY QUESTION: Do embryologists from different European countries agree on embryo disposition decisions ('use' or 'discard') about Day 7 (>144 h post-insemination) and/or low-quality blastocysts (LQB;

Cryostorage management of reproductive cells and tissues in ART: status, needs, opportunities and potential new challenges.

Among the wide range of procedures performed by clinical embryologists, the cryopreservation of reproductive cells and tissues represents a fundamental task in the daily routine. Indeed, cryopreservation procedures can be considered a subspecialty of medically assisted reproductive technology (ART), having the same relevance as sperm injection or embryo biopsy for preimplantation genetic testing. However, although a great deal of care has been devoted to optimizing cryopreservation protocols, the same energy has only recently been spent on developing and implementing strategies for the safe and reliable storage and transport of reproductive specimens. Herein, we have summarized the content of the available guidelines, the risks, the needs and the future perspectives regarding the management of cryopreservation biorepositories used in ART.

Humidified atmosphere in a time-lapse embryo culture system does not improve ongoing pregnancy rate: a retrospective propensity score model study derived from 496 first ICSI cycles.

PURPOSE: To investigate whether high relative humidity conditions (HC), when using a time-lapse system (TLS) with sequential culture media, are beneficial to embryo culture, improving ongoing pregnancy rates. METHODS: We included patients undergoing their first ICSI cycle treatment from April 2021 to May 2022. Patients assigned to dry conditions (DC) or HC were 278 and 218, respectively. We used a GERI TLS, three chambers configured in humidity conditions and three in dry conditions. The effect of HC on ongoing pregnancy rate was assessed by the propensity matched sample, to reduce potential differences between women undergoing either HC or DC and reduce biased estimation of treatment effect. RESULT: After adjusting for several confounding variables and applying the propensity score (PS), no significant differences were observed in the rates of normal (2PN) and abnormal (1PN and 3PN) fertilization, blastulation, top-quality blastocysts, frozen blastocysts, ongoing pregnancies, and miscarriages. The 2-cell (t2) and 4-cell (t4) stages and cell divisions between such stages occurred earlier and were more synchronous in the in DC. CONCLUSION: These results suggest that HC conditions do not improve the rate of ongoing pregnancy and several embryological outcomes, under the conditions used in this study based on a time-lapse system and sequential culture with day 3 medium change-over.

Towards Automation in IVF: Pre-Clinical Validation of a Deep Learning-Based Embryo Grading System during PGT-A Cycles.

Preimplantation genetic testing for aneuploidies (PGT-A) is arguably the most effective embryo selection strategy. Nevertheless, it requires greater workload, costs, and expertise. Therefore, a quest towards user-friendly, non-invasive strategies is ongoing. Although insufficient to replace PGT-A, embryo morphological evaluation is significantly associated with embryonic competence, but scarcely reproducible. Recently, artificial intelligence-powered analyses have been proposed to objectify and automate image evaluations. iDAScore v1.0 is a deep-learning model based on a 3D convolutional neural network trained on time-lapse videos from implanted and non-implanted blastocysts. It is a decision support system for ranking blastocysts without manual input. This retrospective, pre-clinical, external validation included 3604 blastocysts and 808 euploid transfers from 1232 cycles. All blastocysts were retrospectively assessed through the iDAScore v1.0; therefore, it did not influence embryologists' decision-making process. iDAScore v1.0 was significantly associated with embryo morphology and competence, although AUCs for euploidy and live-birth prediction were 0.60 and 0.66, respectively, which is rather comparable to embryologists' performance. Nevertheless, iDAScore v1.0 is objective and reproducible, while embryologists' evaluations are not. In a retrospective simulation, iDAScore v1.0 would have ranked euploid blastocysts as top quality in 63% of cases with one or more euploid and aneuploid blastocysts, and it would have questioned embryologists' ranking in 48% of cases with two or more euploid blastocysts and one or more live birth. Therefore, iDAScore v1.0 may objectify embryologists' evaluations, but randomized controlled trials are required to assess its clinical value.

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

STUDY QUESTION: What is the clinical value of Day 7 blastocysts? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE AND DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTEREST(S): This study did not receive any funding. I.E., A.B.M. and I.H.-V. are employees of Fairtility Ltd. TRIAL REGISTRATION NUMBER: N/A.

Inter-centre reliability in embryo grading across several IVF clinics is limited: implications for embryo selection.

RESEARCH QUESTION: What is the intra- and inter-centre reliability in embryo grading performed according to the Istanbul Consensus across several IVF clinics? DESIGN: Forty Day 3 embryos and 40 blastocysts were photographed on three focal planes. Senior and junior embryologists from 65 clinics were invited to grade them according to the Istanbul Consensus (Study Phase I). All participants then attended interactive training where a panel of experts graded the same embryos (Study Phase II). Finally, a second set of pictures was sent to both embryologists and experts for a blinded evaluation (Study Phase III). Intra-centre reliability was reported for Study Phase I as Cohen's kappa between senior and junior embryologists; inter-centre reliability was instead calculated between senior/junior embryologists and experts in Study Phase I versus III to outline improvements after training (i.e. upgrade of Cohen's kappa category according to Landis and Koch). RESULTS: Thirty-six embryologists from 18 centres participated (28% participation rate). The intra-centre reliability was (i) substantial (0.63) for blastomere symmetry (range -0.02 to 1.0), (ii) substantial (0.72) for fragmentation (range 0.29-1.0), (iii) substantial (0.66) for blastocyst expansion (range 0.19-1.0), (iv) moderate (0.59) for inner cell mass quality (range 0.07-0.92), (v) moderate (0.56) for trophectoderm quality (range 0.01-0.97). The inter-centre reliability showed an overall improvement from Study Phase I to III, from fair (0.21-0.4) to moderate (0.41-0.6) for all parameters under analysis, except for blastomere fragmentation among senior embryologists, which was already moderate before training. CONCLUSIONS: Intra-centre reliability was generally moderate/substantial, while inter-centre reliability was just fair. The interactive training improved it to moderate, hence this workflow was deemed helpful. The establishment of external quality assessment services (e.g. UK NEQAS) and the avant-garde of artificial intelligence might further improve the reliability of this key practice for embryo selection.

Clinical, obstetric and perinatal outcomes after vitrified-warmed euploid blastocyst transfer are independent of cryo-storage duration.

RESEARCH QUESTION: The study aimed to retrospectively evaluate the impact of cryo-storage duration on clinical, obstetric and perinatal outcomes after vitrified-warmed euploid blastocyst transfer. DESIGN: This was an observational study including 2688 vitrified-warmed euploid single blastocyst transfers that was conducted at a private IVF centre between May 2013 and March 2020. It included a total of 1884 women (age 38 ± 3 years) undergoing at least one transfer after preimplantation genetic testing for aneuploidies. The euploid blastocysts transferred were clustered into seven groups according to the cryo-storage duration between vitrification and warming: ≤60 days (n = 646; control group), 61-90 days (n = 599), 91-180 days (n = 679), 181-360 days (n = 405), 361-720 days (n = 144), 721-1080 days (n = 118) and >1080 days (n = 97). The primary outcome was the live birth rate (LBR) per transfer. The secondary outcomes were miscarriage rate, obstetric and perinatal issues. The data were adjusted for confounders through logistic or linear regressions. RESULTS: A significantly lower LBR was reported for transfers performed within 91-180 days (n = 291/679, 42.9%; P = 0.017), 181-360 days (n = 169/405, 41.7%; P = 0.016) and 361-720 days (n = 57/144, 39.6%; P = 0.034) versus ≤60 days (n = 319/646, 49.4%). However, this was mainly due to top-quality embryos being transferred first when more euploid blastocysts were available, thereby leaving lower quality ones for subsequent procedures. Indeed, the multivariate odds ratios adjusted for confounders showed similar results across all cryo-storage duration clusters. No difference was reported also for all secondary outcomes. CONCLUSIONS: Cryo-storage duration even beyond 3 years from blastocyst vitrification does not affect clinical, obstetric and perinatal outcomes.

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives.

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women, for both medical and non-medical indications. The main medical indications are oncologic diseases, gynecologic diseases such as severe endometriosis, systemic diseases compromising the ovarian reserve, and genetic conditions involving premature menopause. This paper describes the whole clinical and laboratory work-up of a fertility preservation treatment by outlining recommendations for objective and evidence-based counseling. Furthermore, it focuses on the effectiveness of the procedure and describes the most appropriate strategies to fully exploit the ovarian reserve and maximize the number of oocytes retrieved in the shortest possible time. The evaluation of the ovarian reserve, the definition of an ideal stimulation protocol, as well as oocyte retrieval, denudation, and vitrification procedures have been detailed along with approaches to maximize their efficacy, efficiency, and safety.

Maternal body mass index associates with blastocyst euploidy and live birth rates: the tip of an iceberg?

RESEARCH QUESTION: Does maternal preconceptional body mass index (BMI) associate with mean blastocyst euploidy rate (m-ER) per patient and live birth rate (LBR) after vitrified-warmed euploid single embryo transfer (SET)? DESIGN: Observational study conducted between April 2013 and March 2020 at a private IVF clinic, involving 1811 Caucasian women undergoing trophectoderm biopsy and comprehensive chromosome testing. The outcomes of 1125 first vitrified-warmed euploid SET were also analysed. Patients were clustered as normal weight (BMI 18.5-25; n = 1392 performing 859 SET), underweight (BMI <18.5; n = 160 performing 112 SET) and overweight (BMI >25; n = 259 performing 154 SET). m-ER per patient was the primary outcome. The secondary outcomes were all clinical outcomes per euploid SET. All data were adjusted for confounders through regression analyses. RESULTS: The m-ER per patient decreases as maternal BMI increases from 17 up to 22-23 before reaching a plateau. A linear regression adjusted for maternal age confirmed this moderate association (unstandardized coefficient B: -0.6%, 95% confidence interval [CI]: -1.1 to -0.1%, P = 0.02). All clinical outcomes were similar between normal weight and underweight women. Overweight women, instead, showed higher miscarriage rate per clinical pregnancy (n = 20/75, 26.7% versus n = 67/461, 14.5%; odds ratio [OR] adjusted for blastocyst quality and day of full blastulation: 2.0, 95% CI: 1.1-3.6, P = 0.01) and lower LBR per SET (n = 55/154, 35.7% versus n = 388/859, 45.2%; OR adjusted for blastocyst quality and day of full blastulation: 0.67, 95% CI: 0.46-0.96, P = 0.03). CONCLUSION: These data indicate a need for future research on more sensitive metrics to assess body fat mass and distribution, as well as on the mechanisms leading to lipotoxicity, thereby impairing embryo competence and/or endometrial receptivity. Overweight women should be informed of their higher risk for miscarriage and, whenever possible, encouraged to lose weight, especially before transfer.

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.

STUDY QUESTION: 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? SUMMARY ANSWER: 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). WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTEREST(S): No funding was received and there are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

The Impact of Unbalanced Maternal Nutritional Intakes on Oocyte Mitochondrial Activity: Implications for Reproductive Function.

Accumulating evidence on the effect of nutrition on reproduction is emerging from both animal and human studies. A healthy dietary pattern and nutrient supplementation, especially during the peri-conceptional period, might be helpful to achieve a live birth, although the mechanisms implicated are not fully understood. The endocrine system and the ooplasmic organelles apparatus, in particular the mitochondria, are clearly key elements during oogenesis and subsequent embryo development, and their proper functioning is associated with nutrition, even beyond maternal aging. Several studies in animal models have reported various adverse effects on mitochondria caused by unbalanced dietary intakes such as high fat diet, high fat high sugar diet, and low protein diet. The alterations produced might include mitochondrial intracellular distribution, content, structure, biogenesis, and functioning. This review summarizes the key role of mitochondria in female reproduction and the effects of different dietary macronutrient compositions on oocyte mitochondrial activity with their possible short-, medium-, and long-term effects.

Blastulation rates of sibling oocytes in two IVF culture media: an evidence-based workflow to implement newly commercialized products.

RESEARCH QUESTION: An evidence-based novel commercially available continuous IVF culture medium in compliance with an efficient quality-management system is proposed. DESIGN: Non-interventional study on sibling oocytes. Intracytoplasmic sperm injection cycles among women aged 42 years or younger that used ejaculated spermatozoa and retrieved four to eight oocytes were included. Sibling oocytes were randomized for culture in the novel Geri-medium or continuous single culture medium (CSCM). Primary outcome measure was blastulation rate per cohort of inseminated oocytes; 1182 oocytes were required to outline down to a 7% difference (power = 80%). RESULTS: A total of 181 cohorts of sibling oocytes were included. Geri-medium (n = 631 oocytes) and CSCM (n = 643 oocytes) resulted in similar blastulation rates (mean ± SD: 42.8% ± 30.1% versus 43.1% ± 29.0%; Wilcoxon signed rank test = 0.77). Blastocysts cultured in the former (n = 275 versus n = 277) showed longer timings during preimplantation development (P < 0.01) and were poorer quality (26% versus 18%; P = 0.03). Euploidy rate was no different in cycles that underwent preimplantation genetic testing for aneuploidy (n = 113) (117/237 [49%] versus 117/249 blastocysts [47%]; P = 0.6). Ongoing implantation rate was comparable in the study arms after euploid (29/47 [63%] versus 14/ 34 [41%]; P = 0.1) or untested (12/31 [39%] versus 7/18 [39%]; P = 0.3) transfers. CONCLUSION: Blastulation rate among cohorts of sibling oocytes cultured in the same incubator is a fast, reliable and comprehensive performance indicator to validate novel commercially available culture medium. The media tested were considered similarly efficient. The differences in blastocyst morphology and developmental timings warrant further investigation, although euploidy and ongoing implantation rates were similar.

Assessment and management of the risk of SARS-CoV-2 infection in an IVF laboratory.

RESEARCH QUESTION: The study set out to identify corrective measures aimed at reducing the risk of aerosol-mediated viral infection within an IVF laboratory. DESIGN: A failure modes and effect analysis (FMEA) was conducted by a multidisciplinary IVF team. A schematic representation of new protocols and procedures adopted during COVID-19 emergency has been defined, including directives about the behaviour to adopt when entering the clinic and the laboratory, in case of face-to-face contact with patients and between staff members. In addition, the risk of cross-contamination between samples belonging to different patients during cell handling and manipulation has been evaluated. Potential failure modes for each phase of the emergency have been analysed, focusing on possible sources of error. Risk priority numbers have been calculated as products of Occurrence × Severity × Detection scores. RESULTS: Except for cell-cell contamination, which was considered highly unlikely, failure modes during patient-staff, staff-staff and staff-cell interactions were estimated as carrrying a moderate to high risk of infection. The main corrective measures entailed precautionary logistic measures, the implementation of additional personal protective equipment and changes in the IVF laboratory procedures and scheduling of the daily routine. Some procedures were also revised, aiming to increase staff's awareness and caution. CONCLUSIONS: Standard laboratory protocols are insufficient to face a virus whose transmission is aerosol mediated. The measures outlined in this FMEA should thus be considered not only for facing this pandemic, but also for the future to promptly manage any aerosol-mediated virus infection, whose impact on the management of an IVF laboratory might be less severe than COVID-19 although not completely negligible.

Which key performance indicators are most effective in evaluating and managing an in vitro fertilization laboratory?

The laboratory is the heart of an in vitro fertilization (IVF) clinic, and a quality management system is critical for its administration. We review the main structural, process, and outcome key performance indicators (KPIs) to provide laboratory managers with concrete tools aimed at enhancing the quality of their work. Three concepts must be stressed when dealing with KPIs in IVF: [1] always consider the three types of indicators (structural, process, and outcome related), [2] carefully adapt the control chart to either promptly identify issues and adopt corrective measures, or redefine the control limits in a process called "progress building," [3] consider that achieving a healthy live birth is a multidisciplinary effort that is subject to several confounders, which must be recognized and accounted for in the analyses. In this regard, future KPIs shared among clinicians and embryologists are desirable to enhance the quality of infertility care for IVF patients.

Definition of a clinical strategy to enhance the efficacy, efficiency and safety of egg donation cycles with imported vitrified oocytes.

STUDY QUESTION: Which is the most suitable clinical strategy in egg donation IVF cycles conducted with imported donated vitrified oocytes? SUMMARY ANSWER: The importation, and allocation, of at least eight vitrified eggs per couple during an egg donation cycle is associated with a high cumulative live birth delivery rate per cycle, as well as the confident adoption of a single blastocyst transfer strategy to minimize the risk of multiple pregnancies. WHAT IS KNOWN ALREADY: IVF using donor eggs is commonly used worldwide to treat women who are unable to conceive with their own oocytes. In 2014, the Constitutional Court (n.162/2014) gave permission for gamete donation to be allowed for ART in Italy. Initially recommended as a therapeutic approach for premature ovarian insufficiency, the use of donated oocytes has become more and more common. In countries such as Italy, fresh oocyte donation is theoretically possible, but practically impossible due to the lack of donors. In fact, the Italian law does not allow reimbursement to the young women, who can only voluntarily donate their eggs. Therefore, Italian IVF centers have established several collaborations with international oocyte cryo-banks. The most popular workflow involves the importation of donated oocytes that have been vitrified. However, recent evidence has questioned the overall efficacy of such an approach. This is because detrimental effects arising from oocyte vitrification and warming might reduce the number of eggs available for insemination, with a consequential reduction in the achievable live birth rate per cycle. STUDY DESIGN, SIZE, DURATION: This was a longitudinal cohort study, conducted between October 2015 and December 2018 at two private IVF centers. Overall, 273 couples were treated (mean maternal age: 42.5 ± 3.5 years, range: 31-50 years; mean donor age: 25.7 ± 4.2, 20-35 years) with oocytes purchased from three different Spanish egg banks. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed an overall analysis, as well as several sub-analyses clustering the data according to the year of treatment (2015-2016, 2017 or 2018), the number of warmed (6, 7, 8 or 9) and surviving oocytes (≤4, 5, 6, 7, 8 or 9) and the cycle strategy adopted (cleavage stage embryo transfer and vitrification, cleavage stage embryo transfer and blastocyst vitrification, blastocyst stage embryo transfer and vitrification). This study aimed to create a workflow to maximize IVF efficacy, efficiency, and safety, during egg donation cycles with imported vitrified oocytes. The primary outcome was the cumulative live birth delivery rate among completed cycles (i.e. cycles where at least a delivery of a live birth was achieved, or no embryo was produced/left to transfer). All cycles, along with their embryological, obstetric and neonatal outcomes, were registered and inspected. MAIN RESULTS AND THE ROLE OF CHANCE: The survival rate after warming was 86 ± 16%. When 6, 7, 8 and 9 oocytes were warmed, 94, 100, 72 and 70% of cycles were completed, resulting in 35, 44, 69 and 59% cumulative live birth delivery rates per completed cycle, respectively. When ≤4, 5, 6, 7, 8 and 9 oocytes survived, 98, 94, 85, 84, 66 and 68% of cycles were completed, resulting in 16, 46, 50, 61, 76 and 60% cumulative live birth delivery rates per completed cycle, respectively. When correcting for donor age, and oocyte bank, in a multivariate logistic regression analysis, warming eight to nine oocytes resulted in an odds ratio (OR) of 2.5 (95% CI: 1.07-6.03, P = 0.03) for the cumulative live birth delivery rate per completed cycle with respect to six to seven oocytes. Similarly, when seven to nine oocytes survived warming, the OR was 2.7 (95% CI: 1.28-5.71, P < 0.01) with respect to ≤6 oocytes. When cleavage stage embryos were transferred, a single embryo transfer strategy was adopted in 17% of cases (N = 28/162); the live birth delivery rate per transfer was 26% (n = 43/162), but among the pregnancies to term, 28% involved twins (n = 12/43). Conversely, when blastocysts were transferred, a single embryo transfer strategy was adopted in 96% of cases (n = 224/234) with a 30% live birth delivery rate per transfer (N = 70/234), and the pregnancies to term were all singleton (n = 70/70). During the study period, 125 babies were born from 113 patients. When comparing the obstetric outcomes for the cleavage and blastocyst stage transfer strategies, the only significant difference was the prevalence of low birthweight: 34 versus 5%, respectively (P < 0.01). However, several significant differences were identified when comparing singleton with twin pregnancies; in fact, the latter resulted in a generally lower birthweight (mean ± SD: 3048 ± 566 g versus 2271 ± 247 g, P < 0.01), a significantly shorter gestation (38 ± 2 versus 36 ± 2 weeks, P < 0.01), solely Caesarean sections (72 versus 100%, P = 0.02), a higher prevalence of low birthweight (8 versus 86%, P < 0.01), small newborns for gestational age (24 versus 57%, P = 0.02) and preterm births (25 versus 86%, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: This retrospective study should now be confirmed across several IVF centers and with a greater sample size in order to improve the accuracy of the sub-analyses. WIDER IMPLICATIONS OF THE FINDINGS: Single blastocyst transfer is the most suitable approach to achieve high success rates per procedure, thereby also limiting the obstetric complications that arise from twin pregnancies in oocyte donation programs. In this regard, the larger the cohort of imported donated vitrified oocytes, the more efficient the management of each cycle. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: None.

The effect of ICSI-related procedural timings and operators on the outcome.

STUDY QUESTION: Do the ICSI-related procedural timings and operators affect the outcomes of an ART cycle? SUMMARY ANSWER: The ICSI-related timings and operators do not associate with the mean blastulation rate per cohort of inseminated oocytes and the cumulative delivery rate per concluded cycle, except for a mild association between the times from induction of ovulation to oocyte denudation and the former outcome. WHAT IS KNOWN ALREADY: In ART, specific timings, protocols and conditions must be complied with to preserve gamete developmental and reproductive competence during the required manipulations. ICSI represents a groundbreaking advancement that has been widely implemented. Nevertheless, the studies that examined the putative impact of ICSI-related procedural timings were mainly conducted in old-fashioned settings or in good prognosis patients. No report addressed issues like operators' skills and experience and uncertainties exist dealing with the effect of cumulus cells in the pre-incubation period in vitro before ICSI. However, all this information is crucial to efficiently plan the daily routine of an IVF lab, fill the existing gaps of knowledge and define proper key performance indicators. STUDY DESIGN, SIZE, DURATION: Observational study conducted at a private IVF clinic (January 2016 to January 2018). We included all consecutive ICSI procedures (n = 1084 infertile couples undergoing 1444 cycles with or without preimplantation genetic testing (PGT); mean ± SD maternal age: 38.1 ± 4.0 years) with fresh autologous oocytes (n = 7999 oocytes, 5.5 ± 3.2 per treatment) inseminated with fresh non-donor ejaculated sperm. All operators and critical procedural timings (induction of ovulation to oocyte denudation, denudation and ICSI) were automatically recorded through an electronic witnessing system. The primary outcome measure was the cumulative delivery rate among both non-PGT and PGT-concluded cycles (i.e. delivery achieved or no supernumerary cryopreserved blastocyst available). The secondary outcome measure was the mean blastulation rate per cohort of inseminated oocytes. All confounders were registered and included in generalized linear models and multivariate logistic regression analyses. PARTICIPANTS/MATERIALS, SETTING, METHODS: Fourteen and 12 operators were involved in denudation and ICSI procedures, respectively. Denudation was performed after 4.1 ± 1.2 h (2-7) of pre-incubation in vitro after oocyte retrieval, and ICSI was started immediately after. Beyond procedural timings and operators, all the putative confounders (patients' and cycles' characteristics) on the primary and/or secondary outcomes were systematically registered and included in the statistical analyses. MAIN RESULTS AND THE ROLE OF CHANCE: The mean time from induction of ovulation to oocyte denudation was 39.3 ± 1.3 h. The mean procedural timings for denudation and ICSI were 8.1 ± 3.8 and 12.6 ± 6.4 min; both these variables were significantly dependent on the number of inseminated oocytes and the operators' skills and experience. The overall mean blastulation rate per cohort of inseminated oocytes was 34.0 ± 27.9%. This outcome was significantly associated with the time from induction of ovulation to oocyte denudation (mean blastulation rate stable in the time interval 38-42 h, but significantly higher for timings <38 h), maternal age (the mean blastulation rate drops especially beyond the age of 40 years) and categorized sperm concentration (highest mean blastulation rate for sperm concentrations ≥15 mil/ml and lowest for cryptozoospermic patients) through a generalized linear model that showed an adjusted r2 = 0.053 (P < 0.01). No association was found for denudation and ICSI timings and operators. Lastly, when adjusted for maternal age and number of inseminated oocytes, both ICSI-related procedural timings and operators did not associate with the cumulative delivery rate among both non-PGT- or PGT-concluded cycles. LIMITATIONS, REASONS FOR CAUTION: This is a single private IVF center study. Its reproducibility should be assessed in different laboratory conditions, with different protocols and in the hands of different operators. Moreover, specific studies are warranted to address the beneficial/detrimental effect of the other putative confounders under investigation (e.g. kind of ovulation trigger, culture media, incubator, etc.). WIDER IMPLICATIONS OF THE FINDINGS: Proactive communication between the embryologists and the clinicians might contribute to a reasoned and more efficient organization of the daily workload and increase the mean blastulation rate, especially when poor prognosis couples (advanced maternal age, reduced sperm count and/or ovarian reserve) are treated. STUDY FUNDING/COMPETING INTEREST(S): No funding. The authors declare no conflict of interest related to the present study.

Human Blastocyst Biopsy and Vitrification.

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow entails a safe and efficient vitrification protocol, due to the turnaround time of the diagnostic techniques and to transfer the selected embryo(s) on a physiological endometrium in a following natural cycle. A biopsy approach encompassing the sequential opening of the zona pellucida and retrieval of 5-10 trophectoderm cells (ideally 7-8) limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions. After proper training, the technique was reproducible across different operators in terms of timing of biopsy (~8 min, ranging 3-22 min based on the number of embryos to biopsy per dish), conclusive diagnoses obtained (~97.5%) and live birth rates after vitrified-warmed euploid blastocyst transfer (>40%). The survival rate after biopsy, vitrification and warming was as high as 99.8%. The re-expansion rate at 1.5 h from warming was as high as 97%, largely dependent on the timing between biopsy and vitrification (ideally ≤30 min), blastocyst morphological quality and day of biopsy. In general, it is better to vitrify a collapsed blastocyst; therefore, in non-PGT cycles, laser-assisted artificial shrinkage might be performed to induce embryo collapse prior to cryopreservation. The most promising future perspective is the non-invasive analysis of the IVF culture media after blastocyst culture as a putative source of embryonic DNA. However, this potential avant-garde is still under investigation and a reliable protocol yet needs to be defined and validated.

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.

STUDY QUESTION: 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)? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: PQBs (