Objective To compare the effectiveness and cost of in-vitro fertilization (IVF) with or without preimplantation genetic testing for aneuploidy (PGT-A) when only one or two blastocysts are obtained. Methods A dataset was gathered from 1829 patients including 368 non-PGT-A and 1461 PGT-A cycles with one or two blastocysts obtained, between April 2013 and July 2022. Patients were matched 1:1 by propensity-score matching for maternal age, number of metaphase-II oocytes inseminated and number of blastocysts obtained, achieving a database of 242 patients per group. The non-PGT-A and PGT-A groups were compared for differences in live birth rate (LBR) per embryo transfer (ET), cumulative LBR per patient, miscarriage rate (MR) per clinical pregnancy, number of days between oocyte retrieval and conclusion of the IVF cycle (primary outcome), mean expenses incurred at the clinic and incremental cost-effectiveness ratio (ICER). Results More than twice as many ETs were conducted in the non-PGT-A group compared with the PGT-A group, yet the cumulative LBR per patient was similar between groups (23.6% (95% CI, 18.5-29.5%) vs 27.3% (95% CI, 21.9-33.4%)). This outcome was achieved with a higher LBR per ET (16.2% (95% CI, 12.6-20.5%) vs 41.5% (95% CI, 33.9-49.4%)) and lower MR per clinical pregnancy (30.1% (95% CI, 21.8-42.6%) vs 13.9% (95% CI, 7.5-24.0%)) in the PGT-A group. The MR per patient was also lower in the PGT-A group (9.5% (95% CI, 6.2-14.1%) vs 4.5% (95% CI, 2.4-8.2%)). The mean duration between oocyte retrieval and IVF cycle conclusion was 131 (95% CI, 113-150) days in the non-PGT-A group vs 74 (95% CI, 61-87) days in the PGT-A group (P < 0.001; power = 99.8%). The ICER of PGT-A for the months saved between oocyte retrieval and conclusion of the IVF cycle was 499 overall, ranging between 170 and 2065 according to the number of blastocysts obtained and/or maternal age. The ICER of PGT-A for prevented miscarriages was 18 968, decreasing to 3525 when calculated among patients aged >= 35 years with two blastocysts obtained. Conclusions When conducted in expert IVF clinics for patients indicated for the procedure, PGT-A is clinically valuable even when only one or two blastocysts are obtained. PGT-A reduces the number of ETs and miscarriages while the cumulative LBR per patient remains unaffected, and allows these outcomes to be achieved in a shorter timeframe. The ICER of PGT-A vs non PGT-A decreased as maternal age increased.

Abstract Study question Does body composition differ between fertile and infertile patients with history of repeated implantation failure (RIF)? A pilot study. Summary answer Compared to fertile, infertile women with history of RIF show differences in tissue distribution and bone mineralization (BM). What is known already It is known that being under/overweight or obese increases the risk of RIF or miscarriage probably due to the key role that adipose tissue exerts in reproduction. Body mass index (BMI) is the most used metric to define anthropometric characteristics; however, it represents a poor indicator of body composition leading to the risk of miscalculating the percentage of fat mass (FM) and underestimating the risk of reproductive failure. This study aims to analyze body composition of infertile patients by DXA, the gold standard for FM calculation and location, providing a 3-dimensional picture of body organ densities. Study design, size, duration This observational pilot study was conducted over 11 months. Sixty-six women were recruited and underwent all assessments. Participants/materials, setting, methods Out of 66 enrolled women, 16 were RIF (according to ESHRE 2023 criteria; here defined infertile women [IW] and aged 38.88±4.62 years), and 50 were women who already had at least one live birth (here defined fertile [FW] and aged 31.74±6.36 years), with varying weight ranges and BMI. The patients underwent an assessment of body composition by DXA Primus, X-ray densitometer; software v1.2.2, Osteosys Co., Ltd. Statistical analysis was conducted using IBM SPSS v21.0. Main results and the role of chance From FM percentage (FM%) analysis, 50% of the IW were normal-weight (NW - BMI 18.5-25, FM%<30), 31.25% were NW-obese (NWO - BMI 18.5-25, FM%≥30), and 18.75% were Obese (BMI≥25, FM%≥30). Of the FW, 48% were NW, 32% NWO, and 20% obese. Based on the Shapiro-Wilk normality test, independent samples t-test analysis or Wilcoxon’s nonparametric tests were adopted. Differences in body composition emerged between the IW and FW groups. IW showed a significant increase in right leg lean (7.38±1.03 vs 6.77±1.13, p < 0.05) and in left total tissue (28.02±5.3 vs 18.94±9.19, p < 0.0001) compared with FW. In contrast, FW showed a significant increase in left trunk tissue (22.85±8.33 vs 14.17±7.03, p < 0.0001), and in left and right trunk BM (367.6±90.47 vs 306.66±69.67, p < 0.005 and 368.35±94.49 vs 313.73±76.98, p < 0.012) versus IW. Limitations, reasons for caution Given the pilot nature of the study, its small sample size may influence the outcomes. Moreover, individual variations beyond dietary factors could contribute to the difference, including age. Additionally, our findings should be confirmed in a broader population. Wider implications of the findings The significant differences observed in IW compared to FW regarding body composition, especially concerning the tissue distribution and BM, encourage further investigations, particularly exploring possible hormonal, metabolic, and genetic correlations, to better understand the distinctive characteristics of this group. Trial registration number ClinicalTrials.gov NCT01890070.

Abstract Study question Is the Magenta-score associated with fresh donor oocytes’ blastulation competence, and can it be used to predict blastocyst rates per cohort of inseminated oocytes (BRpC)? Summary answer Developmentally-competent fresh donor oocytes showed significantly higher Magenta-score, although the number of blastocysts obtained was within the predicted range in only 63% of the cycles. What is known already Currently, many couples undergo donor oocyte treatments. However, despite donors being theoretically fertile, not all oocytes are developmentally competent, and many do not reach the blastocyst stage. Even if subjective and ineffective to predict oocyte competence, visual oocyte morphological assessment is still routinely used to evaluate oocytes. Lately, the rise of artificial intelligence (AI) resulted in promising tools to assess oocytes more objectively in a standardized method, possibly providing novel insights into the prediction of their competence. In this context, Magenta-score (Future Fertility), an AI-based image analysis tool, can potentially prove useful to optimize the management of oocyte donation treatments. Study design, size, duration A Blinded cohort study (June-2023/December-2023) was conducted to assess the primary outcome: association between fresh donor-oocytes’ Magenta-Score and their subsequent blastocyst development (assumed to be 50%). A sample size of 779 achieves 80%-power to detect the difference between the null-hypothesis point-biserial-correlation of 0 and the alternative hypothesis of 0.1 using a two-sided test with 5%-significance. Secondary outcomes were differences (i) predicted - true BRpCs, and (ii) predicted - true numbers of blastocysts obtained. Participants/materials, setting, methods Interim-analysis of 514 fresh-oocytes obtained from 63 donors at 3 centers and allocated (7.7±3, range:3-18) to 67 recipients. Pictures of denuded mature oocytes were acquired before ICSI. Magenta-scores were generated blindly (providing only donor age and total number of mature oocytes in cohort to the software). The software also estimated BRpCs and range of blastocysts obtainable from each cohort. Donors’ characteristics, ovarian stimulation/treatment cycle parameters, and sperm analyses were tested as confounders. Main results and the role of chance The only visual oocyte anomaly associated with lower Magenta-score was irregular shape (N = 484,5.4±2.2 vs N = 30,3.3±2.1, Mann-Whitney-U<0.01). Oocytes that developed into blastocysts (N = 194) had significantly higher Magenta-scores versus oocytes that did not reach this milestone (N = 320) (5.8±2.1 vs 4.9±2.3, Mann-Whitney-U<0.01; Odds-Ratio adjusted for sperm motility and incubator [standard/time-lapse]=1.26, 95%CI:1.13-1.34, p < 0.01; power=99.6%). No association was noted with day of blastulation or morphological quality. Data were concordant across centers. The average predicted-BRpC was 39.4±11.4% versus a true-BRpC of 40.6±22.6% (Person’s correlation: -0.24; mean difference: -1.1±27.6%, 95%CI from -7.8% to + 5.6%, p = 0.74). The average predicted number of blastocysts per oocyte cohort was 3.3±1.6 versus a true average of 2.9±1.7 (Person’s correlation: 0.25; mean difference: +0.4±2.0, 95%CI from -0.04 to + 0.9, p = 0.07). In 21% (N = 14) and 31% (N = 21) of cycles, the tool predicted an equal or lower number of blastocysts, respectively. In 5 cycles, no blastocyst was obtained, but the tool did not predict this unexpected outcome. When testing the predicted range of blastocysts obtained, the true number was within the range in 63% (N = 42) of cases and higher than the maximum predicted number in 13% (N = 9) of cases. No confounder was identified on the BRpC in this population of fresh donor cycles. Limitations, reasons for caution Of note, this is an interim analysis of an ongoing study. Not all mature oocytes recovered from a donor were utilized, thus not all oocytes in a cohort were imaged and included in analysis. Other putative confounders on BRpC should be considered in future studies with a larger sample size. Wider implications of the findings Effective management of an oocyte donation program is critical to comply with expected high success rates while minimizing the number of surplus blastocysts produced. The integration of genomic data, recipient couples’ characteristics, and the benefits of objective standardized AI-powered oocyte scoring is the most promising workflow to optimize this task. Trial registration number Not applicable

Abstract Study question When needed for PGT purposes, is a second biopsy and/or vitrification associated with lower reproductive outcomes? Summary answer Double biopsy and double vitrification (DBDV) is associated with lower live birth rate (LBR) per transfer (4 studies, OR 0.59, 95% CI 0.40-0.87, I2=11%, p = 0.008). What is known already PGT requires trophectoderm biopsy and vitrification to prevent the transfer of embryos affected from monogenic conditions and/or chromosomal defects. In 2-6% of biopsy procedures, the diagnosis might be inconclusive because of DNA amplification failure or non-concurrent results. In these cases, a round of warming, re-biopsy and re-vitrification is required. In other cases (i.e., the IVF center implements PGT and/or the patients develop an indication to this procedure), cryopreserved untested embryos might be warmed, submitted to a first biopsy and then re-vitrified. Study design, size, duration A systematic-search was conducted in PubMed/Scopus up to November-2023 to retrieve all publications focused on double biopsy and/or double vitrification in PGT cycles, including PGT-M, PGT-SR and PGT-A. We searched original works in peer-reviewed journals published in English. PRISMA guidelines were followed. PICO-model and ROBINS-I scoring for bias were adopted. The primary outcome was LBR per transfer. The main secondary outcome was clinical pregnancy rate (CPR) and miscarriage rate (MR) (<22 weeks) per clinical pregnancy. Participants/materials, setting, methods Statistical analysis was carried out using Review Manager 5.4. Categorical data were combined with a pooled odds-ratio (OR) with 95% confidence interval (CI). The random-effect model was used for meta-analysis. Between-study heterogeneity was addressed using I2. P-values <0.05 were considered significant. Main results and the role of chance Overall, we retrieved 3357 studies. Among them, 3322 were excluded because duplicates or not meeting the inclusion criteria. 35 were scrutinized by two authors independently. Discrepancies were solved by a third author. A total of 7 studies were included and considered for meta-analysis. Single Biopsy and Double Vitrification (SBDV) was associated with higher MR (5 studies, OR 1.65, 95% CI 1.13-2.42, I2 = 0%, p = 0.009) when compared with Single Biopsy and Single Vitrification (SBSV) group. CPR and LBR were instead comparable. DBDV was associated with lower CPR (5 studies, OR 0.60, 95% CI 0.45-0.80, I2 = 0%, p = 0.0004) and LBR (4 studies, OR 0.59, 95% CI 0.40-0.87, I2=11%, p = 0.008), and with higher MR (5 studies, OR 1.71, 95% CI 1.04-2.79, I2 = 0, p = 0.03) when compared with the SBSV group. Limitations, reasons for caution These associations issue from retrospective assessment based on a limited sample size, therefore the level of evidence is low/very-low. Of note, re-biopsied and/or re-vitrified euploid blastocysts are often transferred to poorer prognosis women who already failed with control embryos. Lastly, limited evidence exists on gestational/neonatal outcomes. Wider implications of the findings Improved genetic technologies and operators’ training are essential to minimize inconclusive diagnoses and the putative impact of additional embryo manipulations. Although poorer reproductive outcomes derive from re-biopsy and re-vitrification, they might still be worthwhile to avoid transferring affected/aneuploid blastocysts. Trial registration number PROSPERO: CRD42024503678

Abstract Study question Do blastocysts with a diverse history of compaction pattern (complete or partial) have different chances to be aneuploid and to implant? Summary answer Embryos undergoing partial or complete compaction have comparable chances to be aneuploid. Euploid blastocysts with previous history of blastomere extrusion have reduced chances of implantation. What is known already Compaction at the morula stage is an essential requisite for blastocyst formation and involves blastomere flattening and establishment of tenacious cell-to-cell contacts. Incomplete compaction is associated with several upstream and downstream morphokinetic anomalies. It also affects blastocyst yield and quality and clinical outcome. However, evidence on blastocyst chromosomal constitution and implantation potential after preimplantation genetic testing for aneuploidies (PGT-A) is lacking. Study design, size, duration This retrospective cohort study assessed laboratory and clinical outcomes of 1206 blastocysts derived from 483 infertile patients undergoing ART treatment and PGT-A from January 2015 to July 2022. Three age groups (≤34, 35–38 and ≥39 years) were adopted for sub-analyses. Biopsied blastocysts were vitrified and subsequently used in single vitrified-warmed blastocyst transfers (SVBT). Participants/materials, setting, methods Embryo development was monitored by time-lapse technology (TLT), also annotating abnormal cleavages and multinucleation. Patterns of morula compaction were assessed according to previously published categories: (i) full compaction, with all blastomeres undergoing compaction (FCM); partial compaction (partially compacted morula [PCM]), with (ii) blastomeres excluded from the outset (exc-PCM) or (iii) extruded after start of compaction (ext-PCM). Embryos were assessed in relation to embryonic and clinical outcomes. PGT-A data were obtained from trophectoderm biopsies. Main results and the role of chance Average maternal age of the overall patient population was 39.0 years. The full compaction pattern (FCM) was detected in 35.7% of all embryos. Partially compacted morulae showing excluded (Exc-PCM) or extruded (Ext-PCM) cells were also observed (54.4% and 9.9%, respectively) and collectively (64.3%) were the larger fraction (P < 0.0001, among all groups). Of 1206 analysed blastocysts, 551 were euploid, of which 366 were used in SVBT. Aneuploidy rate in the overall blastocyst population derived from FCM, Exc-PCM and Ext-PCM was 50.8%, 55.6%, and 60.0%, respectively (P > 0.05). However, in the younger maternal age group (≤34 years), the aneuploidy rate of the Exc-PCM group was higher compared with FCM (39.8% vs. 25.3%, P = 0.03). In the overall population, the Ext-PCM phenotype was associated with a lower implantation rate (FCM, Exc-PCM and Ext-PCM:47.8%, 44.7%, 55.6%, and 27,6%, respectively (P = 0.04), while miscarriage rates were comparable. Finally, transferred euploid blastocysts not classified according to maternal age implanted with comparable rates, irrespective of their morphokinetic history (abnormal division, multinucleation, no morphokinetic abnormalities). Limitations, reasons for caution The study is limited by its retrospective design. Having been obtained from a single centre, the data require independent validation Wider implications of the findings This study confirms and extends our previous findings on the implications of partial compaction. Importantly, it further consolidates the notion that the embryo can develop through atypical morphokinetic patterns while often preserving its genomic integrity and implantation ability. Trial registration number Not applicable

Abstract Study question Is PPOS rather than conventional GnRH-antagonist a valid strategy in DuoStim protocols? Summary answer PPOS-DuoStim and conventional-DuoStim showed similar mean euploid blastocyst rate (EBR) per inseminated metaphase-II (MII) oocytes. What is known already DuoStim (double stimulation in the same ovarian cycle) is an efficient strategy to increase the number of oocytes retrieved and embryos obtained in the shortest possible timeframe. PPOS is based on the use of exogenous oral progesterone to prevent LH surge during ovarian stimulation (OS). The effectiveness and safety of PPOS have been consistently reported across the last years, supporting it as a clinically-valuable OS protocol. To date, no evidence exists about PPOS application in DuoStim protocols and its association with oocyte competence, defined as EBR per MII-oocytes. Study design, size, duration Retrospective study involving 138 PPOS-DuoStim and 138 matched conventional-DuoStim patients treated at a single clinic between 2021 and 2022 and collecting ≥1 MII-oocyte after both stimulations. Matching was based on maternal age (40.3±2.7 years), number of oocytes collected after the I-stimulation (5.8±3.6 years), and sperm quality (66% normozoospermic). The primary outcome was the EBR per MII-oocytes. All intermediate embryological and clinical outcomes were also compared. Sub-analyses among I- and II-Stimulations were also conducted. Participants/materials, setting, methods DuoStim entailed the same protocol in I- and II-Stimulations with FSH 300IU/day+LH 150IU/day, 5 days spanning the two stimulations and GnRH-agonist to trigger ovulation. The only difference between PPOS-DuoStim and conventional-DuoStim was the administration of medrossiprogesterone acetate10mg/day orally from the first day of stimulation versus flexible GnRH-antagonist from the day the follicles reached a diameter of 13-14 mm. The same laboratory procedures were applied: ICSI, trophectoderm biopsy, comprehensive-chromosome-testing to report uniform aneuploidies and vitrified-warmed single-embryo-transfers. Main results and the role of chance The patients in the two groups were comparable for ovarian reserve markers, BMI, and duration of infertility. All data were comparable in conventional-DuoStim and PPOS-DuoStim (Mann-Whitney U tests were conducted): (i) overall fertilization rate per inseminated MII-oocytes were 73.1±23.2% versus 76.8±17.8% (p = 0.143), (ii) overall blastulation rate per 2PN-zygotes were 46.6±30.4% versus 45.7±25.1%(p = 0.797), (iii) overall euploidy rates per biopsied blastocysts were 24.1±30.9% versus 25.4±31.3%(p = 0.956), (iv) EBR per inseminated MII-oocytes were 9.6±17% versus 9.2±12.7% (p = 0.735). The data among only I- and II-stimulations were also similar. When comparing I- versus II-stimulations, paired t-tests highlighted longer II-stimulations (11±1.9 vs11.6±1.9 days, p = 0.001), resulting in larger cohorts of MII-oocytes (4.3±2.8 vs 5.2±3.3, p < 0.001) with comparable EBR per MII-oocytes (9.7±20.2% vs 11.6±16.2%, p = 0.233), thereby eliciting larger cohorts of euploid blastocysts (0.5±0.07 vs 0.7±0.09, p = 0.005). These data were confirmed also among conventional-DuoStim and PPOS-DuoStim study arms only. From a cycle perspective, among the 138 conventional-DuoStim couples, 31 (22%), 44 (32%) and 59 (43%) obtained ≥1 euploid blastocyst after I-stimulation, II-stimulation and overall, respectively. The same figures among the 138 PPOS-DuoStim were 36 (26%), 44 (32%) and 63 (46%), respectively. No statistical difference was reported (Fisher’s exact test=0.716). Limitations, reasons for caution Retrospective single center design. The current sample size should be doubled in order to reach a 80%-power with a 5%-α to exclude a 5% two-sided difference in the primary outcome. Patients’ compliance and discomfort, as well as clinical, gestational, and perinatal outcomes and cost-effectiveness analyses are needed. Wider implications of the findings PPOS is valuable in unconventional-OS protocols entailing freeze-all, like DuoStim with PGT-A. Future studies should assess if PPOS-DuoStim, by decreasing the number of injections and scans, may improve patient compliance and decrease OS discomfort. Any putative impact on follicular recruitment and oocyte yield should also be determined. Trial registration number not applicable

Abstract Study question Is there any association between PCOS phenotype-D and oocyte competence defined as euploid blastocyst rate (EBR) per cohort of inseminated oocytes? Summary answer Although higher fertilization and blastulation rates were reported in PCOS phenotype-D women versus idiopathic infertile patients, the EBR per cohort of inseminated oocytes was comparable. What is known already Several studies report poorer oocyte quality and cumulative-live-birth-rate (CLBR) in hyperandrogenic PCOS phenotypes, as defined according to the Rotterdam criteria. Increased androgen concentrations in follicular fluids are indeed associated with elevated serum-LH levels, which can block dominant follicle development inducing atresia and negatively impact fertilization rates and embryo development. Limited evidence exists in phenotype-D PCOS patients, namely women characterized by oligomenorrhea, ovarian PCO morphology and absence of hyperandrogenism. Here, we report the embryological and clinical outcomes in these women during ICSI cycles with preimplantation genetic testing for aneuploidies (PGT-A) at the blastocyst stage. Study design, size, duration Retrospective propensity score matched (PSM) case-control study. Among PGT-A cycles conducted by naïve patients with own oocytes (2013-2021), we excluded FNA/TESE. 58 phenotype-D PCOS patients were identified according to the Rotterdam criteria. These patients were matched for maternal age, number of cumulus-oocyte-complexes (COCs), and sperm quality, with 58 patients with idiopathic infertility, regular cycles, normal ovarian morphology, and no sign of clinical hyperandrogenism. The primary outcome was EBR per cohort of inseminated oocytes. Participants/materials, setting, methods GnRH-antagonist ovarian stimulation, ICSI, blastocyst biopsy, qPCR/NGS-analysis to assess uniform aneuploidies and vitrified-warmed single blastocyst transfers were conducted. The PCOS and control groups were similar for age (35.6±3.0 versus 36.0±3.9 years), COCs (23.1±6.7 versus 23.5±7.6), sperm factor, primary/secondary infertility, hormonal values, BMI (23.8±5.3 versus 22.0±2.8), and duration of infertility (3.6±2.2 versus 3.6±2.0 years). All intermediate embryological and clinical outcomes were assessed as secondary endpoints. Linear/logistic regressions adjusted for confounders were conducted to confirm statistically-significant differences. Main results and the role of chance No difference was reported in maturation rates, but women with PCOS showed better fertilization rates per cohort of inseminated oocytes (76.9±15.2% versus 67.3±20.4%, Mann-Whitney-U-test<0.01), and blastulation rates per cohort of 2PN-zygotes (52±20.4% versus 43.7±20.7%, Mann-Whitney-U-test=0.03). Eventually, PCOS patients obtained more blastocysts than controls (5.8±3.1 versus 4.5±2.9, Mann-Whitney-U-test=0.02), despite similar numbers of metaphase-II oocytes in the groups (16.5±6.1 versus 16.5±7.0). All differences remained significant after adjusting for maternal age and sperm factor. Nevertheless, the euploidy rates per cohort of biopsied blastocysts were similar, thus involving comparable EBR per cohort of inseminated oocytes (21.7±14.1% versus 17.2±16%, Mann-Whitney-U-test=0.12). More patients obtained ≥1 euploid blastocyst in the PCOS versus the control group (N = 56/58, 96% versus N = 47/58, 81%, Fisher’s-exact-test=0.02), independently from maternal age, semen quality, and number of inseminated oocytes (Odds-Ratio: 6.5, 95%CI:1.3-33.7, p = 0.03). Lastly, all clinical outcomes per first vitrified-warmed euploid transfer were similar, thereby involving comparable LBRs (N = 28/54, 51.9% versus N = 26/47, 55.3% in the PCOS and control groups, p = 0.8; Odds-Ratio adjusted for blastocyst quality and day: 1.19, 95%CI:0.5-2.7, p = 0.68). Limitations, reasons for caution Although clinical hyperandrogenism was absent in both groups, biochemical hyperandrogenism was not assessed. Nonetheless, this pilot study paves the way for future more detailed investigations. 24% of cycles in the PCOS group were not concluded, therefore a CLBR comparison would be biased and was not reported. Wider implications of the findings The lower developmental competence in the control group suggests oocyte quality issues, other than chromosomal, underlying their “unexplained infertility” condition. In phenotype-D PCOS women, IVF is effective. Since >50% of this subgroup of women had already failed multiple ovulation-induction and/or intrauterine-insemination attempts, an early referral to IVF deserves further investigations. Trial registration number Not applicable

Is blastocyst expansion-speed, examined through Artificial-Intelligence (AI) between time of blastulation (tB) and time of expanding blastocyst (tEB), associated with embryo blastulation and reproductive competence? Blastocyst expansion-speed-assay (ESA) was significantly associated with euploidy and live-birth rates, being discordant with clinical embryologists’ ranking in 50% of embryonic cohorts. Blastocyst expansion is one of the earliest morphogenetic events in mammalian development. It is essential for the establishment of the blastocyst layout and requires trophectoderm integrity. Several studies suggested that expansion is a valuable feature for ranking and prioritizing blastocysts for transfer based on their developmental competence. Time-lapse technology (TLT) implementation in IVF allows a deeper understanding of blastocyst expansion dynamics. In this study, we combined TLT and AI to develop a blastocyst-ESA and investigate its association with embryo blastulation and reproductive competence. Retrospective study including 2184 blastocysts cultured in EmbryoScope incubators during 786 PGT-A cycles across 2013-2020. Videos were analyzed through an AI-powered tool (CHLOETM, Fairtility). The expansion-speed was calculated as [(Δ embryo proper area at tEB – embryo proper area at tB) / (Δ tEB - tB)]. ESA was tested for its association with embryo quality, euploidy and live-birth rate (LBR) in 548 vitrified-warmed single euploid transfers. A simulation of ESA putative clinical effectiveness was conducted. ICSI, trophectoderm biopsy of fully-expanded blastocysts without day3 zona-drilling, and qPCR/NGS to assess full-chromosome uniform aneuploidies were performed. tB and tEB, expressed as hours-post-insemination (hpi), embryo proper area (in µm2) at both these stages, and blastocyst quality (EQ-Score from 0 to 1) were all automatically recorded through CHLOE. Possible confounders (e.g., maternal age, male factor, and cause of infertility) were considered. Regression analyses were conducted to adjust the data. The average ESA was 705±458 µm2/hour. Higher ESA was associated with higher EQ-Score. Euploid blastocysts showed higher ESA than aneuploid (761±465 µm2/hour versus 667±449 µm2/hour; p < 0.01). ESA was also associated with LBR per euploid blastocyst transfer (LB: 873±438 µm2/hour versus 736±467 µm2/hour; p < 0.01). Of note, maternal age showed no association with ESA. Multivariate regressions outlined a + 5.1%-increase in the chance of euploidy and a + 6.3%-increase in chance of LB, every +100 µm2/hour-increase in ESA. ESA and embryologists were discordant in grading top-quality embryos in each cohort in 50% of the cases. In 59% of the 352 cohorts where both euploid and aneuploid blastocysts were obtained, ESA would have ranked the former embryos as top-quality. In the 216 cycles with ≥2 euploid blastocysts obtained and ≥1 transferred, ESA would have prioritized (i) a competent embryo in 46% of the cases, (ii) an incompetent embryo in 20%, but (iii) in 33% its value could not be assessed (i.e., the top embryo according to ESA has not been transferred yet). When compared to the embryologists, ESA would have been (i) equally-effective in 49% of the cases, (ii) more effective in 12-24%, and (iii) less effective in 5%-26%. Retrospective single-center study. Both continuous and sequential media were used, although they were not associated with the outcomes under investigation. To assess the clinical value of ESA, a prospective study among first transfers is warranted. ESA is an automated, unbiased, and easily-applicable measurement that certainly deserves further appraisal. If validated in prospective studies, AI-based selection algorithms could include this assessment to increase their predictive power. None

BACKGROUND AND AIM In the context of medically assisted reproduction (MAR), care providers are faced with a myriad of stressors from organizational challenges (e.g., intense time pressures and workload) to external factors such as patient unrealistic expectations. Occupational stress should be kept under control to minimize a progressive worsening of professional well-being, job dissatisfaction, increased anxiety, and behavioral disorders. Also, the quality of patient care (efficacy and safety) might suffer from the consequences of occupational stress. A few national studies investigated the occupational demands of clinical embryologists, suggesting a high risk of burnout syndrome. Our study aimed to explore the topic of occupational well-being by investigating the experiences of embryologists working at Italian MAR centers. METHODS Qualitative interview study of 18 Italian embryologists working at both public and private centers. Participants were recruited among SIERR (Italian Society of Embryology, Reproduction, and Research) members. The interviews were recorded and transcribed. Thematic analysis was utilized to identify the main themes and sub-themes. RESULTS The interviewees were concordant that excessive workload might affect their psychophysical well-being, especially in view of a salary perceived as unsatisfactory with respect to the high responsibility and training involved by this job. In general, only public sector senior embryologists reported a manageable workload, proportionate to their salary. A key issue identified was the shortage of qualified embryologists, sometimes replaced with less specialized personnel. This might worsen a work environment often perceived challenging because of complex intra-team dynamics, management of interpersonal relationships, and unclear definition of the roles. A competitive and isolating atmosphere can slow down professional growth and limit positive networking. Of note, the participants emphasized the importance of patient counselling for their professional well-being, advocating for a more intense interaction with the couples. CONCLUSIONS Although being limited by a small sample size, this study suggests that embryologists’ workload, compensation, and professional well-being themes should be carefully addressed at a national and international level. In fact, this is crucial for high-quality patient care as a greater recognition of their role might positively influence MAR performance as well.

Abstract Study question How to develop a robust strategy to pinpoint women exhibiting potential oocyte maturity defects, making them candidates for in-depth genetic investigations and rescue-IVM interventions? Summary answer Oocyte immaturity rate warning-limit was 51%. Within three retrievals, 7.8%, 1.5%, and 0.3% of the patients might exceed it once, twice and three times, respectively. What is known already Oocyte maturity involves cytoplasmic and nuclear aspects. While cytoplasmic maturity is challenging to define and monitor in IVF, nuclear maturation is evaluated through germinal vesicle breakdown and first polar body extrusion. Consensus suggests 10-20% immaturity rates post-OS is acceptable, but some patients exceed. From a diagnostic standpoint, women surpassing these rates may be eligible for additional genetic assessments; from a clinical standpoint, instead, exploring rescue-IVM is promising. However, guidelines for both practices are currently lacking. Study design, size, duration Retrospective study including retrievals with ≥1 cumulus-oocyte-complex (COC; years:2008-2022). The weighted-mean immaturity-rate (20%) and the outliers for COCs retrieved (>22) were defined on the whole dataset (N = 16155). Confounders upon immaturity-rates were outlined among first retrievals with ≥5 COCs (N = 7963). Warning immaturity-rate limit was calculated as “weighted-average+2SD” among first retrievals with 5-22 COCs (N = 7523). Conservative and estimated prevalence of facing immaturity-rates higher than the warning limit across multiple retrievals were also calculated. Participants/materials, setting, methods All retrievals were conducted 35.5±0.5 hours after trigger and cumulus-cells were removed to assess oocyte maturity 3.7±1.2 hours later. Rescue-IVM was not conducted at our center, and immature oocytes were discarded. OS, time between pick-up and denudation, and maternal characteristics were tested as putative confounders through linear regressions. Main results and the role of chance Among all retrievals conducted between 2008 and 2022, the weighted-mean oocyte immaturity-rate was 20%, suggesting that normally ≥1 immature oocyte(s) might be obtained with ≥5 COCs. Use of agonist trigger versus hCG (unstandardized coefficient-B: -2.1%,95%CI from -2.8% to -1.4%,adjusted-p<0.001), length of OS (unstandardized coefficient-B:-0.6%,95%CI from -0.8% to -0.4%,adjusted-p<0.001), and ratio COCs:follicles >16 mm at ovulation induction (unstandardized coefficient-B:+4.6%, 95%CI from +3.8% to + 5.3%,adjusted-p<0.001) were associated with the immaturity-rates in a multivariate linear regression. Among first retrievals with ≥5 and ≤22 COCs, the immaturity-rates warning limit was defined as 51%. In 3.6% (N = 286) of the 7962 first retrievals with ≥5 COCs the immaturity-rates were ≥51%. The same prevalence for second and third retrievals were 3.8% (N = 86/2232) and 2.1% (N = 14/667). The conservative prevalence of patients with immaturity-rates ≥51% once, twice and three times among three consecutive retrievals were 4.5% (N = 361/7962), 0.3% (N = 23/7962), and 0.03% (N = 2/7962). The same rates, assuming all patients would conduct three consecutive retrievals, were 7.8% (N = +257), 1.5% ( = +93), and 0.3% (N = +21). Out of the 667 patients that indeed conducted 3 retrievals between 2008-2022, these rates were 7.6% (N = 51), 0.9% (N = 6) and 0.4% (N = 3), supporting the reliability of the predicted estimates. Limitations, reasons for caution Both germinal-vesicles and metaphase-I immature oocytes were not deemed clinically useful here, although this is controversial. Well-designed studies to systematically monitor rescued oocytes’ developmental, chromosomal, and reproductive competence are still needed, especially in patients subject to high immaturity-rates. Wider implications of the findings Oocyte immaturity-rates ≥51% with ≥5 COCs especially when consistently experienced across multiple retrievals, is a statistically-sound criterion to candidate patients to whole-exome-screening. This aims at outlining gene associations with defective oocyte maturation. The clinical effectiveness of rescue-IVM should be primarily studied in these women (prevalence ≤1%). Trial registration number None

Background Artificial Intelligence entails the application of computer algorithms to the huge and heterogeneous amount of morphodynamic data produced by Time-Lapse Technology. In this context, Machine Learning (ML) methods were developed in order to assist embryologists with automatized and objective predictive models able to standardize human embryo assessment. In this study, we aimed at developing a novel ML-based strategy to identify relevant patterns associated with the prediction of blastocyst development stage on day 5. Methods We retrospectively analysed the morphokinetics of 575 embryos obtained from 80 women who underwent IVF at our Unit. Embryo morphokinetics was registered using the Geri plus® time-lapse system. Overall, 30 clinical, morphological and morphokinetic variables related to women and embryos were recorded and combined. Some embryos reached the expanded blastocyst stage on day 5 (BL Group, n = 210), some others did not (nBL Group, n = 365). Results The novel EmbryoMLSelection framework was developed following four-steps: Feature Selection, Rules Extraction, Rules Selection and Rules Evaluation. Six rules composed by a combination of 8 variables were finally selected, and provided a predictive power described by an AUC of 0.84 and an accuracy of 81%. Conclusions We provided herein a new feature-signature able to identify with an high performance embryos with the best developmental competence to reach the expanded blastocyst stage on day 5. Clear and clinically relevant cut-offs were identified for each considered variable, providing an objective tool for early embryo developmental assessment.

Abstract Study question Are Artificial Intelligence (AI)-powered tools more efficient than traditional non-invasive assessments to prioritize euploid blastocysts for transfer? Summary answer Only one third of cycles yielded ≥3 blastocysts with different diagnoses. In these cycles, AI and traditional grading prioritized euploid embryos with comparable performance. What is known already While embryo morphology and developmental pace to blastocyst are associated with chromosomal and reproductive competence, their evaluation remains subjective and lacks reproducibility. The introduction of Time-lapse technology (TLT) in IVF has provided valuable insights into preimplantation development but has not enhanced embryologists’ reproducibility. Recently, AI models integrated with TLT offered the potential for automating and standardizing assessments. Whole-chromosome testing for uniform aneuploidies still is the strongest predictor of embryo (in)competence. In fact, if transferred, >98% of aneuploid blastocysts fail to result in a live-birth. AI tools strive to non-invasively predict blastocyst (an)euploidy, yet their clinical utility requires intra-cohort testing. Study design, size, duration Retrospective blinded analysis of 786 PGT-A cycles (maternal-age:38.9; years:2013-2020; 2184 blastocysts). For static assessment, 3 embryologists used Gardner’s grading at the time of biopsy (t-biopsy). For morphodynamic assessment, time of blastocyst-expansion (tEB) and embryo-area (embA at tEB) were also annotated. For AI assessment, scores were generated with three commercially-available models. All approaches were compared for their effectiveness in ranking euploid blastocyst(s) as top-quality among cohorts with ≥3 blastocysts and ≥1 euploid and ≥1 aneuploid (N = 279/786,35.5%). Participants/materials, setting, methods Embryologists ranked AA-blastocysts as top-quality (score=1), CC-blastocysts as worst-quality (score=9), prioritizing trophectoderm over ICM, with t-biopsy as third criterion. Morphodynamic assessments followed the same hierarchy but using tEB and embA in place of t-biopsy. For AI, higher scores corresponded to higher priority. For all assessments, coefficients of variation (CV=SD/mean) were calculated to assess data dispersion within cohorts with ≥3 blastocysts (N = 363/786,46.2%). Intra-cohort CVs were tested for their association with the likelihood of prioritizing euploid blastocysts. Main results and the role of chance Intra-cohort CVs were 0.71±0.35, 0.28±0.14, 0.58±0.26, and 0.56±0.34 for Gardner’s score, AI model-1, -2, and -3, respectively. Spearman’s correlations between Gardner’s score and AI model-1, -2, and -3 Intra-cohort CVs were 0.32, 0.34, and 0.14, respectively. AI model-1 and -2 were strongly correlated (0.63), but poorly with model-3 (0.21 and 0.22). Euploid blastocysts were prioritized in 163 cohorts by static assessments (N = 163/279,58.4%), 179 (64.2%) by morphodynamic, 174 (62.4%), 190 (68.1%), and 145 (52%) by AI model-1, -2, and -3. A maternal age-adjusted 0.1-larger Gardner’s grading intra-cohort CV correlated with higher likelihood of prioritizing euploid over aneuploid blastocysts (multivariate-OR:1.1, 95%CI:1.03-1.19, adjusted-p=0.005). No association was reported with AI models intra-cohort CVs. Notably, blastocysts affected from aneuploidies compatible with implantation were prioritized over euploid in 59 cohorts (N = 59/279,21.1%) by static assessment, 54 (19.4%) by morphodynamic, 52 (18.6%), 48 (17.2%), and 54 (19.4%) by AI model-1, -2, and -3. A maternal age-adjusted 0.1-larger Gardner’s grading intra-cohort CV correlated with lower likelihood of prioritizing aneuploid blastocysts compatible with implantation over euploid (multivariate-OR:0.9, 95%CI:0.83-0.98, adjusted-p=0.010). No association was reported with AI models intra-cohort CVs. Limitations, reasons for caution While representing the real-life scenario of a large PGT-A program, this study is a single center retrospective analysis in advanced maternal age women with a limited number of cycles with ≥3 blastocysts showing both euploid and aneuploid. Other AI-powered models exist, some specifically trained to predict euploidy. Wider implications of the findings Trophectoderm biopsy and testing aim to prevent the transfer of chromosomally-abnormal blastocysts. Although traditional/AI-based grading correlate with euploidy, their goal is prioritizing embryos for transfer, irrespective of the IVF setting (with or without PGT-A). Future research should concentrate on emphasizing live-birth prediction over misleadingly considering them as non-invasive PGT-A surrogates. Trial registration number None

Abstract Study question Is ejaculatory abstinence (EA) associated with fertilization and blastocyst development in ICSI cycles with embryo culture in time-lapse incubators? Summary answer There is no association between length of EA and fertilization, blastocyst development or morphology. What is known already Semen analysis is pivotal in medically assisted reproduction (MAR), serving as a significant indicator of male partners’ health. The period of EA is a relevant factor that can affect sperm quality. Numerous studies described EA association with clinical outcomes, while its impact on embryological results remains inconclusive. Recent meta-analyses, despite a conflicting literature, highlighted positive effects of shorter abstinence periods. A recent study suggested that prolonged abstinence might reduce fertilization rate in ICSI cycles without time-lapse imaging, thereby reducing usable zygotes’ rate Study design, size, duration Retrospective study including all oocytes (N = 7164) from 1148 ICSI cycles (N = 902 couples) cultured in time-lapse incubators at a single IVF center between January 2020 and December 2022. Only non-donor fresh gametes were included (Mean Maternal age: 38.5±4 years; Paternal Age: 41±5.6 years). The primary embryological outcomes were fertilization rate and type, and blastocyst development per inseminated oocyte and per 2PN-zygote. Secondary outcomes were euploidy, day of blastocyst development and Gardner’s morphology. Participants/materials, setting, methods Semen analysis was conducted according to WHO. All oocytes were cultured up to day7 in EmbryoScope or GERI incubators with continuous media and refresh in day5 (if needed). If requested, PGT-A was performed to assess full-chromosome uniform aneuploidies. Univariate and multivariate logistic regressions were conducted. Possible confounders were assessed among ovarian stimulation, maternal, paternal, sperm, and IVF cycles characteristics. Associations were confirmed with Generalized estimating equations (GEE) to account for repeated measurements among oocytes’ cohort. Main results and the role of chance Logistic regressions and GEE were conducted to assess associations between possible confounders and blastocyst development per inseminated metaphase-II oocyte. Maternal age, sperm concentration, and motility A+B were associated. The same confounders were reported on blastocyst development among 2PN-zygotes. The days of EA (1-8 days) did not show any association with fertilization (p = 0.198) or blastocyst development per metaphase-II oocyte (p = 0.495). No association was reported with abnormal fertilization, neither combining all abnormalities in a single group, nor clustering them as 1PN, 3PN, 3.1PN, and multi-PN. An increasing trend was reported in blastocyst development among 2PN-zygotes according to the length of EA (1-day: 17/37,45.9%; 2-days: 190/424,44.8%; 3-days: 1405/2574,54.6%; 4-day: 746/1410,52.9%; 5-days: 450/827,54.4%; 6-days: 70/133,52.6%; 7-days: 46/101,45.5%; 8-days: 85/162,52.5%), but this was not significant in a multivariate logistic regression (p = 0.887). Length of EA showed no association with euploidy (p = 0.424), nor with day of development (p = 0.966) and Gardner’s inner cell mass (p = 0.204) or trophectoderm (p = 0.428) morphology. Limitations, reasons for caution This study is inherently limited by its retrospective design. The limited number of cycles included was balanced using time-lapse incubators to improve the accuracy of fertilization check and benefit from an undisturbed embryo culture environment. Artificial Intelligence will be implemented to automate developmental timings’ annotation and objectify embryo quality assessment. Wider implications of the findings The length of EA seems not associated with embryological outcomes, adding to the controversy on this topic. Here, we included only ICSI cycles with blastocyst culture conducted in time-lapse to enhance the quality of the evidence. Larger datasets and diverse settings are now required, especially enriched with poor prognosis patients. Trial registration number not applicable

Abstract Study question To what extent do clinical and laboratory strategies affect the main measures of IVF success? Summary answer Across 10 years, the implementation and increasing adoption of antagonist protocols combined with agonist-trigger, freeze-all, blastocyst culture, PGT, and multicycle counseling approach improved IVF outcomes. What is known already The primary indicator of IVF success is the cumulative-live-birth-rate per intention-to-treat (CLBR per ITT). Since reproductive competence is inherent to every gamete and embryo, IVF treatments cannot guarantee success. However, they can maximize the likelihood of success with the safest approaches available. Furthermore, there are other outcomes to consider for a more thorough assessment of IVF effectiveness and efficiency, including time in treatment (=LB achieved / no LB achieved and no embryo available for transfer; TiT), miscarriage rate, and prevalence of multiple-births. Moreover, a family planning perspective should be strengthened in IVF aiming at ≥ 2 healthy children, whenever feasible. Study design, size, duration Longitudinal observation of 6610 couples undergoing ≤6 ovarian-stimulations (OS) for IVF at a private clinic within 3 years. Couples were clustered in 11 groups according to the year of their first treatment (years: 2010-2020) and compared for (i) CLBR within 3 years, (ii) prevalence of ≥ 1 miscarriage and ≥1 twin-delivery, (iii) TiT (from first-OS to the last IVF procedure before treatment conclusion), (iv) prevalence of ≥ 2 singleton-deliveries within 6 years. Participants/materials, setting, methods The naïve patients included increased from ≈500 in 2010 to ≥ 600 between 2013 and 2020. The maternal age was stable around 38±4 years with ≈20% prevalence of severe male factor. OS was conducted through long, short flare-up, mild, antagonist, progestin-primed, or DuoStim protocols with hCG/agonist-trigger. All patients underwent ICSI on fresh own eggs and cleavage/blastocyst culture, fresh transfer/freeze-all, untested/euploid transfers, single/multiple-embryo transfers. Main results and the role of chance Antagonist-protocols (≈20% in 2010 and ≈80% in 2020) combined with agonist-trigger (0.4% and 76.8%) superseded long/short flare-up (≈60% and <10%) and hCG-trigger (99.6% and 23.2%). DuoStim, introduced in 2015, reached 15%-prevalence by 2020. The MII-oocytes collected at first-retrievals increased from 5±3 (2010) to 7.2±4.9 (2020). The prevalence of first-cycles without embryos raised from 3% to 14% by 2020 primarily due to an evident cleavage-to-blastocyst culture switch (2010:96% versus 4%; 2020:1% versus 99%). Similarly, PGT-A and freeze-all utilization respectively increased from 3% to 80% and from 12% to 93%. Although TiT increased from ≈2 cycles in ≈140 days (2010-2013) to ≈1.6 in ≈160 days (2014-2020), the 3-years CLBR improved from 32% (2010) to 42% (2020), a difference confirmed also adjusting for maternal age and overall number of MII-oocytes collected (multivariate-OR:1.03,95%CI:1.01-1.05;adjusted-p=0.001). This was paralleled by the halving of women suffering ≥1 miscarriage (≈12% to < 6%;p=0.002) and drop in women with ≥1 twin-delivery (7.5% to 0.5%;p<0.001). The latter achievement was due to single-embryo transfer rates increasing from 26% (2010) to 98% (2020). Accordingly, the women starting in 2017 and obtaining ≥2 LBs within 6-years were 9.9%, 98% of which after consecutive singleton-deliveries; these figures were 6.6% and just 22% among women starting in 2010(p = 0.004). Limitations, reasons for caution This study suffers from the inherent limitations of a retrospective design. Temporal changes, other than clinical strategies and laboratory advancements (e.g., personnel), might have influenced the results, although no critical infrastructural changes were implemented. Gestational and neonatal follow-up data are missing in this study. Wider implications of the findings Clinical and laboratory advancements enhanced IVF efficacy and efficiency over time also fulfilling family planning desire. Future strategies and improved workflows should focus on reducing drop-out and time-to-LB. As larger oocyte cohorts led to higher CLBR throughout maternal age, tailored-multicycle approach might represent a valuable option to this end. Trial registration number none

Abstract Study question Impact on clinical outcomes of day-6 extended culture and reduced culture media volume applied to non-invasive PGT-A Summary answer The implementation of changes in culture conditions to accommodate niPGT-A has no impact on blastocysts viability or pregnancy outcomes. What is known already The recent identification of embryo cell-free DNA (cfDNA) in the culture media has created the possibility of a non-invasive approach for aneuploidy testing in blastocysts. Concerns have however arisen at two levels. Firstly, the concordance rates that cfDNA results have with the full blastocyst or a trophectoderm (TE) biopsy and secondly, the impact on clinical outcomes of the changes in the embryo culture protocol that are required to implement niPGT-A in the IVF laboratory. Concordance rates of niPGT-A to invasive PGT-A have gradually improved, however the impact of culture protocol changes is not as well understood. Study design, size, duration Six IVF clinics participating in a trial examining concordance rates for invasive and non-invasive PGT-A on day-6/7 blastocysts were involved (Rubio et al., 2020). Patients were recruited from April 2018 to December 2020. In the study group, 428 PGT-A cases following the niPGT-A protocol for embryo culture were included. The control group consisted of 1,392 regular PGT-A cases that did not participate in the study and underwent the standard PGT-A culture protocol. Participants/materials, setting, methods In the control group, embryos were biopsied and vitrified on day 5, 6 or 7. In the study group, niPGT-A culture conditions started on day 4, when embryos were washed and cultured individually in 10μl of fresh media. On day 6/7 blastocysts were biopsied, vitrified and media collected for niPGT-A analysis. Clinic A cultured all embryos up to day 6/7, whereas in clinics B-F a minority of day-5 blastocysts were biopsied and transferred. Main results and the role of chance Statistical comparisons were performed between the study and the control groups. Mean female age, number of oocytes, fertilization rates and number of blastocysts biopsied were not significantly different for the study and the control group. Overall, the niPGT-A protocol did not have any detrimental effect on blastocyst quality, euploidy rates or informativity rates. Deferred single embryo transfers (SET) were performed in both groups. Regarding the overall pregnancy outcomes, no significant effect was observed on clinical pregnancy rate, miscarriage rate or ongoing pregnancy rate (OPR, ≥12 weeks) in the study group compared to the control group when stratified by day of biopsy. Clinic A showed no difference in OPR per transfer regardless of culture protocol (62.3% in control group day 5/6/7; 60.8% in control group day 6/7; and 65.6% in study group day 6/7). For the remaining clinics (B-F), a comparison was made separately between day-5 and day-6 blastocysts that were transferred and cultured according to the standard and to the niPGT-A protocol. No significant differences in OPR were observed, neither between control group day 5 (60.6%) and study group day 5 (58.7%), nor between control group day 6 (44.7%) and study group day 6 (41.9%). Limitations, reasons for caution The limitations are intrinsic to the retrospective nature of the study, and also to the fact that the study was conducted in invasive PGT-A patients and not in niPGT-A cases alone. Wider implications of the findings This study shows that changing current IVF laboratory practice to adopt niPGT-A protocols has no impact on the number of blastocysts available for transfer and overall clinical outcomes. Whether removal of the actual invasive biopsy step leads to further improvements in pregnancy rates awaits further studies. Trial registration number ClinicalTrials.gov (NCT03520933)