IVF Center Research Articles

P-757 Clinical and neonatal outcome of single vitrified-warmed blastocyst transfer with complete zona pellucida removal using laser-assisted hatching

Abstract Study question Complete zona pellucida (ZP) removal using laser assisted hatching can improve clinical and neonatal outcome in single vitrified-warmed blastocyst transfer (SVBT) cycle? Summary answer Implantation rate, clinical pregnancy rate and live birth rate can be improved by complete removal of zona pellucida, whereas no impact on neonatal outcomes. What is known already Assisted hatching (AH) technique widely used in assisted reproductive technology for improving clinical outcome in frozen embryo transfer (FET), which could impair successful embryo hatching out of the zona pellucida (ZP) due to ZP hardening during vitrification-warming. The zona thinning and drilling methods of AH doesn’t affect clinical, live births outcomes and it could increase monozygotic twin birth, risk of placenta associated diseases. But recent studies showed that the complete removal of ZP may increase the chance of blastocyst adhesion and outgrowth afterwards, which required to be proven. Study design, size, duration Retrospective study data collected from July 2015 to November 2019 included SVBT in the Ojinmed IVF center. We divided into 2 groups as follows: case group 160 (randomly selected complete removal of ZP) and control group 160. The clinical outcomes were implantation rate (IR), clinical pregnancy rate (CPR), live birth rate (LBR), miscarriage and neonatal outcomes including monozygotic twin, birth baby sex, birth weight, height and gestational weeks. All parameters were compared between two groups. Participants/materials, setting, methods 320 vitrified blastocyst transfer cycles in 215 patients underwent a clomiphene-based minimal ovarian stimulation protocol or drug-free natural cycles. All embryos were vitrified-warmed by Cryotop method (Kitazato, Japan). Complete removal of ZP selected an survived blastocysts after warming and used an immediate laser drilling (Laser: 1.8-2.2 ms, Octax NaviLaseTM) in perivitelline space. And gently glass pipetting done for complete removal of ZP. Statistical analysis performed by Chi-square test and logistic regression analysis using STATA 12. Main results and the role of chance A total 320 SVBT cycles, including ZP completely removed 160 cycles and control 160 cycles. In analysis of the patient's age, fertility causes and fertilization methods were not significant (p < 0.05) between two groups. The IR (55.2 % and 66.2 %, p < 0.01), CPR (39.3 % and 52.5 %, p = 0.01) and LBR (29.3 % and 43.7 %, p < 0.01) are highly correlated between the control and the completely removal of ZP group, respectively. In multivariable logistic regression analysis showed the completely removal of ZP group had significance for IR (aOR 0.54, CI 95 % 0.31-0.93, p = 0.02), CPR (aOR 0.56, CI 95 % 0.32-0.96, p = 0.03) and LBR (aOR 0.56, CI 95 % 0.32-0.99, p = 0.048), which adjusted by patient age, blastocyst grade and vitrification day. Miscarriage rate didn’t show significance (23.8% and 21.4%, p = 0.73) between groups. The neonatal outcomes including monozygotic twin, sex of the births, average gestation week and birthweight were non significant (p < 0.05) between two groups (49 for control group and 66 for case group) children. Limitations, reasons for caution The result of the current retrospective study is limited to data from a single IVF center. Wider implications of the findings Our study suggests that complete removal of ZP using laser assisted can improve clinical outcomes including implantation, clinical pregnancy and live birth after SVBT. And it may doesn’t affect to miscarriage rate and neonatal outcome. Trial registration number not applicable

P-676 Comparison of hCG triggering versus hCG in combination with a GnRH agonist: a prospective randomized controlled trial

Abstract Study question Does co-administration of GnRH agonist and Human chorionic gonadotropin(dual trigger) in PGT cycles improve the number of usable blastocysts per patient compared to hCG alone? Summary answer Using the dual trigger has no effect on the number of usable blastocysts undergoing the GnRH-ant protocol for PGT compared to triggering with hCG alone. What is known already HCG is used at the end of controlled ovarian hyperstimulation as a surrogate LH surge to induce final oocyte maturation. Recently, based on retrospective studies, the co-administration of GnRH agonist and hCG for final oocyte maturation (dual trigger) has been suggested to improve IVF outcome and pregnancy rates. Study design, size, duration A prospective, randomized, open-label controlled clinical trial (ChiCTR-ICR-2000031342), enrolled patients attending our university affiliated Infertility and IVF center (the Reproductive and Genetic Hospital of CITIC-Xiangya, China) between August 2020 and June 2021. This sample size achieves with a power of 80%, at a significance level(α) of 0.05. A sample size of 160 patients, 80 randomized to each group, was chosen to allow for those 10% dropped out. Participants/materials, setting, methods The inclusion criteria for participating were: women age 20–35years, AMH≥1.2ng/ml and /or AFC ≥5, Couples with PGT-SR preimplantation genetic testing for chromosomal structural rearrangements (patients with abnormal chromosomal structure only in male or female), undergoing one of their first PGT cycle attempts. Patients fulfilled the criteria on the trigger day were randomly assigned to receive hCG or the dual trigger for final oocyte maturation. The primary outcome was the number of usable blastocysts per patient. Main results and the role of chance 160 patients were included in the study. The age (29.1 years versus 29.4 years), BMI (22.1 kg/m2 versus 21.9 kg/m2) and the AMH (5.3 ng/ml versus 5.3 ng/ml) were comparable between the two groups. Based on PP analysis, there were no statistical difference in the number of eggs retrieved (17.1 versus 15.9), the MII oocytes (13.3 versus 12.5), the number of usable blastocysts per patient (5.0 versus 4.6) and top-quality blastocysts per patient (3.4 versus 3.2) between the two groups. Limitations, reasons for caution None Wider implications of the findings The enhanced response observed with the dual trigger might lead to better IVF outcomes were it used more widely. Trial registration number ChiCTR-ICR-2000031342

P-645 Intra-ovarian injection of autologous platelet-rich plasma (PRP) in poor prognosis IVF patients within 1 year of treatment: a cohort study

Abstract Study question Does intraovarian injection of platelet-rich plasma (PRP) change parameters of ovarian function in patients with evidence of diminished ovarian reserve? Summary answer Over one year of follow-up there was no evidence of a positive effect of PRP treatment on ovarian function. What is known already Several authors have previously reported positive outcomes and improved response in ovarian function after treatment with PRP. However, thus far, no randomized controlled trials and few cohort studies have been published. Study design, size, duration This is a longitudinal cohort study following ovarian response among 80 patients with low functional ovarian reserve (LFOR) defined by AMH < 1.1 ng/mL, FSH > 12 mIU/mL or at least one prior IVF cycle with ≤ 3 oocytes for one year before and one year after intraovarian treatment with PRP. Participants/materials, setting, methods 1.5 cc of PRP was injected into the cortex of ovaries in an average 12 injections per ovary. Beginning one month after their PRP treatment, patients underwent one or more cycles of ovulation induction for IVF. Outcome measures were endocrine response, and numbers of oocytes and embryos produced in by patients in response to a maximal gonadotropin stimulation before and after PRP treatment. Main results and the role of chance In this study women between ages 28 and 54 failed to demonstrate statistically significant outcome benefits from intra-ovarian PRP. Two very poor-prognosis 40-year-old patients with prior failed IVF cycles at other IVF centers that never reached embryo transfer, however, achieved pregnancy in an IVF cycle following PRP treatment for an ongoing pregnancy rate of 4.7% if a patient following PRP achieved retrieval of at least 1 oocyte with IVF. Limitations, reasons for caution This is an observational study of patients who performed poorly in past ovulation induction cycles, thus any improvement observed could have been accounted for by regression to the mean. The occurrence of two pregnancies in this group could be secondary to eventual success following a string of previous failures. Wider implications of the findings Even extremely poor prognosis will occasionally achieve a successful outcome from IVF. Uncontrolled case series cannot provide sufficient evidence to support routine use of an intervention like PRP. Future studies demand the use of well- controlled randomized trials. Trial registration number NCT04275700

P-442 A new option to thaw slow-frozen human ovarian tissue in cancer patients: efficacy and safety of the combination of different cryopreservation kits

Abstract Study question Is follicular viability of slow-frozen human ovarian tissue preserved if rapid thawing is performed using a solution containing extracellular cryoprotectant only? Summary answer Follicular viability is preserved even using thawing solutions containing extracellular cryoprotectant only, combining kits with different composition. What is known already Ovarian tissue cryopreservation is an alternative option to oocytes or embryos for fertility preservation in cancer patients facing gonadotoxic treatments. To date, each brand producing kits approved for the slow-freezing of human ovarian tissue recommends the use of its own thawing kit. However, a potential single protocol based on the use of any extracellular cryoprotectant has already been proposed for human oocytes and embryos. The current study aims at finding alternative options to thaw cryopreserved human ovarian tissue when the original kit was withdrawn from the market and only one CE-marked kit was available, even with different composition in cryoprotectants. Study design, size, duration Ovarian tissue cryopreservation of ten cancer patients (18.3 ± 7.6 years) undergoing fertility preservation between 2001 and 2012 was performed following a slow-freezing protocol with 1.5M 1,2 PROH and 0.5M Sucrose. Once deceased, for each patient, cortical fragments were prospectively thawed and equally allocated into two groups: i) fragments thawed using 0.5-1M 1,2 PROH and 0.3M Sucrose (PROH+S Group, n = 73); ii) fragments thawed following an adjusted protocol with 0.5M Sucrose only (S Group, n = 73). Participants/materials, setting, methods Post thawing follicular density/mm2, integrity (%) and the presence of interstitial oedema were assessed by histological and ultrastructural analysis performed after formalin fixation and haematoxylin/eosin staining. Follicular viability was evaluated by the expression of markers of proliferation (Ki67) and of vascularization (CD31) by immunohistochemistry after a 24h culture in Iscove’s modified Dulbecco’s medium at 37 °C and 6% CO2. A paired comparison was performed referring to the fresh tissue of the same patient as control. Main results and the role of chance The histological evaluation performed after thawing revealed that follicles were predominantly primordial (91%), with no follicles larger than the proliferating primary stage. A significant reduction of follicular density per mm2 was observed in both study groups (14.2 ± 12.0 vs. 15.1 ± 14.0 for PROH+S and S Group, respectively; p = 0.4) compared to the fresh tissue (27.2 ± 31.6; p = 0.04) as well as a remarkable decreased of the proportion of intact follicles (39.3 ± 17.1 vs. 25.5 ± 9.8; p = 0.2) compared to the fresh tissue (98.1 ± 1.4; p = 0.002). Thawed samples equally showed interstitial oedema and increased stromal cell vacuolization and chromatin clumping. Ki67 positive staining of active proliferating cells revealed a comparable proportion of viable follicles between thawed samples (46.3 ± 20.8 vs. 28.3 ± 27.9 for PROH+S and S Group, respectively; p = 0.2). Finally, the expression of the endothelial marker CD31 in the thawed samples suggested an equivalent number of blood vessels per mm2 (43.8 ± 34.3 vs. 41.7 ± 44.8; p = 0.6). Limitations, reasons for caution Single centre study with a limited sample size. Only 24h of in vitro culture was assessed. The use of the freezing medium corresponding to the Sucrose only solution was not tested. Clinical outcomes after ovarian tissue transplantation should be evaluated before drawing final conclusion. Wider implications of the findings First evidence of the feasible application of a “Universal Warming” protocol, irrespective of brand and cryoprotectants, for the rapid thawing of slow-frozen human ovarian tissue. IVF centres would be provided with alternative options to thaw ovarian tissue for restoring reproductive potential in cancer patient undergoing ovarian transplantation. Trial registration number Not applicable

P-679 Comparison of hormone replacement treatment (HRT) and true-natural cycle (t-NC) protocols for endometrial priming: An analysis of 1,815 warmed blastocyst transfer cycles

Abstract Study question Does the ongoing pregnancy rate (OPR) of HRT with or without GnRH-agonist suppression and t-NC protocols differ in patients undergoing warmed blastocyst transfer? Summary answer HRT, with or without GnRH-agonist suppression, and t-NC protocols are associated with comparable OPRs in patients undergoing warmed blastocyst transfer. What is known already Despite the worldwide increase in frozen embryo transfer cycles, the most optimal protocol for priming of the endometrium is debated. Although HRT offers flexibility, recent evidence points tot-NC being superior to HRT regarding safety, i.e., maternal, obstetric, and neonatal outcomes.However, there are still conflicting data regarding pre-clinical losses and reproductive outcomes when comparing the two protocols. Study design, size, duration In this longitudinal prospective study, 1,815 consecutive patients undergoing 1,815 warmed blastocyst transfer cycles at the Anatolia IVF Centre, Ankara, between 2015-2021, were included. HRT with pituitary suppression was the protocol of choice during 2015- 2017, whereas HRT without suppression and t-NC were more commonly employed during the latter part of the period. Participants/materials, setting, methods All patients with an available day-5/6 vitrified blastocyst(s) were included. Each patient was included only once. The three protocols were t-NC and HRT - with or without suppression. The prerequisites for t-NC was being a local patient with regular menstrual cycles. For t-NC, neither human chorionic gonadotropin (hCG) nor luteal phase support was administered. The primary outcome measure was OPR, defined as pregnancy >12 weeks of gestation. Main results and the role of chance Of the 1,815 cycles,124 were t-NC, 477 were HRT with suppression, and 1,214 were HRT without suppression. For the stimulated cycles leading to FET, no difference was seen among the three groups regarding female age, body mass index, duration of infertility, number of previous embryo transfer attempts, ovarian stimulation protocol, estradiol levels on the day of hCG trigger, number of oocytes retrieved, number of preimplantation genetic testing-aneuploidy, freeze-all cycles and number of embryos transferred. The positive pregnancy test rates of the HRT protocol with or without suppression were higher when compared with that of t-NC (63.7%, 66.6%, and 58.1%,respectively; p = 0.05). The respective figure for clinical pregnancy rates were 56.6%, 60.8% and 55.6% (p = 0.07). However, the pre-clinical (biochemical) loss rates (11.9%, 10.9%, and 4.9%, respectively; p = 0.05), as well as the miscarriage rates (11.9%, 10.9%, and 4.9%, respectively; p = 0.04), were higher in the HRT groups with or without suppression compared to those of t-NC. The OPRs of t-NC, HRT with or without suppression were comparable (53.2%, 45.1%, and 49.0%, respectively; p = 0.73). The protocol for endometrial priming was not an independent predictor of ongoing pregnancy at logistic regression analysis when potential confounders were used as covariates (OR = 0.998; 95%CI 0.669-1.490, p = 0.99). Limitations, reasons for caution The longitudinal study design and the lack of obstetric and perinatal outcome data are limitations. Wider implications of the findings Compared with t-NC, the HRT protocol with/without suppression is associated with higher positive pregnancy test rates albeit increased pre-clinical and clinical loss rates, resulting incomparable OPRs. When compared with t-NC, the HRT protocol could be associated with enhanced endometrial receptivity at the expense of decreased selectivity. Trial registration number not applicable

P-513 Quality of life assessment of women undergoing in vitro fertilization treatment in Kazakhstan

Abstract Study question What is the quality of life of Kazakhstani women with infertility and what additional factors can affect it? Summary answer The FertiQoL questionnaire showed the low quality of life of Kazakhstani women and a significant affect some factors. What is known already Studies showed that infertility problems are among the saddest events in people's lives. It is reported that women undergoing IVF procedures are experiencing various types of psychological burdens like stress, anxiety and depression. Study design, size, duration This cross-sectional study involved women of reproductive age undergoing an IVF cycle at a large IVF center in Kazakhstan in the period from September 1, 2020 to September 31, 2021. Participants/materials, setting, methods The survey was carried out using the Google Form program and was sent to patients of large IVF clinic of Kazakhstan by the attending physician for an online survey. Out of 500 women who were given questionnaires, 453 women completely filled out. Questionnaire results data were entered into SPSS version 26.0 for aggregation and statistical analysis. Two-sample Student's t-test or one-way analysis of variance (ANOVA) was used to test for differences between groups. Main results and the role of chance Total FertiQoL was 59.6±11.3. 292 women with primary infertility and 161 with secondary infertility. According to the main indicators as Total FertiQoL and Core FertiQoL, the results of the scales were significantly higher (p < 0.001). The source of income for infertility treatment affects the level of Treatment FertiQoL. Cronbach's α coefficients of the FertiQoL subscale averaged 0.8. Cronbach's α coefficients of the FertiQoL subscale averaged 0.8. We examined the satisfaction scores by FertiQoL, dividing them into two groups according to the presence of children (Table 3). FertiQoL scores were statistically significantly higher in the group with children than in the group with primary infertility in all areas (p < 0.05), except for Relation, Tolerability (p = 0.65, p = 0.59). Limitations, reasons for caution This study cross-sectional and was conducted in the countries of the Commonwealth of Independent States (CIS) for the first time. We want to continue research in cooperation with other CIS countries. Wider implications of the findings The duration of infertility, age and low economic status negatively affect the quality of life of women. The availability of subsidies from the state for infertility treatment improves the quality of life. The health expenditure percentage of GDP may affect the overall level of satisfaction of women. Trial registration number IRB-A108

O-121 Uncovering the value of day 7 blastocysts using artificial intelligence on time lapse videos

Abstract Study question Which is the clinical value of day 7 blastocysts? Summary answer Ending embryo culture at 144 hours-post-insemination (hpi) would involve7.3%- and 4.4%-relative reductions in the 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 at ironing 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, 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 Trophectoderm biopsy without hatching and comprehensive-chromosome-testing were performed. Blastocysts were clustered in six groups based on the time-of-biopsy every 12hr from <120hpi (control) to > 168hpi. Blastocyst quality, time-of-expanding-blastocyst (tEB) and duration of expansion were annotated through AI and confirmed manually. The main outcomes were euploidy-rate and LB-rate (LBR) per transfer. Lastly, patients obtaining (euploid) blastocysts, LBs, and supernumerary blastocysts, were reported based on a hypothetical 144hpi cut-off, and all relative reductions calculated. Main results and the role of chance 14.6% of the blastocysts reached full expansionbeyond 144hpi (5.9% between 144-156hpi, 7.9% between 156-168hpi, and 0.8% >168hpi). Slower blastocysts were of a worse quality based on the evaluation of both embryologists and AI. Both longer tEB and a longer duration of expansion concurred to day7 development, quite independently of embryo quality. The lower euploidy rate among day7 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 132-144hpi (N = 76/208, 36.5% versus N = 114/215, 53.0% in the control, multivariate-OR: 0.61, 95%CI 0.40-0.92, adjusted-p=0.02), and a second step for blastocysts biopsied in the range 156-168hpi (N = 3/21, 14.3%, multivariate-OR:0.24, 95%CI 0.07-0.88, adjusted-p=0.03). Nevertheless, when the cut-off was set at 144hpi, no significant difference was reported. In this patient population, ending embryo culture at 144hpi would have caused 10.6%-, 7.3%-, 4.4%-, 13.7%-, and 5.2%-relative reductions in the number ofpatients obtaining blastocysts, euploid blastocysts, LBs, supernumerary blastocysts without a LB and after a LB, respectively. Limitations, reasons for caution Gestational and perinatal outcomes were not assessed, and a cost-effectiveness analysis was not performed. We encourage the production of these data in other clinical settings and regulatory contexts. Wider implications of the findings Day7 culture shall be supported following a careful case-by-case evaluation. Patients shall be aware of their lower competence, yet day7 blastocysts are valuable for poor-prognosis couples, couples less compliant towards other attempts in case of failures, and couples wishing for second children. AI may improve the generalizability of these evidence. Trial registration number Not applicable

P-228 Higher oxidation levels in follicular fluid correlate with better outcome in ICSI treatments

Abstract Study question Can the oxidative status of the follicular fluid be a valid biomarker of the quality of the oocyte cohort and clinical outcome in ICSI treatments? Summary answer The follicular fluid (FF) of oocyte cohorts that achieved at least one pregnancy, ongoing pregnancy and life birth have higher oxidative stress levels. What is known already The role of oxidative stress in female infertility is yet unclear, due to the numerous factors that participate in the production and activity of reactive oxygen species (ROS), and the different techniques used for its detection. The Fertissimo TCL AnalyzerTM (Carmel Diagnostics, Israel) uses thermochemiluminiscence to assess the oxidative status (OS) of biological samples by counting the photons emitted per second (cps) after a heat-catalyzed oxidative reaction. Using this technique, a negative correlation was found between ROS levels in FF and patient age (Wiener-Megnazi et al, 2004), but its potential as a biomarker of clinical outcome is unclear. Study design, size, duration Prospective observational study performed in a single IVF center during one year. 173 women were subjected to controlled ovarian stimulation as part of an autologous (103 patients) or ovum donation ICSI treatment (70 donors). The OS of the follicular fluid (FF) obtained in the oocyte retrieval was assessed and compared with the characteristics of the treatment and clinical outcome. Participants/materials, setting, methods The first tube of follicular fluid obtained in the oocyte retrieval was centrifuged (10 minutes at 600g) and the supernatant was analyzed in the Fertissimo TCL AnalyzerTM. OS results were normalized per volume and summarized as TCL-Score (average photon counting from 50-280s) and Ratio (slope of the reaction time curve). TCL parameters were compared with the demographics, number of oocytes retrieved and clinical outcome of the cohort using Pearsons Correlation and Anova tests Main results and the role of chance FF from donors had higher TCL-Score (44.27±14.41cps vs 40.59±11.65cps, P = 0.066) and Ratio (367.38±89.47 vs 329.88±78.12, P = 0.004) than patients. A significant inverse correlation was found between TCL Ratio and the age of the women (Pearsons Correlation Coefficient (PCC)=-0.207; P = 0.006). A direct significant correlation was found between the body mass index (BMI) and the TCL-Score (PCC=0.186, P = 0.014) and Ratio (PCC=0.186, P = 0.014). TCL-Score (PCC=0.173, P = 0.023) and Ratio (PCC= 0.275, P <0.001) also had a significant positive correlation with the number of mature oocytes retrieved. In donors, TCL Ratio was negatively correlated with the fertilization rate (PCC=-0.350, P = 0.009) and positively correlated with the blastocyst formation rate at day 5 (PCC=0.289, P = 0.032), but said correlation was not found in FF of patients (PCC(Ratio-Fertilization rate)=0.066, P = 0.554; PCC(Ratio- Blastocyst rate)=-0.005, P = 0.965). However, in patients, a statistically significant negative correlation was found between TCL-Score and the good quality blastocyst rate (PCC=-0.234, P = 0.038), which was not statistically significant in donors (PCC=-0.131, P = 0.342). Finally, TCL Ratio levels resulted significantly higher in FF of oocyte cohorts that resulted in at least one pregnancy (362.77±86.60 vs 321.90±77.60, P = 0.005), one ongoing pregnancy (365.99±84.10 vs 322.66±80.94, P = 0.003) and one life birth (364.93±83.74 vs 330.07±84.17, P = 0.017). Limitations, reasons for caution Some miscalibration issues were met in the course of the analysis, which may hinder its reproducibility. Stimulation protocol or sperm quality was not considered in the analysis. A larger scale, controlled trial should be performed to validate results. Wider implications of the findings Presented results are in tune with previously published data using similar and different techniques for assessing oxidative and antioxidative capacity in FF samples. The analysis of the oxidative status of the FF could be a valid biomarker of clinical outcome of ICSI treatments and as indicator for antioxidant therapy. Trial registration number APOTIP/2020/015 Generalitat Valenciana.

P-282 Blastocyst transfer in advanced maternal age: Single versus double embryo transfer

Abstract Study question Is there a role for elective single blastocyst transfer (eSBT) in advanced maternal age? Summary answer Double blastocyst transfer (DBT) versus eSBT resulted in higher live birth rates except for those undergoing their first cycle. What is known already Women with advanced maternal age are generally considered poorer prognosis patients with lower live birth rates compared to their younger counterparts. This often results in a higher likelihood of double embryo transfer in this group. This is not without risk as multiple pregnancy is associated with significant maternal and neonatal morbidity especially with advancing maternal age. In older women with blastocysts available for transfer, it is unclear who should be recommended double versus elective single transfer. The concept of the “good-prognosis” older woman remains elusive. Study design, size, duration This was a retrospective observational study looking at 511 IVF/ICSI cycles between January 2010 and January 2020. Treatment cycle details and clinical outcomes were entered prospectively into a dedicated clinic database. Data was retrieved and analysed using SPSS V25. Participants/materials, setting, methods The study was conducted in a large London IVF centre. Data was collected on women aged 40 or above undergoing an IVF/ICSI cycle with eSBT (Group 1, n = 79) or DBT (Group 2, n = 430). Women with more than three previous IVF attempts were excluded. eSBT was defined as a single blastocyst transfer with at least one further blastocyst available for cryopreservation. Subgroup analysis was performed for those undergoing their first cycle. Main results and the role of chance Data from 511 cycles was analysed. The mean age was 40.6±1.2years. The live birth rate was 27%. Group 1 was marginally younger (40.2±0.6 v 40.8±1.2, p < 0.005) and was more likely to be undergoing their first IVF cycle (84% v 68%, p = 0.003). Those in Group 1 had more eggs collected (13.6±7.3 v 11.3±5.5, p = 0.009), more zygotes (8.9±4.7 v 7.3±3.6) formed and more blastocysts frozen (3.4±2.6 v 1.1±1.7, p < 0005). More women in Group 1 had a top quality blastocyst (expansion of > 2 and inner cell mass and trophectoderm of AA, AB, BA or BB) transfer (91% v 71%, p < 0.005). After logistic regression controlling for maternal age, number of previous IVF cycles and blastocyst quality, Group 1 had a lower likelihood of livebirth (aOR 0.550, 95%CI 0.306-0.988) but with a significantly lower multiple pregnancy rate (0% v 24%, p = 0.024). Importantly, for those undergoing their first IVF cycle (n = 359), there was no difference in live birth rate in the two groups (aOR 0.617, 95%CI 0.329-1.156) after controlling for age and blastocyst quality but Group 2 had a higher multiple pregnancy rate (24% v 0%, p = 0.020). Limitations, reasons for caution This study is limited by its retrospective nature putting it at risk of information bias as it relied on accurate documentation of studied variables into the patient database. The study did not examine cumulative birth rates of fresh and subsequent frozen cycles in Group 1. Wider implications of the findings Women should have individualised counseling about number of blastocysts to transfer taking into account their circumstances. Those undergoing their first IVF attempt and with a blastocyst available for transfer, should be counselled that DBT is associated with a higher multiple pregnancy rate with no increase in overall live birth rate. Trial registration number Not Applicable

P-215 Use of hyaluronan-based solution as an alternative to polyvinylpyrrolidone to improve blastulation in ICSI

Abstract Study question This study aimed to analyze whether hyaluronan as an alternative to polyvinylpyrrolidone (PVP) for sperm immobilization, aspiration, and injection into the ooplasm improves ICSI outcomes. Summary answer The use of hyaluronan solution as an alternative to PVP improves the blastocyst formation rate and good quality blastocyst formation rate compared to PVP. What is known already The PVP solution facilitates ICSI procedures, such as sperm immobilization, aspiration, and injection into the ooplasm. However, it has been reported that injection of a small amount of PVP along with the sperm into the ooplasm negatively affects subsequent embryo development. Hyaluronan is a natural component of the extracellular matrix of the cumulus-oocyte complex. Balaban et al. (2003) reported that hyaluronan can replace PVP during ICSI procedures in the early embryo transfer cycle without adversely affecting pregnancy outcomes. This study analyzed whether using hyaluronan-based solution for the ICSI procedure would improves blastocyst development compared with ICSI using PVP. Study design, size, duration This retrospective study analyzed clinical medical reports at Takahashi Women’s Clinic, Japan. We included 4002 oocytes retrieved from 411 patients under 39 years of age who underwent ICSI using autologous oocytes between December 2019 and August 2021. Of the oocytes used for ICSI, 1909 underwent sperm aspiration and injection into the ooplasm with hyaluronan (Sperm Slow; Origio), and 2093 oocytes with 7% PVP (NakaMedical). We did not perform preimplantation genetic testing-aneuploidy for any cycles. Participants/materials, setting, methods We used PVP droplets for sperm selection under 400× magnification in both groups. In hyaluronan-ICSI, the selected sperm were transferred to a hyaluronan droplet and washed three times. The sperm was then aspirated with hyaluronan, and ICSI was performed. In PVP-ICSI, all procedures were performed using PVP. The ICSI and embryo transfer outcomes were compared between hyaluronan-ICSI and PVP-ICSI by logistic regression analysis considering patient age, BMI, and basal level of anti-mullerian hormone. Main results and the role of chance Normal fertilization rates were 74.3% (1556/2093) in PVP-ICSI and 75.5% (1442/1909) in hyaluronan-ICSI. There was no significant difference in the normal fertilization rate between PVP-ICSI and hyaluronan-ICSI groups (p = 0.437, aOR:1.06, 95% CI: 0.92–1.22). We cultured 1323 2PN embryos in PVP-ICSI and 1237 2PN embryos in hyaluronan-ICSI until the blastocyst stage. Blastocyst formation rates were 48.1% for PVP-ICSI and 52.3% for hyaluronan-ICSI, and this difference was statistically significant (aOR, 1.20; 95% CI: 1.02–1.40; p = 0.024). Moreover, the good grade (Gardner criteria ≧BB) blastocyst formation rates were significantly higher in the hyaluronan-ICSI group (36.9% and 41.0%, aOR: 1.21, 95% CI: 1.03–1.42, p = 0.022). During the study period, we performed 163 and 169 cryo-thawed blastocyst transfer cycles in PVP-ICSI and hyaluronan-ICSI, respectively. The clinical pregnancy rate (50.9% vs. 54.4%, aOR: 1.19, 95% CI: 0.77–1.83, p = 0.443) and miscarriage rate (19.3% vs. 13.0%, aOR: 0.66, 95% CI: 0.3–1.44, p = 0.295) after embryo transfer were not significantly different between PVP-ICSI and hyaluronan-ICSI. Limitations, reasons for caution The study was conducted at a single IVF center, and the oocytes included in this study were collected from patients aged < 39 years. Embryo transfer result is based on ongoing pregnancy, while the live birth data for all pregnancies are not yet available. Wider implications of the findings Hyaluronan facilitates ICSI procedures such as sperm immobilization, aspiration, and injection. Moreover, the hyaluronan improves blastocyst development. The present study indicates that using hyaluronan as an alternative to PVP during the ICSI procedure is recommended. Trial registration number not applicable

P-528 Time-lapse monitoring: an adjunct tool to select embryos for preimplantation genetic testing

Abstract Study question Does embryo aneuploidy have any impact on embryo morphokinetic events in a time-lapse imaging (TLI) culture system? Summary answer Aneuploidy had a significant impact on early and late embryo morphokinetic events. Mosaic and aneuploid embryos behaved similarly in terms of morphokinetics. What is known already The preimplantation genetic testing (PGT) emerged as an approach to analyse embryos’ DNA for determining genetic abnormalities, distinguishing chromosomally normal embryos, considered to have high developmental potential, from their aneuploid siblings. Although shown to be efficient and clinically relevant, the invasive nature of the technique limits its success. Given the challenges associated with invasive embryo biopsy, ongoing studies are now seeking for non-invasive assessments of the embryo DNA. Timelapse imaging (TLI) may allow the identification of morphokinetics events affected by aneuploidy, which could be a powerful tool for improving embryo selection for transfer, without the detrimental effects of embryo biopsy. Study design, size, duration This case-control study was performed in a private university–affiliated IVF center, between 2019 March and 2020 December. Kinetic data were analyzed in 957 embryos, derived from 316 patients undergoing ICSI cycle with PGT-A. Kinetic markers from the point of insemination were recorded. Generalized linear models followed by Bonferroni post hoc were used to compare timing of specific events in euploid (n = 352), aneuploid (n = 583) and mosaic embryos (n = 22). The post hoc achieved power was > 80%. Participants/materials, setting, methods Embryos were cultured in the EmbryoScope incubator, which recorded the following kinetic markers: timing to pronuclei appearance and fading (tPNa and tPNf), two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), morulae (tM), start of blastulation (tSB) and blastulation (tB). Durations of second and third cell cycles (cc2 and cc3) and timing to complete synchronous divisions s1, s2, and s3 were calculated. The KIDScore ranking was recorded. Main results and the role of chance Aneuploid embryos showed significantly longer tPNf, t2, t3, t4, t6, t7, t8, tM, tSB, tB, s2 and s3 compared to euploid embryos. The KIDScore day 5 also rank was significantly lower in aneuploid embryos compared to euploid ones. As for mosaic embryos no significant differences were observed in the comparisons of means of morphokinetic events and KIDScore rank, but regression analyses showed an increased chance of mosaicism among embryos with longer tPNf, t2, t3, t6, t7, tM, tB, as well as a inverse association with KIDScore rank, when compared with euploid embryos. Embryos were hierarchically distributed into quartiles according to the KIDScore rank: Q1 ≤3.9, Q2 4-5.6, Q3 5.7-7.5, and Q4 ≥7.6. Euploidy incidence was significantly different among the KIDScore quartiles, in which the percentage of euploidy increased directly proportional to the kidscore rank. For patients >35 years old, the logistic regression analysis demonstrated that comparing with embryos in the Q4, embryos in the Q1, Q2, and Q3 have a decreased chance of being euploid. For patients ≤35 years old, comparing with embryos in the Q4, embryos with KIDScore D5 in the Q1 and Q2, but not in Q3, have a decreased chance of being euploid. Limitations, reasons for caution Our study is limited by its retrospective nature and small casuistic, despite adequate power has been achieved. The reason for the lack of consistency with some previous findings can be explained by variations in culture systems, studied population, embryo stage for biopsy and wide range of tests used for PGT. Wider implications of the findings Our evidence suggests that TLI monitoring may be an adjunct approach to select embryos for PGT, however, cautious investigation is still needed. The mechanism by which embryo aneuploidy may alter the cleavage timing of embryos is undefined, but disruptions of mitosis, cell-cell interactions and blastocyst differentiation are possible candidates. Trial registration number not applicable

O-173 Artificial intelligence blastocyst ploidy distinction through morphokinetics data

Abstract Study question Is it possible to estimate blastocyst ploidy with an artificial intelligence (AI) algorithm built using embryo morphokinetics data? Summary answer AI was able to estimate blastocysts status in euploid or aneuploid with an accuracy of 0.99 (training) and 0.71 and 0.70 AUC (blind-test), respectively. What is known already Morphokinetic parameters are well associated with implantation rates, thus ensuring time-lapse as a useful tool to enhance embryo ranking and selection. However, regarding embryo ploidy status, clinics still rely exclusively on molecular/genetic testing. Our previous studies has indicated that euploid blastocysts are faster at time of pronucleous fading (tPNf) and time to blastulation (tB). Considering the amount of data derived from the morphokinetic annotations and the promising results that AI approach is bringing to the field, this study aims to verify AI accuracy to distinguish euploid and aneuploidy blastocysts using 17 morphokinetics parameters and prospectively compared to embryo biopsy results. Study design, size, duration This is a prospective cohort study including 402 embryos (cultured in a time-lapse incubator, EmbryoscopePlus, Vitrolife) from 140 patients undergoing IVF treatment with preimplantation genetic testing for aneuploidy (NGS platform) after inform consent form signature between July 2019 and September 2021. Morphokinetics annotations were analyzed by AI in an association of the technique of artificial neural networks (ANN) and genetic algorithms (GA) for ploidy assessment. Participants/materials, setting, methods Morphokinetic parameters were manually annotated up to full-expanded blastocyst time. Time intervals and time ratios were calculated, resulting in 17 variables for the IA analysis. Of the 402 embryos data, 252 were randomly divided into training, validating and testing (70%, 15% and 15%, respectively) of the ANN. The remaining 150 data were used for the blind test. The area under the curve (AUC) of the receiver operating characteristic curve was measured to obtain predictive power. Main results and the role of chance From 402 blastocysts biopsied, 185 were euploid and 217 aneuploid. The AI algorithm was trained with 176 embryos (AUC for both euploid and aneuploid= 0.99), tested with 38 embryos (AUC for euploid=0.62 and aneuploid=0.61), validated with 38 embryos (AUC for euploid=0.82 and aneuploid=0.83). For the blind-test 150 embryos were used (AUC for euploid=0.70 and aneuploid=0.71). Blind-test database was checked only after AI algorithm was tested. Limitations, reasons for caution The development of this AI algorithm was built from a single IVF center database. The absence of an extent dataset for the blind-test does not allow us to transpose this algorithm for clinical use at this moment. Wider implications of the findings The use of artificial intelligence for embryo assessment is a promising tool in IVF laboratories. In our model, using only morphokinetics, a considerable predictive power to evaluate euploid (0.71 AUC) and aneuploid (0.70 AUC) embryos was achieved, indicating a potential use as a non-invasive approach for embryo ranking and selection. Trial registration number Not applicable

P-667 Follitropin delta for ovarian stimulation in daily practice in a Dutch IVF centre

Abstract Study question This study reports individualized follitropin delta dosing in a real life setting in patients undergoing IVF treatment. Summary answer The use of follitropin delta, in real-world clinical setting with a broad and varied patient population, shows results aligned with the ESTHER-1 registration trial (NCT01956110). What is known already Individualized follitropin delta dosing based on anti-Müllerian Hormone (AMH) and body weight (BW) is as effective as treatment with Chinese hamster ovary (CHO) derived follitropin alfa, and leads to a more predictable ovarian response and improved safety. Results of the ESTHER-1 trial show: similar pregnancy rates and less frequent excessive ovarian response compared to women treated with follitropin alfa. Moreover, the incidence of ovarian hyperstimulation syndrome (OHSS) was reduced, fewer poor responses occurred in women with AMH <15 pmol/L and fewer excessive responses in women with AMH ≥ 15 pmol/L. Study design, size, duration Descriptive analysis from real-world data obtained in the Erasmus University Medical Centre. Since its introduction in 2017, 2369 treatment cycles have been performed with follitropin delta in our center. In 499 patients, with a regular ovulatory menstrual cycle, follitropin delta was used in a GnRH-antagonist protocol in their first IVF cycle in 2017. Subsequent fresh embryo transfers were done on day 5. EMC patients were compared to patients in the ESTHER-1 trial using follitropin delta. Participants/materials, setting, methods Women are prescribed follitropin delta according to the dose algorithm based on the patient’s BW and serum AMH levels as measured by the automated Elecsys® AMH plus immunoassay (Roche Diagnostics). Results of the treatment were collected from the clinical records. The outcomes of the first IVF cycles with follitropin delta are reported. Data were expressed as mean (SD) or percentages and statistically analyzed using t-test and chi-square test. Main results and the role of chance EMC patients were significantly older (33.9 (4.5) vs. 33.4 (3.9) years (p < 0.01) and had higher AMH serum levels (21.5 (13.2) vs. 19.4 (14.6) pmol/l (p < 0.001) compared to those in the ESTHER-1 trial. Less patients started IVF in a naïve cycle (75.4% vs. 100% (p < 0.001) and BW was higher (68.6 (11.1) vs. 64.7 (10.7) kg (p < 0.01). Retrieved number of oocytes (10.6 (5.7) vs. 10.0 (5.6) (p < 0.05), number of cleavage stage embryos (6.1 (3.6) vs. 5.4 (3.7) (p < 0.001), number of cryopreserved embryos (2.3 (2.3) vs. 1.9 (2.4) (p < 0.001) and proportion of women with embryos cryopreserved (72.4% vs. 63.4% (p < 0.01) were all significantly higher in the EMC patients. Proportion of women with 8-14 oocytes was similar (47.3% vs. 43.3% of patients (NS). Women with AMH <15 pmol/L had more oocytes retrieved (8.9 (4.5) vs. 8.0 (4.3) (p < 0.01). Women with AMH ≥15 pmol//L had similar numbers of oocytes retrieved (11.4 (6.0) vs. 11.6 (5.9) (NS). More cycles were cancelled with an insufficient number of follicles in the overall population (7.4% vs. 3.8% (p < 0.01). Hospitalization due to OHSS was similar (0.6% vs. 0.3% (NS). Positive hCG test (40.7% vs. 38.6% (NS) and ongoing pregnancy rate per started cycle (32% vs. 30.7% (NS) were similar. Limitations, reasons for caution This is a retrospective analysis of the use of follitropin delta in patients in daily clinical practice. As this population was drawn from daily clinical practice, it is a heterogeneous population in which no strict in- or exclusion criteria were applied. Wider implications of the findings Individualized follitropin delta treatment of a broad patient population as presented here shows clinically similar results in ovarian response compared to the previously reported ESTHER-1 trial. Moreover, pregnancy rates were also similar even though patient characteristics were slightly different. Trial registration number not applicable

P-769 Endometrial thickness is not associated with birthweight as well as serum levels of pregnancy-associated plasma protein-A: a retrospective analysis of 560 newborns following frozen cycles

Abstract Study question Does endometrial thickness (EMT) affect birthweight and maternal serum evels of pregnancy-associated plasma protein-A (PAPP-A) in singleton pregnancies following frozen-thawed blastocyst transfer (FET)? Summary answer EMT does not significantly affect birthweight and maternal serum evels of PAPP-A in singleton pregnancies following FET. What is known already Several studies have shown an association between EMT and low birth weight in pregnancies resulting from fresh embryo transfers. Supraphyiological levels of steroid hormone during ovarian stimulation may be responsible for poor fetal growth. Few studies have investigated the relationship between EMT and fetal growth potential in frozen-thawed embryo transfers. Study design, size, duration This retrospective study included women who underwent first-trimester combined screening for aneuploidy after frozen-thawed blastocyst transfer and subsequently delivered a singleton live birth after 24 weeks of gestations between January 2016 and July 2020 in a private IVF center. Participants/materials, setting, methods A total of 560 women met the inclusion criteria and were stratified into three groups based on the EMT percentiles: <7.7 mm (<10th percentile), 7.7–12 mm (10-90th percentile), and >12 mm (>90th percentile). The primary outcome measures were mean birth weight and mean PAPP-A level. A multivariable linear regression analysis was carried out to identify the association between birth weight and EMT after adjusting for variables found at < 0.1 level in the univariate analysis. Main results and the role of chance The mean birth weight was 3127 ± 783 g in those with EMT <7.7 mm 3225 ± 644 g in those with EMT 7.7-12 mm, and 3256 ± 599 g in those with EMT >12 mm (p = 0.577). The mean PAPP-A levels and PAPP-A<0.5 rates were similar in the groups, as well. In multivariate analysis, the number of previous ART attempts and maternal serum levvels of PAPP-A were found to be significant predictors of birth weight (p = 0.001 and p = 0.013, respectively). EMT was not a predictor of birth weight in both univariate and multivariate analyses. The multivariate logistic regression analysis indicated that the number of previous ART attempts was an independent factor for low birth weight (OR:1.39, 95%CI:1.09-1.76, p = 0.006). Limitations, reasons for caution The study has inheret limitations due to its retrospective nature. Further studies are needed to investigate the relationship between EMT and birth weight along with maternal serum levels of PAPP-A. Wider implications of the findings Our study provided a new angle of view for evaluating the association between EMT and neonatal birth weight by analyzing this association along with maternal serum levels of PAPP-A as a marker for placental function, suggesting that EMT is not an independent factor for neonatal birth weight. Trial registration number NA

O-260 Improved implantation and pregnancy rates in frozen-thawed embryo transfer using a modified-natural ovulatory regimen

Abstract Study question Are the outcomes of pregnancy significant different between modified-natural (mN) cycle frozen-thawed embryo transfer (FET) and hormone terapy (HT) cycle FET? Summary answer Implantation and pregnancy rates are significantly higher in mN cycle FET as compared to HT cycle FET. What is known already The use of FET has dramatically increased the cumulative pregnancy rate and reduced the cost and time to pregnancy in assisted reproductive technology (ART). Three different regimens can be used to perform FET, the spontaneous ovulatory (natural) cycle; the one in which the endometrium is artificially prepared with HT; and cycles in which ovulation is induced. In our center, for the past three years, we have performed HT cycle FET as well as a modified-natural regimen, which includes the administration of 250 µg recombinant hCG (r-hCG) upon the visualization of one follicle measuring ≥ 18 mm in diameter. Study design, size, duration Women undergoing FET post intracytoplasmic sperm injection (ICSI), between Jan/2019 and August/2021 in a private university-affiliated IVF center were included (mN cycle FET group, n = 142 and HT cycle FET group, n = 1180). Generalized linear models adjusted for potential confounders, followed by Bonferroni post hoc were used to compare clinical outcomes between groups. Participants/materials, setting, methods In mN cycle FET group, after menses, the endometrial development was monitored, and patients received 250µg r-hCG upon the visualization of one follicle ≥ 18mm in diameter, and 600mg daily of micronized progesterone for five days, until embryo transfer. In HT cycle FET group, after menses the patients received 200µg of transdermal 17 β-estradiol every three days. When endometrium showed proliferative morphology and thickness of at least 7.5mm, 600mg of progesterone was daily administered. Main results and the role of chance Mean female age was similar between the groups (36.0 years ± 0.5 for mN cycle FET and 35.6 years ± 0.2 for HT cycle FET groups, respectively). The implantation rate was significantly higher in mN cycle FET group compared to HT cycle FET group (45.3% ± 0.76 vs. 38.6% ± 0.22, p < 0.001, respectively). The pregnancy rate was also significantly higher in mN cycle FET group compared to HT cycle FET group (66.0% vs. 52.4%, p = 0.017, respectively). No significant differences were observed between the groups in miscarriage rate (10.7% for mN cycle FET and 15.7% for HT cycle FET groups, respectively, p = 0.279). Limitations, reasons for caution Retrospective nature of this study and the small sample size may be a reason for caution. Wider implications of the findings Our study suggests use of an hCG trigger may improve outcomes in natural cycle FET. In patients undergoing FET, triggering ovulation can significantly reduce the number of visits necessary for scheduling embryo transfer without an adverse effect on outcomes. Ovulation triggering can increase both patient convenience and cycle cost effectiveness. Trial registration number Not applicable

O-076 Genomic and morphokinetic blueprint of mosaic embryos is evidence supporting their distinct clinical category

Abstract Study question Is there a correlation between blastocyst mosaicism, morphokinetic and morphologic characteristics and can it inform the clinical outcome of mosaic embryo transfers? Summary answer Mosaic (diploid/aneuploid) embryos have a distinct genetic, morphokinetic and morphologic signature that distinguishes them from euploid and abnormal embryos and supports their unique clinical identity. What is known already Advances in technologies for preimplantation aneuploidy testing (PGT-A) as the ultimate test for embryo selection has increased the resolution and sensitivity to detect intermediate chromosomal copy number as low as 20%. This has brought to light a biological phenomenon that has created clinical dilemma. Current recommendations support mosaic embryo transfer but with conflicting standpoints ranging from complete dismissal of mosaic embryos to arguing their existence. Time-lapse imaging technologies allow collection of morphokinetic data as a non-invasive assessment tool for embryo development. The aim of this study was to correlate genomic and morphokinetic data with clinical outcomes of mosaic embryos. Study design, size, duration This is a retrospective cohort study that took place in a single academic IVF centre, where we analysed the results from high resolution PGT-A, morphokinetic, morphologic and cell division features of 4113 embryos cultured in the time lapse imaging incubator (Embryoscope™) from 2016-2021. Associated patient data and transfer outcome of 770 euploid and 147 mosaic embryo transfers were analyzed. Participants/materials, setting, methods High resolution NGS PGT-A was performed using the Illumina platform and BluGnome and NxClinical for data analysis. Clinically relevant findings were aberrations >10Mb and mosaicism from 25%-75%. Time-lapse imaging was performed with an Embryoscope™. Comparison of clinical, morphological, morphokinetic characteristics of euploid, aneuploid, low-level mosaic (25%-<50% aneuploid cells in trophectoderm biopsy), and high-level mosaics (50% -75%) was done with R-statistical package, Mathlab and SSPS software were used, and p < 0.05 with CI 95% was considered significant. Main results and the role of chance Aneuploidy was detected in 26.2%, euploidy in 56.7% and diploid-aneuploid mosaicism in 17.1% of embryos included in this study. Compared with euploid embryos, mosaic embryos had significant delays in t2 (27.04±0.35vs.26.4±0.17 h-post-ICSI(hpi), p = 0.0029), which was not correlated with transfer outcome. Embryos with whole chromosome mosaicism (WCM) had longer t2 compared to those with segmental chromosome mosaicism (SCM) (27.29±0.59vs.26.4±0.3hpi,p=0.013). The time to first mitosis was also longer for mosaic vs. euploid embryos (4.57± 0.59vs.3.4±0.25hpi,p=0.000056) and for mosaic embryos with WCM vs SCM embryos (4.64± 0.9 vs 3.5 ±0.5hpi,p=0.04). Mosaic embryos had longer tB &tSB than euploid embryos (110.12±0.69vs.109.13hpi±0.4,p=0.01). Embryos with WCM had longer tB &tSB than those with SCM (111.07±1.09vs.108.7±0.8hpi,p=0.0006). Time to blast was longer for mosaic vs euploid embryos (13.69±0.52vs.12.7±0.4hpi,p=0.0035) and for mosaic WCM vs. SCM embryos (14.3±0.7vs.12.8±0.5hpi,p=0.00084). Both euploid and mosaic embryos that implanted had shorter time to tB&tSB than mosaic embryos that didn’t (p = 0.029; p = 0.007 and p = 0.002; p = 0.006 and p = 0.029 respectively); and mosaic embryos that implanted had shorter time to first mitosis than euploid embryos that didn’t (p = 0.00228). High level mosaic embryos had significantly more multinucleation at 2-cell stage (MN2) vs. euploid (p = 0.04,OD1.58-95%CI[1.01-2.45]),aneuploid (p = 0.02,OD1.7-95%CI[1.08-2.8]), as well as MN at 4-cell stage (euploid-p=0.0007,OD2.6-95CI1.4-4.7] and aneuploid-p=0.039,OD1.8-95CI1.02-3.4]). Limitations, reasons for caution Although this is the largest study to date that evaluated genomic, morphokinetic and outcome data of mosaic transfers, the numbers are still low to analyze the impact of specific chromosomal aberrations on morphokinetics and pregnancy outcome. The retrospective nature and cohort design limited the generalizability of our results. Wider implications of the findings Mosaic embryos have significant developmental potential and result in birth of healthy babies. Our study reveals the genomic interconnection of genomic and morphokinetic features of mosaic embryos and provides further evidence that mosaic embryos are a distinct category that requires specific clinical attention. Trial registration number Not applicable

P-098 Male age does not affect sperm motility rate after thawing and laboratory outcomes of transnational oocyte donation programs

Abstract Study question Can outcomes of transnational egg donation treatments, which involves the shipping of cryopreserved partner semen, be affected by advanced paternal age? Summary answer Male age does not affect percentage of motile spermatozoa after thawing. Advanced paternal age does not affect IVF laboratory outcomes of transnational egg donation treatments What is known already Studies on advanced paternal age are limited and a clear definition of advanced paternal age is still missing. The separation between advanced paternal and maternal age effects is hampered by the collinearity of maternal age with paternal age, since older males are often associated with older women. Many studies have focused on egg donation treatments, to overcome this problem. However, these studies reported conflicting results, such as a putative effect of advanced paternal age on ICSI outcomes, while some others not. This study analyzes paternal age in transnational egg donation program, which involves the shipping of cryopreserved partner semen Study design, size, duration A multicentric retrospective cohort study involving 101 couples and 327 blastocysts was performed from June 2018 up to December 2021. Two Italian IVF centers involved a Spanish center to perform a transnational fresh oocyte donation program. The aim of the study was to analyze the putative effect of male age on (i) semen parameters, (ii) fertilization rate and (iii) blastocyst rate. Participants/materials, setting, methods Donations of both male and female gametes were excluded. We performed sperm cryopreservation of the male partner of the recruited couples. Frozen samples were shipped with a authorized courier, which certified the safety of the whole procedure. The day of donor’s egg retrieval, semen samples were thawed and ICSI was performed on fresh donated oocytes. Resulted blastocysts were vitrified and shipped to the centers where the frozen embryo transfers in the recipient patients were performed. Main results and the role of chance The mean of male partners age was 42.6 ± 6.5 (min 26- max 63). Overall fertilization rate was 81.6% (686/840), and blastocyst rate was 47.6% (327/686). Male age was analyzed as a continuous variable and divided into five groups calculated for 5-year intervals. No differences in terms of fertilization rates nor blastocysts rates were found (85.3% and 43.7% for <35-years; 84.7% and 54.0% for 36-39-years; 79.7% and 44.2% for 40-44-years; 89.6 and 48.1% for 45-49-years; 74.5% and 56.5 for >50-years). Although advanced paternal age has been correlated to the decrease of most of sperm parameters, in the present study we found that only progressive rapid motility was significantly affected by advanced paternal age (coefficient -0.261 , P = 0.009). To evaluate the predictive value of advanced paternal age and sperm parameters on fertilization and blastocysts formation, we performed a multiple linear regression analysis. We found that sperm parameters and male age in our sample are not predictive of the number of fertilized oocytes nor the number of obtained blastocysts. Importantly for the setting of the study, the percentage of progressive motile sperm at the thawing was not influenced by paternal age. Limitations, reasons for caution The main limitation of the study is the small sample size, which might mask the paternal age effect on the sperm parameters, as the volume, which tend to decrease in the classes of age, but did not reached the statistical significance. The multicentric nature of the study represent a strength Wider implications of the findings The discomfort for couples that need oocyte donation in countries where there is a low availability of egg donors is often underestimated. This study demonstrate that, independently of male age, the shipping of frozen sperm and the ICSI of freshly donated oocytes represent a valid option for these couples. Trial registration number not applicable

O-205 Application of a single “Universal warming protocol” for vitrified donor oocytes: A prospective, multicenter study

Abstract Study question Is it possible to use different commercial thawing kits with donor oocytes that have been vitrified with a Kitazato vitrification kit? Summary answer The Omni thawing kit showed higher survival rates of donor oocytes. However, fertilization, blastulation, and chemical pregnancy rates were similar in all three thawing kits. What is known already Vitrification is the best method for cryopreserving metaphase II (M2) oocytes and blastocysts. Oocyte vitrification and subsequent thawing is performed with the use of different commercial kits. Currently, the thawing kit usually corresponds to the same provider as the vitrification kit used. However, this may create issues when using cryopreserved oocytes among different clinics with different vitrification kits and, furthermore, not all lab professionals are accustomed to using other commercial kits. Using a “single warming protocol” would provide a solution to these drawbacks and would improve the compatibility of exchanging gametes and embryos among collaborating centers. Study design, size, duration This is a longitudinal, comparative and prospective study conducted between January 2017 and December 2020 at four different IVF private centers. Overall, we vitrified a total number of oocytes (n = 2172) with a single Kitazato commercial vitrification kit at a Spanish Egg Bank, and three independent IVF centers used different thawing kits (Kitazato, Irvine or Omni) as a warming protocol. Participants/materials, setting, methods The study included oocytes from altruistic anonymous egg donors aged 18-35 years, subject to the rigorous standards to become an oocyte donor. The primary outcome was survival and fertilization rate; secondary outcomes included blastulation rate, and chemical pregnancy rate. Main results and the role of chance A total of 2172 M2 oocytes were retrieved and subsequently vitrified using the Kitazato protocol and the Cryotop device. All vitrified donor M2 oocytes were thawed at independent assisted reproduction centers using the following thawing kits: Omni Vitrolife (n = 655), Kitazato (n = 1021), and Irvine (n = 496). Oocytes thawed using the Omni kit had a 94% (617/655) survival rate and a 73% fertilization rate. Kitazato thawing kits had an 86% (877/1021) survival rate and a 77% fertilization rate. Lastly, Irvine thawing kits had an 85% (423/496) survival rate and a 74% fertilization rate. Survival rate results were statistically significant among different thawing kits (P-Value = <0.01). Fertilization rates were not statistically significant. Blastulation rates with Omni, Kitazato and Irvine thawing kits were 57%, 52%, and 58%, respectively (Not statistically significant). Lastly, chemical pregnancy (HCG-Beta positive) rates with Omni, Kitazato and Irvine thawing kits, results were 65%, 54%, and 76%, respectively (Not statistically significant). Limitations, reasons for caution The study was carried out in centers with long experience collaborating together, but many different professionals intervened in the process, without the same conditions in all labs. Wider implications of the findings With increasing requirements for cryopreserved oocytes due to the difficulties of donor-recipient synchronization, popularization of treatments from abroad, and the need to import or send donor oocytes for recipient phenotypes, it has become of paramount importance to use a single universal warming protocol irrespective of cryopreservation kits. Trial registration number NOT APPLICABBLE

P-076 Standard IV cannula aspiration of testicular sperm: A novel, efficient and minimally invasive aspiration technique

Abstract Study question Can we aspirate enough testicular tissue for sperm retrieval in non-obstructive azoospermia (NOA), using a wide bore 14-G Standard IV cannula in comparison to micro-TESE? Summary answer Standard IV cannula Aspiration (SIVCA) can yield an ample amount of testicular tissue sufficient for sperm retrieval through a single puncture site on the scrotum. What is known already The current conventional method of testicular sperm aspiration is fine needle aspiration (FNA). FNA has the advantage of being a cost-effective and minimally invasive procedure compared to open testicular sperm extraction (TESE). But FNA with its conventional 23-G needle may not always yield enough testicular tissue for sperm retrieval. Furthermore, FNA may require multiple punctures on the scrotum to retrieve enough tissues from different areas of the testis. Study design, size, duration A 24 months prospective cohort study conducted at a specialized IVF center. A total of 130 men aged from 22 to 53 years old (35.03 +/- 9.04) with NOA and normal testicular volume (≥ 12ml) were enrolled in the study. The men had testicular biopsies taken at the day of their partners’ ovum pick-up. On each patient, the testes were randomized to undergo SIVCA on one testis followed by micro-TESE on the contralateral testis. Participants/materials, setting, methods After local anesthesia, a wide bore 14-G standard IV cannula was introduced near the lower pole of the testis. The needle was withdrawn and the catheter introduced into the testicular tissue. A 20-ml syringe was secured to the catheter and constant negative pressure applied and secured with a clamp. Back and forth motions were performed covering as many areas of the testis as possible. Micro-TESE was then performed on the contralateral testis. Main results and the role of chance Sperm retrieval rates (SRR) were compared between the two techniques using McNemar χ2 test. A P-value of less than 0.05 was considered to be statistically significant. Out of a total of 130 cases, an adequate tissue sample could successfully be aspirated using SIVCA in 122 cases, showing a 0.93 probability of success. In those 8 cases where SIVCA had not yielded sufficient tissue and were reverted to micro-TESE, 2 cases were positive for sperm retrieval. Whenever tissue aspiration was successful with both methods, both SIVCA and micro-TESE showed similar success rate with respect to SRR (56.2 vs 57.3%; McNemar P = 1.000). Limitations, reasons for caution Both FNA and SIVCA are not easy to perform on small sized testes. Moreover if testicular tissue is fibrotic or sclerotic, it may be difficult to aspirate and micro-TESE would be the ideal choice to yield enough testicular tissue. Further blinded randomized control studies with larger datasets are recommended. Wider implications of the findings The use of a peripheral IV wide bore cannula is suitable for the aspiration of adequate amounts of testicular tissue for sperm searching whilst requiring only a single puncture on the scrotum. We recommend routinely starting off with SIVCA to search for sperm before reverting to micro-TESE. Trial registration number Unique Protocol ID: SIVCA Protocol registration Records number will be made available to the public through the ClinicalTrials.gov web site within 2 to 5 days of today 31/1/2022

P-359 Effect of estradiol pretreatment on antagonist ICSI cycles outcome

Abstract Study question Does estrogen pretreatment in antagonist ICSI cycles improves the outcome? Summary answer Pretreatment with estrogen in antagonist cycles reduced the days of stimulation, the number of hMG ampoules and enhanced the implantation rate. What is known already It has been previously reported that, programming of antagonist cycles with E2 only, started in the luteal phase and continued beyond the menses till the first day of stimulation, yielded similar results as the long agonist protocol. E2 pre-treatment in the luteal phase has an inhibiting role on follicular growth through its negative feedback on the FSH increase during the luteal follicular transition phase. This effect stops as soon as E2 administration is terminated. Study design, size, duration This was a prospective randomized controlled trial performed in a private IVF centers from June 2020 to February 2021. One hundred and fourteen infertile couples undergoing intracytoplasmic sperm injection embryo transfer cycles (ICSI-ET) were included. Participants/materials, setting, methods Group A includes 57 patients were pretreated with a dose of 4 mg estradiol valerate (progynova 2 mg; Bayer) once daily starting 10days before expected menses. Ovarian stimulation was started one day after the last estradiol dose, in a dose ranging from 225 to 375 IU of hMG. While in group B , 57 patients received stimulation in a dose of 225 to 375 IU of hMG starting on cycle day 2 without estradiol pretreatment. Main results and the role of chance The daily dose of hMG was significantly lower in The study group (P = 0.048), the stimulation days were significantly higher in the control group (P = 0.049), the total number of hMG ampoules was significantly higher in the control group (P = 0.041), and the implantation rate was higher in the study group (P = 0.045) , also the pregnancy rate (positive pregnancy test) was higher in the study group (p = 0.044) but there was no significant statistical difference detected as regards the clinical pregnancy rate between both groups (P = 0.180) , the serum estradiol on day of hCG ( p = 0.055), serum progesterone level on hCG day (P = 0.71) and the endometrial thickness (p = 0.780) . Also there is no significant statistical difference between the two groups as regarding number of follicles on day of hCG. (P = 0.803), the number of mature oocytes (P = 0.703) and no significant difference in the number of good quality embryos (p = 0.532) and the ratio of mature oocytes to number of follicles on day of hCG (P = 0.835), with no significant statistical difference between both groups. Limitations, reasons for caution The limitation of this study is the small number of patients as we included only 114 patients. Wider implications of the findings Luteal estradiol in antagonist cycles improved the outcomes of ICSI procedure by reducing the number of gonadotropin ampoules used for COH and reduced duration of stimulation, also the implantation rate is improved by luteal estradiol administration. Trial registration number NCT05197374