Fertilization Rate Research Articles

P-079 Bilateral versus unilateral orchidopexy: IVF/ICSI-ET outcomes

Abstract Study question Does unilateral and bilateral cryptorchidism infertility have an impact on IVF/ICSI-ET results? Summary answer Unilateral and bilateral orchidopexy do not affect the final live birth rate in IVF. What is known already A study was compared ICSI-ET outcomes between bilateral and unilateral cryptorchidism in NOA patients. Although 90.36% of their study population have undergone orchidopexy to improve their conditions at around 9 to 10 years old, the fertilization rates of bilateral and unilateral cryptorchidism group were only at 46.8% and 46.5% respectively, which were slightly below the average ICSI fertilization rate. The other study showed that patients of bilateral cryptorchidism had lower implantation rate. Apart from the mentioned study, no other study has attempted to evaluate the IVF/ICSI-ET outcomes in infertile men who had undergone orchidopexy. Study design, size, duration A retrospective cohort study at a tertiary hospital, including a total of 99 infertile men who underwent orchidopexy to treat cryptorchidism and subsequently underwent their first IVF/ICSI-ET cycle from January 2014 to January 2022. Participants/materials, setting, methods Men are divided into unilateral fixation group and bilateral fixation group based on the laterality of the cryptorchidism and testicular fixation surgery they have received. Choose fertilization rate and live birth rate as parameters for evaluating the results. Main results and the role of chance The sperm concentration and viability were significantly higher in unilateral orchidopexy group than in bilateral orchidopexy group (28.09 ± 27.99 vs 7.99 ± 14.68, P=0.001; 33.34 ± 22.52 vs 11.95 ± 17.85, P=0.001). Unilateral orchidopexy group showed lower demand for ICSI (66.07% vs 95.35%, P<0.001). Interestingly, both groups exhibited similar rates of fertilization, clinical pregnancy, live birth and birth defect. Boy birth ratio was lower in bilateral orchidopexy group as compared to unilateral orchidopexy group (27.27% vs 58.62%, P=0.026). Limitations, reasons for caution This study was limited in that the data source captured only patients from a single medical center, which could lead to selection bias, small sample size and limited external validity to the findings. Wider implications of the findings Our study suggested that blastocyst-stage ET is a good strategy for patients with history of orchidopexy. Trial registration number not applicable

P-656 Comparative clinical outcome following individualized follitropin delta dosing in Japanese women undergoing progestin-primed ovarian stimulation for in vitro fertilization /intracytoplasmic sperm injection

Abstract Study question Is ovarian stimulation with follitropin delta (FD) in its individualized regimen as efficacious as follitropin alfa and human menopausal gonadotropin (FAHMG)? Summary answer Ovarian stimulation with individualized FD dosing resulted in a significantly higher fertilization rate and number of blastocysts with good quality compared to conventional FAHMG dosing. What is known already Previous randomized controlled trials conducted in Japan, China, Europe, and both North and South America have demonstrated that ovarian stimulation using the individualized FD dosing regimen, which is based on serum anti-Müllerian hormone (AMH) levels and body weight, effectively modulated the ovarian response without compromising pregnancy and live birth rates. Study design, size, duration A retrospective study was conducted by reviewing the medical records of 1,720 IVF/ICSI cycles in 1,272 Japanese patients. The primary endpoint of the study was the ongoing pregnancy rate, assessed at 8 weeks of gestation, in the frozen-thawed embryo transfer. Participants/materials, setting, methods The FD treatment consisted of a fixed daily dose individualized according to each patient’s initial AMH level and body weight (AMH <2.04 ng/ml: 12 μg; AMH ≥2.04 ng/ml: 0.19 to 0.10 μg/kg; min-max 6–12 μg). The FAHMG dose was determined by the antral follicle count (AFC), with the following protocol: for AFC <25, 300 IU/day; for 40> AFC ≥25, 225 IU/day; and for AFC ≥40: 150 IU/day). A progestin-primed ovarian stimulation (PPOS) protocol was applied. Main results and the role of chance Multiple regression analyses (MRA) showed that the use of FD was negatively correlated with the number of oocytes retrieved (NOR) (P = 0.001), and positively correlated with the fertilization rate (FR) (P = 0.02). NOR was comparable between FD and FAHMG (10.9 ± 6.4 versus 11.8 ± 7.8) when the Mann-Whitney test (MW) was applied (P = 0.111), but significantly higher in FAHMG when the patient is younger than 40 years old with AMH ≥2 ng/ml (P < 0.01). The total gonadotropin use was significantly (P < 0.001) reduced from an average of 167.1 ± 46.9 μg (2789.9 ± 782.9 IU) FAHMG to 102.6 ± 31.6 μg FD. FR was significantly higher in FD compared with FAHMG (78.7% versus 74.1%, P < 0.05) on MW. Additionally, MW indicated a significantly higher number of good-quality blastocysts in FD (2.9±2.6) versus with FAHMG (2.4±2.5) (P < 0.05). The blastocysts obtained from FD also showed a significantly higher implantation rate (50.1% versus 39.7%, P < 0.01) and a comparable ongoing pregnancy rate (28.7% versus 25.7%, P = 0.292) compared to those from FAHMG as determined by chi-square test. The higher implantation rate in FD may be attributed to the greater number of good-quality blastocysts obtained compared with FAHMG. Limitations, reasons for caution This study only covered the clinical outcome of women undergoing PPOS protocol with frozen-thawed blastocyst transfers. Wider implications of the findings The present study shows that FD in its individualized fixed-dose regimen has the potential to improve the success rate in frozen-thawed embryo transfer across all ages and with a lower gonadotropin consumption compared to conventional FAHMG dosing. Trial registration number not applicable

O-018 Utilisation of second successive ejaculate as a way to overcome poor semen parameters for couples who wish to undergo conventional-IVF

Abstract Study question Can a second successive-ejaculate (SSE) be an option for patients with poor sperm parameters (PSPs) on ovum pick-up (OPU)-day who prefer to undergo conventional-IVF (c-IVF)? Summary answer SSE can be a valid option for patients with PSPs who do not wish to undergo ICSI as an assisted reproductive technology (ART) treatment. What is known already ART is applied when first-line treatment for infertility (timed intercourse or intrauterine insemination) is unsuccessful. The male partner is requested to follow ejaculatory abstinence, which according to the WHO guidelines is 2-7 days, while ESHRE recommends 3-4 days. Among these couples there are cases that require ICSI due to semen analysis with PSPs on the day of OPU. However, some couples are reluctant to undergo ICSI and prefer c-IVF as a treatment method. For those cases, we request a second semen sample on the same day (time interval: 1-4 h) and c-IVF is performed using the two ejaculates combined. Study design, size, duration This is a single-centre retrospective study employing 212 semen from male partners with PSPs in the first semen collection on the OPU-day and subsequently, an SSE was requested in order to perform c-IVF according to patients’ wishes. The data were collected from May 2006 to October 2023 in our clinic (SSE-group). The Control group included 271 semen samples from men where c-IVF was conducted using one ejaculate from January to December 2021 in our clinic. Participants/materials, setting, methods The sperm parameters were analysed according to the WHO manual for the examination and processing of human semen and compared between the first and second ejaculates in the SSE-group. Fertilisation rate (FR), percentage of clinically utilised embryos and clinical pregnancy rate were compared between the SSE-group and the Control group. The SSE-group had 254 ART-cycles and the mean maternal age was 36.8±4.9y.o. The Control group included 432 ART-cycles and the mean maternal age was 38.1±5.2y.o. Main results and the role of chance In SSE-group the semen volume of the first ejaculate was significantly higher compared to the second ejaculate (P< 0.0001). The sperm concentration and sperm motility were significantly higher in the second ejaculate compared to the first one (P<0.0001). In the same fashion, the total-motile sperm-count was significantly higher in the second ejaculate compared to the first (P=0.01). All the semen parameters in the Control group were significantly higher compared to both first and second ejaculates from the SSE-group (P<0.00001). The normal FR was significantly higher in the Control group compared to the SSE–group (P<0.01). On the other hand, there was no significant difference in the percentage of embryos that could be utilized (cryopreserved + freshly-transferred) between the two groups (P=0.218). Pregnancy rate for fresh embryo transfers (ETs) was significantly higher in the SSE-group compared to the Control (P=0.005), while there was no significant difference between the two groups for pregnancy rate for frozen/thawed ETs (P=0.728). The main strength of our study is that the utilisation of the SSE combined with the first ejaculate did not decrease the pregnancy rate for the couples with PSPs that wished to follow c-IVF as their treatment and it could actually provide an effective option for them. Limitations, reasons for caution SSE may improve the sperm parameters and give the opportunity to infertile couples to undergo c-IVF according to their wish. However, since the FR was lower in the SSE-group, it is of paramount importance to provide sufficient explanation to those couples and obtain their consent when requesting an SSE. Wider implications of the findings Overall, SSE had positive effect to a large number of parameters evaluated in this study. Additionally, the rate of utilised embryos was similar between the two groups. Two successive-ejaculates combined can be employed without any negative impact for couples with PSPs on the OPU-day that are reluctant to undergo ICSI. Trial registration number not applicable

P-025 Mitochondrial activity in sperm is associated to blastocyst development: a double-donation study

Abstract Study question Do oxidative parameters in sperm affect oocyte fertilization or blastocyst development in double-donation ICSI cycles? Summary answer While intracellular oxidative stress in sperm does not suppose an impact on fertilization or embryo development, mitochondrial activity is related to increased blastocyst development rates What is known already Reactive oxygen species(ROS) are molecules causing a detrimental effect on sperm quality and function, especially when they are imbalanced with respect to the sperm and seminal plasma antioxidant capacity. Previous research have extensively demonstrated that sperm affected by oxidative stress(OS) show reduced fertilizing capacity, and lead to embryos with impaired development after conventional IVF. The role of OS in sperm in the case of ICSI is, however, controversial because many factors may cause a bias. Not only could this explain the inconsistent results reported thus far, but would also support the need of testing healthy donor gametes to reduce variability. Study design, size, duration This research was devised in the context of a collaboration between a sperm bank and a public university, and was set as a blinded, retrospective study that included samples from 45 sperm donors used in a total of 55 oocyte donation cycles. Intracellular levels of total ROS and of superoxides (O2•-), oxidative stress and mitochondrial activity (MA) were evaluated in frozen-thawed sperm donor samples, and related to the embryo outcomes obtained from the same samples. Participants/materials, setting, methods Samples from selected sperm donors were used to fertilize oocytes through ICSI in oocyte donation programs, and fertilization and blastocyst rates were recorded. Sperm viability and intracellular levels of total ROS and superoxides were determined through a combined assay (2’,7’-Dichlorofluorescin diacetate/Hydroethidine/Sytox Red); oxidative stress and sperm viability were assessed through co-staining with CellRox Far Red and propidium iodide (PI); and MA (mitochondrial membrane potential) and sperm viability were evaluated after staining with DiOC6(3) and PI. Main results and the role of chance The average and standard deviation for fertilization and blastocyst rates were 76.8%±17.3% and 67.5%±22.8%, respectively. Taking the viable sperm population into account, percentages of sperm with high ROS and low O2•- were 17.6%±15.9%, those with high ROS and high O2•- were 0.5%±0.4%, percentages of sperm with low ROS and low O2•- were 25.9%±9.2%, and those with low ROS and high O2•- were 54.5%±21.1%. The evaluation of oxidative stress with CellRox revealed that the percentage of viable sperm with high OS was 63.1%±16.5%. Neither intracellular levels of ROS and superoxides, nor oxidative stress in viable sperm were found to be correlated to fertilization or blastocyst rates (P>0.05). In spite of this, the percentage of viable sperm with high MA, which was 20.7%±10.5%, was observed to be positively correlated to blastocyst rates (Rs=0.276; P=0.04). While sperm mitochondria do not participate in further embryo development, a greater mitochondrial activity in post-thawed donor sperm could indicate a healthier sperm function. Mitochondrial activity could, therefore, be used as a predictive biomarker for embryo development. Limitations, reasons for caution The sample size is the main limitation of this study, despite the efforts made to include a high number of sperm donors. Wider implications of the findings Results obtained in the present study show that ICSI evades the detrimental effects of sperm oxidative stress on fertilization and embryo development. Yet, sperm mitochondrial activity could be used as a biomarker to test the ability of a sperm donor to generate healthy embryos that develop up to blastocyst stage. Trial registration number Not applicable

P-228 Competence of injected MI versus MII sibling oocytes in terms of normal fertilization, blastocyst development and euploidy

Abstract Study question Is the injection of immature MI oocytes as effective as injection of their siblings mature MII oocytes regarding normal fertilization, blastocyst development and euploidy? Summary answer MI oocytes show a lower rate of fertilization, a similar blastulation rate, and a slightly lower rate of euploidy compared to their MII sibling oocytes. What is known already Although several studies have been performed on immature MI oocytes evaluating their developmental competence after intracytoplasmic sperm injection (ICSI), all have been performed on oocytes that extruded the first polar body by the time of injection. ICSI is typically performed on MII oocytes which have completed the process of maturation. Some laboratories also inject MI oocytes, nevertheless, they are generally not used due to the lack of required molecular and cellular changes to support normal development. However, there is limited research on the reproductive potential of oocytes in MI. Consequently, our current practices are founded on assumptions. Study design, size, duration This is a retrospective, observational study of 168 oocytes injected in MI and 839 sibling oocytes injected in MII from 82 ICSI cycles with Preimplantation Genetic Testing for Aneuploidies (PGT-A) performed in a single IVF clinic between April 2018 and December 2023. All PGT-A cycles included in this study contained at least one MI oocyte injected. Participants/materials, setting, methods After oocyte retrieval and denudation, the oocytes were classified as MII, MI or GV. MI oocytes with extruded polar body by the moment of ICSI were reclassified as MII. All MII and MI oocytes were inseminated and the embryos that reached blastocyst stage were biopsied on day 5, 6 or 7. Normal fertilization rate, blastocyst formation rate and euploidy rate were compared between MI-derived embryos and their MII-derived siblings. Main results and the role of chance A total of 1,094 oocytes were retrieved from 83 ICSI cycles with PGT-A, comprising 839 (76.7%) MII, 168 (15.4%) MI y 87 (7.9%) VG. The normal fertilization rate was significantly higher in oocytes injected at MII compared to MI (76.9% vs 53.0%, P < 0.001). The rates of abnormal fertilization in MII and MI oocytes were as follows: 1 PN 0.8% vs 0.6%, P = 0.342; 3PN 2.6% vs 1.2%, P = 0.075; 0 PN 19.7% vs 45.2%, P < 0.001. Interestingly, the blastulation rate of zygotes with normal fertilization was very similar between both groups (46.4% vs 43.8%, P = 0.708), as well as the proportion of biopsied blastocysts on day 5 (74.2% vs 71.8%, P = 0.858), on day 6 (23.1% vs 25.6%, P = 0.946), and on day 7 (2.7% vs 2.6%, P = 0.594). The euploidy rate of the 298 biopsied MII-derived blastocyst was 36.2%, compared with 23.1% of the 39 MI-derived blastocysts biopsied (P = 0.077). Despite the limited sample size, it is noteworthy that three out of the four euploid blastocysts derived from MI oocytes, which were transferred in single embryo transfers, successfully led to pregnancy resulting in one healthy live birth, one ongoing pregnancy and one miscarriage at 9 weeks of pregnancy. Limitations, reasons for caution While this study provides valuable insights into the developmental competence of MI oocytes, it is not exempt from certain limitations, such as its retrospective nature, limited sample size and ethical considerations regarding the injection of putative immature oocytes. Wider implications of the findings MI oocytes showed lower fertilization rates compared to their MII siblings. However, once fertilized, MI-derived embryos had similar blastulation rates and a trend towards lower euploidy rates. Our study suggests that MI oocytes may result in successful pregnancies and live birth, but further research is needed to validate these findings. Trial registration number Not applicable

P-026 Retrospective descriptive analysis of 1300 ICSI-AOA cycles: evaluating clinical outcomes and child health in the context of artificial oocyte activation (AOA)

Abstract Study question How does the outcomes of ICSI-AOA and child health differ from those reported in the literature? Summary answer ICSI-AOA clinical outcomes, including fertilization, embryo quality, pregnancy, live birth rates, and anomaly rates in children, align with those reported for ICSI alone. What is known already The use of AOA to artificially induce calcium rises by employing calcium ionophores has been shown as an effective approach in overcoming fertilization failure following ICSI, especially in couples facing severe male factor infertility. However, a significant gap exists in reporting live birth outcomes. On the other hand, uncertainties surrounding the health implications for children born through this method necessitate further investigation due to the limited sample sizes in previous reports. This descriptive analysis fills that gap by assessing 1300 cases, providing crucial insights into the clinical outcomes and health status of babies resulting from ICSI-AOA treatment. Study design, size, duration This retrospective study analyzes 1300 cases of ICSI-AOA between 2018 and 2020. Clinical outcomes in this study includes: fertilization, embryo quality, pregnancy, and live birth rates. Questionaries regarding the children health were included from birth to two years. Participants/materials, setting, methods The study included 1300 cases that referred to Isfahan Fertility and Infertility Center (Isfahan, Iran) and underwent ICSI-AOA due to: 1) cases with testicular biopsy (N = 458), 2) cases with 99-100% sperm head abnormal morphology and/or 3) previous low fertilization or total fertilization failure after ICSI (N = 842). In all cases, to induce AOA, oocytes were subjected to a 10-minute exposure of 10 micromolar ionomycin following ICSI. Main results and the role of chance From 1300 couples, 11,756 mature, 2,012 immatures, 193 degenerate, and 76 uninjectable oocytes were obtained. Fertilization were 59.69%, and 52.18% for mature and immature oocytes, resulting in cleavage rates of 96.93% and 93.80%, respectively. On day 3, 25.14% grade A, 31.49% grade B, and 43.36% grade C were observed. In 321 couples, 571 fresh embryos were transferred on day 3, resulting in clinical pregnancy and live birth rates of 20.82%, and 19.55%, respectively. For 32 couples with 44 fresh ET on day 5, rates were 9.38%, and 9.38% respectively. Out of 3270 vitrified embryos on day 3, 1743 were thawed (337 couples) and 1147 were transferred in 643 ET cycles. Clinical pregnancy and live birth rates were 21.61%, and 20.37%, respectively. 1527 embryos remain frozen. Out of 2,974 day 5-embryos, 2,571 did not reach blastocyst, leaving 403 vitrified embryos in 699 cases. Among them, 197 embryos remain frozen (80 cases) while 206 were transferred, with clinical pregnancy and live birth rates of 22.11%, and 21.80%, respectively. Cancellation rate, encompassing failed fertilization, cancellations on multiple days, and after freeze-thawing, was 20.16%. Among 266 live births, seven children showed anomalies, including achondroplasia, combined immunodeficiency, left eye deviation, intrauterine growth restriction, hydrocephaly, epilepsy, and umbilical hernia. Limitations, reasons for caution The main limitations of this study were the lack of comprehensive information on previous cycles, the absence of an ICSI control group, and the limited long-term follow-up on the physical and mental health of children born through the ICSI-AOA method beyond age two. Wider implications of the findings ICSI-AOA yields helpful clinical outcomes in high fertilization, pregnancy, and live birth rates in infertile couples, especially those with severe male factor infertility similar to ICSI in literature. Furthermore, presently, there seem to be no real concerns about health of babies born through this method, aligning with ICSI studies. Trial registration number IR.ACECR.ROYAN.REC.1401.038

O-186 A novel micro-insemination method, assisted sperm fusion insemination, increases the embryo utilization rate compared to conventional intracytoplasmic sperm injection

Abstract Study question Does a novel micro-insemination method, assisted sperm fusion insemination (ASFI), improve fertilization and embryonic development compared to conventional intracytoplasmic sperm injection (C-ICSI)? Summary answer ASFI showed comparable fertilization and blastocyst formation rates to C-ICSI, while achieving significantly higher embryo utilization rates. Furthermore, there were no degenerated oocytes after ASFI. What is known already We have reported that ASFI yielded fertilization and blastocyst rates similar to those obtained with rescue ICSI performed at 6 hours after in vitro fertilization (IVF). In ASFI, fertilization is achieved by pressing a zona pellucida (ZP)-bound sperm onto the oocyte membrane without breaking the oocyte membrane. However, there was a risk of artificial polyspermy because the sperm were pressed against the unfertilized oocytes after IVF. Moreover, it is uncertain whether fertilization occurred with sperm that penetrated into the oocyte via IVF or that were pressed against the oocyte membrane. Clarifying this requires performing ASFI of the oocyte without IVF. Study design, size, duration We prospectively evaluated the effectiveness of ASFI in a sibling oocyte study including 14 subjects from which at least two metaphase II (MII) oocytes and one immature or degenerated oocyte were retrieved. Between December 2022 and October 2023, 119 MII oocytes were obtained in 22 oocyte retrieval cycles and used in the study. Sibling MII oocytes were randomly assigned to a C-ICSI or ASFI group. The present study was approved by the local ethics committee. Participants/materials, setting, methods In the ASFI group, 10,000 motile sperm were incubated with at least one immature or degenerated oocyte for 3 hours in 100 μL of medium per patient. After incubation, the motile sperm bound to the ZP were aspirated directly using an injection pipette. The heads of these ZP-bound sperm were pressed onto the membrane of MII oocytes for 10 seconds using the injection pipette. C-ICSI was conducted using the conventional method. Main results and the role of chance The mean and standard deviation of age of the women and number of MII oocytes per cycle were 39.9 ± 2.2 years and 5.5 ± 1.8, respectively. All sperm collected from the ZP adhered to the MII oocyte membrane after ASFI. Furthermore, time-lapse imaging revealed that all sperm adhering to the MII oocyte membrane were subsequently incorporated into the oocyte. Comparison of the ASFI and C-ICSI groups did not reveal any significant differences in 2 pronuclear embryo rate (ASFI group: 85.4% vs. C-ICSI group: 71.8%), degeneration rate (0% vs. 5.6%), good-quality embryo rate at day 3 (63.4% vs. 52.9%), blastocyst formation rate (67.6% vs. 51.9%), or good-quality blastocyst (Grade 3BB and above by the Gardner criteria) formation rate (29.4% vs. 15.4%). On the contrary, the embryo utilization rate (defined as the total number of embryos transferred and cryopreserved divided by the number of MII oocytes) was significantly higher in the ASFI group compared to the C-ICSI group (47.9% vs. 25.4%, P < 0.05). Limitations, reasons for caution This study was limited by using the ZP for sperm selection. To expand its applicability, a method to identify sperm capable of fusing with an oocyte without using the ZP must be established. Additionally, since our sample size was small, larger studies are required to confirm our findings. Wider implications of the findings ASFI is expected to enhance the survival rate of oocytes and to increase the number of embryos available for implantation, not only compared to rescue ICSI but also C-ICSI. ASFI could aid in understanding fertilization mechanisms by enabling direct observation of oocyte-sperm fusion during the process. Trial registration number UMIN000050184

O-301 Progesterone Primed Ovarian Stimulation (PPOS) for DuoStim combined with PGT-A in poor prognosis patients: a valuable strategy

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

P-274 Humid or dry culture with medium renewal: impact on A-Grade Day 5 blastocysts. Randomized prospective cohort study

Abstract Study question Could the humid embryo culture impact the development of top-graded blastocysts? Summary answer While no differences were observed in the overall blastocyst development rate, humidity conditions emerged as a significant factor influencing the achievement of A-Grade D + 5 blastocysts. What is known already In recent years, several publications have highlighted the advantages of humid culture (HC) over dry culture (DC) (<45% relative humidity, RH). HC, with its lower risk of media evaporation and consequent osmolality changes. Nevertheless, to the best of our knowledge, this is the first prospective study addressing this aspect through oocytes from the same cohort. Study design, size, duration We have been conducting a randomized control trial (RCT) consisting on 379 MII oocytes from 30 patients from August 2022 until January 2024 (and ongoing). After ICSI procedure, sibling oocytes were divided following 1:1 ratio into both humid (>70% HR) and dry (0% HR) chambers of a Geri incubator (Genea Biomedx, Australia). Ejaculated sperm ICSI cycles with >5 fresh MII were included in the analysis. Time-lapse cleavage timings were manually gathered. Participants/materials, setting, methods Extended culture to the blastocyst stage (D + 5, D + 6 or D + 7) was performed in 80μL of single step G-TL medium (Vitrolife, Sweden). HC remained undisturbed while medium was renewed on D + 3 in DC by changing the culture dish. Fertilization, overall blastocyst development and A-Grade Day 5 blastocysts rates (ASEBIR’s morphology criteria) were compared. Statistical analysis consisted on Chi-squared test, t-student test and logistic regression for computation of Odds Ratio (OR, 95%CI). Main results and the role of chance While no statistically significant differences were observed in the fertilization rate (DC: 76.4%; HC: 69.1%, NS) or the overall usable blastocyst development rate (DC: 48.6%; HC: 53.1%, NS), noteworthy distinctions emerged in the proportion of top-graded embryos based on ASEBIR’s morphology criteria. Specifically, the A-Grade Day 5 blastocyst development rate displayed significant disparities when comparing all fertilized embryos in each culture type (DC: 19.9%; HC: 32.3%; p < 0.05) and also among all usable blastocysts (DC: 40.8%; HC: 60.9%, p < 0,05). The Odds Ratio for achieving A-Grade Day blastocysts depending on type of humidity conditions was 1.87 (1,099 – 3,235) for the humid culture. Moreover, time-lapse imaging facilitated the detection of direct cleavage presence, which was similarly distributed between both incubation types (DC: 27/146; HC: 23/130, NS). However, only 3 embryos showing direct cleavage and cultured in HC reached the blastocyst stage at Day 6, while in dry incubator, 10 embryos with direct cleavage were able to develop to usable blastocysts, three of them being A-Grade on Day 5. Limitations, reasons for caution This study is currently ongoing, aiming to increase the sample size. Future analyses will explore clinical outcomes, especially for underrated embryos, day 6 blastocysts, and those with morphodynamic phenomena. While A-grade blastocyst rate variations may not impact pregnancy outcomes directly, they could influence cumulative success rates. Wider implications of the findings These data highlight that day+3 medium renewal doesn’t equalize conditions to humid culture. Ongoing efforts involve validating these insights through an expanded sample size and extensive outcome analysis. Investigating the long-term impact on clinical outcomes and refining culture protocols can amplify the relevance of these findings in assisted reproductive technologies. Trial registration number pending_protocolOK

P-254 Optimizing clinical outcomes in failed ICSI Donor cycles: evaluating the efficacy of Assisted Oocyte Activation

Abstract Study question Does Assisted Oocyte Activation (AOA) confer benefits to couples undergoing donor ICSI cycles with a history of poor clinical outcomes and poor sperm quality? Summary answer AOA demonstrates efficacy in enhancing reproductive outcomes in donor ICSI cycles, in cases where oocyte activation deficiency (OAD) is attributed to a sperm factor. What is known already Oocyte activation is a spatial-temporal process caused by a series of intracellular calcium oscillations (Ca2+)triggered by the entry of sperm into the oocyte. Insufficient Ca2+ oscillations, primarily attributed to the absence of sperm-specific phospholipase C zeta (PLCζ) can result in OAD that leads to unsuccessful fertilization and poor embryo growth. Ionomycin and calcimycin, are the most common activating agents used to restart meiosis by raising intercellular Ca2+. As far as our knowledge extends, there aren’t studies reporting clinical outcomes on the use of AOA in patients who undergo cycles both without and with this treatment in donor oocytes. Study design, size, duration This study includes 38 couples undergoing ICSI cycles with donor oocytes from January 2020 to December 2023. After a previous cycle with a fertilization rate of < 50% and poor clinical outcomes, these couples underwent a subsequent attempt with AOA. Maternal age of recipients was 42±4.7 and 42±5 years in cycles without and with AOA (P=NS). Fertilization, blastocyst quality, implantation, clinical pregnancy rates (CPR) and pregnancy loss were compared between the cycles with and without AOA. Participants/materials, setting, methods Oocytes were exposed to a ready-to-use solution of Ca2+-ionophore A23187 (GM508 Cult-Active, Gynemed, Germany) for 15 min after sperm injection. Embryos were cultured in a time-lapse technology (Geri®) andevaluated according to the Gardner blastocyst grading system. Embryo transfer was performed on Day 5 or Day 6. Vitrified and fresh ejaculated sperm samples were used. T-test and Fisher’s Exact test were used for statistical analysis. P-value was considered statistically significant at a threshold of < 0.05. Main results and the role of chance A total of 38 men underwent ICSI cycles alongside their female partners, utilizing donor oocytes. The men’s average semen parameters were as follows: concentration of 15.3±25 x 106/mL, motility of 20.1±15%, and 2.0±1% morphology. In the initial 41 cycles without the AOA procedure, an average and standard deviation of 5.8±3 donor oocytes were injected, resulting in a fertilization rate of 33.9% (81/239) and a blastocyst rate of 30.8% (25/81). Among these cryopreserved embryos, 72% (18/25) were identified as top-quality, characterized by A and/or B quality of both inner cell mass and trophectoderm. Couples (N = 13) who underwent a transfer had an implantation rate of 19% (4/21), with CPR of 28.5% (4/14), which resulted in a pregnancy loss of 75% (3/4). Subsequently, these couples underwent 47 ICSI cycles with AOA, injecting an average of 5±1.7 donor oocytes (P=NS). This led to a significantly higher fertilization rate of 74.2% (176/237, P < 0.0001) and a blastocyst rate of 51.7% (91/176, P < 0.01), with 61% (56/91) identified as top-quality embryos (P=NS). Couples (N = 31) who underwent a transfer had a notable increase in implantation rate to 52% (25/48, P < 0.05), accompanied by a significant rise in CPR, reaching 71.4% (25/35, P < 0.01). Importantly, pregnancy loss decreased to 26% (6/23, P=NS). Limitations, reasons for caution The number of patients included in this study is limited. Although we excluded that OAD could be oocyte-related by using donor gamete, PLCζ or mouse oocyte activation tests were not performed to confirm sperm-related OAD. All patients, diagnosed with teratozoospermia, likely exhibit abnormalities causing PLCζlocalization issues or its absence. Wider implications of the findings In our study, sperm’s inability to generate accurate calcium signaling pathways correlates with poor clinical outcomes, even with donor oocytes. Consequently, for couples facing Oocyte Activation Deficiency attributed to sperm factors, implementing the AOA procedure significantly improves fertilization, blastocyst formation, implantation, and clinical pregnancy rates. Trial registration number na

O-300 Dydrogesterone versus GnRH-antagonists to supress the LH surge in IVF/ICSI cycles

Abstract Study question Does Dydrogesterone block premature Luteinaizing hormone surge and ovulation during the controlled ovarian stimulation without compromising of IVF/ICSI outcomes? Summary answer Dydrogesterone and GnRH-antagonist have equal effectivness for prevention of premature luteinizing hormone surge and ovulation without any adverse effect on laboratory and clinical results. What is known already Premature LH surge and premature ovulation during the controlled ovarian stimulation is one of the main causes of cycle cancellation.Traditionally, during in-vitro fertilization for controlled ovarian stimulation to prevention of premature LH surge clinicians use GnRH antagonists. Recent data indicates that premature LH surge may be prevented also by progestins, since they have a role in suppression of preovulatory LH surge. In past, progesterone was not used during ovarian stimulation due to the negative effect on endometrial receptivity, while nowadays advanced techniques give possibility to use it as an alternative to a GnRH antagonists using the freeze-all strategy. Study design, size, duration In this prospective, randomized study were participated sixty -seven egg donors. Sudy was conducted on the basis of Clinic Leadermed (Georgia, Tbilisi) from October of 2021 to November 2023. The Ethics Committee of clinic Leadermed approved this study and appropriate informed consent was obtained from all participants. Participants/materials, setting, methods Recombinant gonadotropin was administered for control ovulation stimulation in oocyte donors. According to the ovarian response HMG was added. Intervention group (group I, n = 34) received 20 mg of Dydrogesterone from the first day of stimulation till trigger day and the control group (group II, n = 33) received GnRH-antagonist when the leading follicle reached 14 mm in diameter till trigger day. Triptorelin acetate 0.3 mg was administered when at least 3 follicles reached ≥18 mm in diameter. Main results and the role of chance There were no statistically significant differences in the oocyte donors age, BMI and AMH levels between two groups. None of the participants experienced a premature LH surge and ovulation. There was no statistically significant difference in the number of MII oocytes (23,7 vs 25.9 p = 0.3). The duration of stimulation and total dose of gonadotropins was similar in both groups (9.6 vs 9.9 days, p = 0.41), (2269.2 IU vs 2423.4 IU, p = 0.08) respectively. There was no difference in fertilization rates (85.62% vs 83.94%, p = 0.41), as well as blastulation rates and top-quality blastocysts (65.78% vs 69.72%, p = 0.17; 54.50% vs 51.75%, p = 0.21). Regarding clinical pregnancy rate, the results were similar, with no significant differences between two groups (66.28% vs 68.10%, p = 0.8). Limitations, reasons for caution The limitation of the study is that embryotransfer was performed as in patients (age till 49years), as well as in surrogate mothetrs (age till 41 years). Wider implications of the findings Dydrogesterone and GnRH-antagonist has equal effectiveness for prevention of premature LH surge without affecting of IVF outcomes. It is a financially profitable and easy to use which can be used as in oocyte donors as well as in patients where embryo transfer is not planned in the current cycle. Trial registration number non-clinical trial

P-023 Gene expression in human sperm as biomarkers and tools for the assessment of male infertility

Abstract Study question Can gene expression as AURKA, HDAC4, and CARHSP1 be used for the diagnosis of sperm quality exploring male infertility? Summary answer The comprehensive outcome of gene expression involving AURKA, HDAC4, and CARHSP1 serves as a key to classifying sperm and predicting their functionality. What is known already Diagnostic male infertility is largely based on semen parameters according to the World Health Organization (WHO) reference values. While these parameters assess sperm quality, they do not provide insights into sperm quality and have a limited predictive value on natural fecundity, fertilization rates, and assisted reproductive technology (ART) outcomes. Study design, size, duration This prospective study investigates the gene expression involved in mitosis, epigenetic regulation, and early embryo development. From February to June 2023, we included all men aged from 20 to 60 years having sperm parameters analysis in our unit center. A total of 277 semen samples were collected, 129 (46.4%) exhibiting three normal sperm parameters according to WHO criteria. Among these, 32 samples showed oligo-astheno-teratozoospermia (OAT), while 116 samples had 1 or 2 abnormal parameters. Participants/materials, setting, methods The mRNA expression levels of three candidate genes (AURKA, HDAC4, and CARHSP1) were quantified in fresh ejaculate semen using real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The thresholds for the spermatozoa function index (SFI) were established by assessing the combined expression of these target genes, considering the morphology and the capability to reach a blastocyst stage. The sperm were ranked according to the concentration (million/ml), motility (%) and morphology (%). Main results and the role of chance We assess the quality of spermatozoa for three biomarkers by analyzing their mRNA expression levels. Receiver operator characteristics curves (ROC) indicate a combined index value expression. This model establishes an index threshold > 320 for an “over-index value,” distinguishing spermatozoa with a normal capacity, an index threshold < 290 for an “under-index value,” and 290 < index < 320 for an “intermediate-index value”. Among the 277, we find 112 (40.4%) have an over-expression index; 161 (58.1%) have an under-expressed index and 4 (1.4%) have an intermediate-index. Interestingly, among the 129 samples with the three normal WHO parameters, we show that 82 (63.5%) have an over-index value, 44 (34.1%) have an under-index value, and 3 (2.3%) display an intermediate-index value. In addition, all the 32 OAT samples have an under-index. These findings suggest that the OAT sperm population implies a potential epigenetic network connected with our three target genes. Furthermore, even in cases with normal sperm parameters, there may be an under-expressed index, indicating lower functionality in terms of fertilization and early embryo development. Limitations, reasons for caution The present method was validated only on ejaculate semen in men free of any chronic disease and any anti-oxidant treatment. This prospective study requires further validation in the largest population. Wider implications of the findings This study’s results could highlight understanding a chaotic early embryo development in ART and used to explore male infertility; particularly in unexplained infertility. Trial registration number not applicable

P-581 Optimizing efficiency and enhancing patient convenience in DuoStim cycles: A study exploring the continuous application of corifollitropin alpha

Abstract Study question Corifollitropin alfa in DuoStim cycles: is weekly continuous dosing a viable alternative to daily FSH administration for comparable oocyte yield? Summary answer Employing corifollitropin alfa on a weekly basis throughout a DuoStim cycle achieves comparable oocyte numbers to daily FSH while significantly reducing the number of injections. What is known already The DuoStim protocol is frequently employed in patients with poor response or low ovarian reserve. However, its utilization has recently been proposed in patients who require elective oocyte/embryo freezing with the aim of maximizing the number of gametes/embryos obtained in a menstrual cycle. While its popularity is growing, the prolonged stimulation period involves the administration of a high number of injectable medications adding to the treatment burden. Study design, size, duration A non-inferiority, paired-matched study was planned with a minimum sample size of 13 patients per group, based on a tolerated difference of ± 1 oocyte, SD: 1, 80% power and type 1 error: 5%. Study group (n = 30): patients undergoing DuoStim from November 2022–December 2023. The control group (n = 30) was selected from patients undergoing DuoStim in years 2021-2022 following a standard daily-FSH administration. Controls were 1-to-1 matched based on AMH (±0.4 pmol/l) and age. Participants/materials, setting, methods The study group received consecutive injections of 150µg corifollitropin alfa every eight days, along with uninterrupted oral administration of natural progesterone 200 mg/day (LH surge prevention) throughout the follicular and luteal phases. The control group employed a daily-FSH/flexible GnRH-antagonist protocol. GnRH-agonist trigger was used in the follicular phase, whereas either hCGr and/or GnRH-agonist triggering was allowed in the luteal phase. Main results and the role of chance Baseline characteristics: age (years), AMH (pmol/l), and BMI (Kg/m2) were comparable between control vs study group (39.7vs40.3, 6.1vs6.1, 24.4vs23.9; p0.05, p0.5 and p0.9, respectively). In terms of the primary objective: total COCs and MII yield (follicular + luteal phase); the standard daily-FSH protocol vs study group achieved a similar total oocyte and MII yield (10.9±5.5 vs 10.4±7.0, p0.6 and 8.7±4.8 vs 7.8±5.6, p0.4, respectively). Additionally, all secondary outcomes (fertilisation rates, number of blastocysts, days of stimulation and gonadotrophin cost) showed no significant differences between the groups. Notably, the study group experienced a remarkable reduction in the number of injections needed to complete a DuoStim cycle [3.8±1.1 vs 30.9±12.9, p < 0.001; MD -27.1±12.5, (95%CI, -49 to -8)]. Limitations, reasons for caution The secondary outcomes revealed comparable fertilisation rates and blastocyst numbers between the two groups. Nevertheless, it is important to interpret the laboratory results beyond oocyte collection cautiously, considering the smaller sample size (n = 23/group). Larger participant cohorts are necessary to validate these findings and mitigate the risk of type II errors. Wider implications of the findings This protocol involving long-acting FSH in combination with natural progesterone offers a viable alternative for patients undergoing DuoStim. It enables comparable results in terms of egg retrieval while minimizing the need for injectables, without increasing the treatment duration or costs. Consequently, it alleviates emotional and therapeutic burdens experienced by patients. Trial registration number NCT05815719

P-165 Benefits of ultra-low oxygen tension (2%) in patients with low quality embryos or previous arrest: the patient as its own control

Abstract Study question Is reduction in oxygen tension from 5% to 2% after D3 of extended culture beneficial for blastocyst development in patients with previous failed culture? Summary answer Ultra-low oxygen tension in extended culture improves blastocyst development (overall blastulation rate OBR), quality (top blastulation rate TPR) and increases the pregnancy rate (PR). What is known already The practice ESHRE guidelines recommends human embryo culture at low oxygen concentrations (2-8%). Traditionally atmospheric conditions (20% O2) were used but better outcomes were observed at low monophasic 5% O2. Human embryo leaves the oviduct (5-7% O2) to cross the uterus-tubal junction around D3-4 and reaches the uterin ultra-low O2 tension (2%). This decreasing gradient coincides with the shift from pre-compaction oxidative phosphorylation in the oviduct, to post-compaction glycolysis in the uterus. Few studies with ultra-low 2% O2 incubation suggested that the best O2 level may be stage-dependent and its reduction after D3 may be a more physiological approach. Study design, size, duration Prospective observational study in a single private hospital during 2023. 71 patients experienced a failed cycle of extended culture (n = 29 cIVF, n = 37 ICSI and n = 5 Sibling oocytes) with a poor embryo quality and low OBR, under standard monophasic 5% O2 incubation. In the subsequent cycle for these 71 patients (n = 24 cIVF and n = 46 ICSI and n = 1 Sibling oocytes) the O2 level was reduced to 2% from D3 onwards. Participants/materials, setting, methods After a failed extended culture, considered as the control cycle, the patients were enrolled for the study. Blastocysts were rated by Gardner’s classification. In the control cycle embryos were cultured under monophasic 5% O2 until D6. In the subsequent cycle embryos were cultured under 5% O2 until D3 and in ultra-low 2% O2 from D3 to D6. Sage 1-Step™ media was used for both cycles. The statistical analyses were done by chi-square and fisher test. Main results and the role of chance 71 patients were included in the study regardless of age, indication or technique. Each patient was its own control. The mean age was not different between the 2 groups (36.5±4 vs 37.2±4) but the attempt rank was logically statistically higher in the study cycle (2.1±1.4 vs 3.4±1, p<0.001). Oocyte maturity (75% vs 76.4%) and fertilization rate (66.8 vs 70.3%) were similar between groups. The average embryo quality at D3 was poor in both groups. OBR and TBR (40.7% vs 52.4%, p=0.004 and 15.8% vs 37.5%, p<0.001) were statistically higher after culture in ultra-low O2 conditions. Extended culture failure with absence of usable blastocyst (57.7% vs 19.7%, p<0.001) was statistically lower in ultra-low incubation group and the pregnancy rate PR per cycle (3.6% vs 25%, p=0.004) was seven fold higher after ultra-low culture. Limitations, reasons for caution The small size of the sample needs to be expanded in order to confirm the encouraging findings in a larger population. The study will continue during 2024 and will compel the live birth rate and the cumulative live birth rate. Wider implications of the findings Providing embryos with the most physiologic culture system would improve IVF cost-effectiveness and a better infertility management. Embryo energetic adaptation to suboptimal conditions results in impaired viability and lower pregnancy rates. In our study extended culture of low-quality embryos under 2% O2 after D3 yielded better culture and pregnancy outcomes. Trial registration number NA

P-359 Ten years’ experience using cryopreserved oocytes from In Vitro Maturation (IVM) or Controlled Ovarian Hyperstimulation (COH) cycles for fertility preservation in oncologic indications

Abstract Study question Is IVM a relevant option for fertility preservation in cancer patients, considering biological/clinical outcomes post-thawing, compared to COH? Summary answer This study shows that despite a significantly altered embryo morphology within the IVM group, the clinical outcomes remain comparable to those of COH. What is known already Fertility Preservation (FP) techniques are proposed to patients with cancer to provide the possibility of childbearing using their own gametes. Currently, oocyte vitrification after COH is the standard option. IVM serves as an alternative when COH is contraindicated, urgent cancer therapy is required, or in conjunction with ovarian cortex cryopreservation. Limited live births have been reported from frozen-thawed oocytes in IVM cycles of cancer survivors. Scarce data are available for comparing biological/clinical outcomes of frozen-thawed oocytes from IVM versus COH cycles in cancer survivors. Study design, size, duration This bicentric retrospective cohort study aimed to analyze the outcomes of all oocyte warming cycles in 73 cancer survivors having undergone oocyte vitrification for FP after COH and/or IVM. All of them had oocyte retrieval before the administration of gonadotoxic treatment and returned after being cured, with their oncologist agreement, for assisted reproduction treatments, between November 2012 and January 2024. Participants/materials, setting, methods Seventy-seven warming attempts followed by ICSI respectively from 37 COH and 40 IVM cycles were analyzed. Four patients benefited from an IVM and COH cycle. Survival, fertilization, top and good-quality embryos, defined at day-2 respectively as 4 and 3-5 adequate-sized blastomeres, without multinucleation with < 20% of cytoplasmic fragments, implantation, biochemical (hCG>100 UI/mL), clinical (intrauterine sac with fetal heartbeat) and live birth rates were compared using appropriate statistical tests. Main results and the role of chance The mean age and antral follicle count at the time of FP was similar in both groups. The indications for FP were breast cancer (n = 61), hematologic malignancies (n = 7), ovarian cancer (n = 3), other (n = 2). The number of cryopreserved oocytes tended to be higher in the COH group, when compared to the IVM group (8.5±7.3 vs 5.9±4.04; p = 0.16). The mean oocyte maturation rate after IVM was 68.6±23.1%. Oocyte survival, and fertilization rates were similar in COH and IVM groups, respectively (76.4±27.0% vs 76.1±22.0%; p = 0.76) and (67.1±34.3% vs 65.3±25.4%; p = 0.36). We reported a significantly better embryo quality at day-2 in the COH group when compared to the IVM group, with respectively top-quality embryos (44.0±34.2% vs 26.1±33.4%, p = 0,04) and good-quality embryos (62.2±38.0% vs 42.8±40.8%, p = 0.049), and also a significantly higher number of useful embryo in the COH group (2.7±2.6 vs 1.5±1.2, p = 0.03). Regarding clinical outcomes, the results were similar in COH and IVM groups, in terms of implantation rates (12.2±30.4% vs 15.5±35.6%; p = 0.86), biochemical (32.3% (10/31) vs 17.9% (6/28); p = 0.39), clinical pregnancies (25.8% (8/31) vs 17.9% (4/28); p = 0.54), live birth rates ( 25.8% (8/31) vs 14.3% (4/28); p = 0.34). Limitations, reasons for caution Statistical power to compare IVF outcomes after COH and IVM is limited by the few women who returned to use their frozen oocytes. The subjective nature of embryo morphology assessment. Wider implications of the findings This study is the largest evaluating frozen-warmed oocytes from IVM cycles in cancer patients, with 5 live births newly reported. A higher oocyte-yield may be necessary in IVM as the number of useful embryos is significantly lower. Novel approaches are being developed to improve maturation rates following IVM. Trial registration number Not applicable

P-683 Progestins vs flexible GnRH antagonist for LH suppression during controlled ovarian stimulation in POSEIDON groups 3 and 4 patients: Analysis of effectiveness and cycle outcomes

Abstract Study question How do cycle outcomes differ between controlled ovarian stimulations using progestins vs flexible GnRH antagonists for LH suppression in women with diminished ovarian reserve (DOR)? Summary answer While progestin use is associated with significantly higher blastocyst rate than GnRH antagonists in POSEIDON group 4, the latter gives better results in group 3. What is known already Controlled ovarian stimulation (COS) in patients with DOR carries the additional risk of premature luteinizing hormone (LH) surge and ovulation.GnRH antagonists are popular for LH suppression in COS because of their convenient, quicker onset of action, flexibility to use GnRH agonist as a trigger and continue as double stimulation, allowing rapid pooling of embryos. Ease of oral administration, combined with cost-effectiveness and retention of all the benefits of GnRH antagonists makes progestins an attractive alternative to GnRH antagonists for COS in patients with DOR who often need multiple stimulation cycles to enable the pooling of embryos. Study design, size, duration An open-label, multicentric, randomized controlled study was conducted over 1 year from January 2023 to December 2023. 140 women aged 21- 42 years and with AMH <1.2 ng/dl were randomized into 2 groups in a 1:1 ratio, using either oral progestins or GnRH antagonists for LH suppression. The two groups were further comparative analyzed under POSEIDON 3 and 4 subgroups. Those with BMI >40kg/m2 and with partners having severely compromised semen parameters were excluded. Participants/materials, setting, methods In Group A (PPOS): Medroxyprogesterone acetate 10 mg orally was given daily with gonadotropins (hMG 300- 375 IU) from cycle day 2 until trigger. In Group B (Antagonist), patients had daily GnRH antagonist, cetrotide 0.25 mg, subcutaneous administration started when the dominant follicles reached 12-14 mm size during controlled ovarian stimulation. This was continued until the trigger. Following oocyte retrieval and ICSI, embryos were cultured until D5 and vitrified fortransfer in subsequent HRT cycles. Main results and the role of chance Patients in Group A, using PPOS, were noted to have significantly better fertilization rates than those in Group B, using flexible GnRH antagonists (0.82 + 0.2 vs 0.66 + 0.4, p-value <0.01). Though the number of top-quality embryos (TQE) frozen was not significantly different between groups A and B (p-value = 0.44), a significant difference in the outcome was noted when comparatively analyzed under the subgroups of POSEIDON groups 3 and 4. While patients in POSEIDON group 4 got significantly more top-quality embryos (TQE) when progestins were used for LH suppression (1 + 0.11 vs 0.77 + 0.41, p-value <0.01), those in POSEIDON group 3 got significantly more top-quality embryos when GnRH antagonist was used (0.94 vs 2, p-value <0.01). Patients in Group A and B were found to have no significant difference in the following outcomes: incidence of premature LH surge (4.3% vs 7.1%, p value= 0.71), number of days of stimulation (10.26 + 2.18 vs 10.26 +2.94, p value= 0.99), total gonadotropin dosage used (3448.64+ 1007.98 vs 3087.41 + 1260.56, p value=0.06), total number of oocytes retrieved (5.84 + 3.3 vs 4.9 + 3.4, p value=0.09) and total cycle cancellation rate (30.1% vs 35.7%, p value= 0.11). Limitations, reasons for caution The small sample size of the cases limits the current statistical power of the study. The results being encouraging need to be confirmed in a larger patient population and followed for a longer duration to enable calculation of implantation and clinical pregnancy rates for all patients. Wider implications of the findings Oral progestins are an effective and cheaper, patient-friendly alternative to injectable GnRH antagonists during COS in women with DOR. Considering the significantly higher TQEs formed, PPOS can be preferred in older, POSEIDON group 4 patients, whereas flexible GnRH antagonist protocol can be our choice in the younger, POSEIDON group 3. Trial registration number Not applicable

P-009 Impact of sperm preparation methods on embryo development rate and embryo ploidy status: ICSI egg donation as a model

Abstract Study question Do different sperm separation techniques result in distinguishable contributions to embryo development rates and ploidy status? Summary answer CA0 sperm separation device, devoid of centrifugation, yielded a higher euploidy rate than conventional swim-up (50% vs. 74%, p = 0.02), notably observed in abnormozoospermic cases. What is known already Sperm DNA fragmentation (SDF) emerges as a key paternal risk factor associated with embryo aneuploidy, increasing the risk of ART failures. Factors like advanced male age, prolonged ejaculatory abstinence, and the detrimental effects of centrifugation during sperm separation jeopardize sperm DNA integrity. A recent centrifugation-free sperm separation device (CA0), employing live sperm sorting technology, exhibits efficiency in producing highly motile sperm with minimized SDF. This retrospective cohort study solely focusing on ICSI egg donation cycles to assess the influence of sperm separation methods (conventional swim-up vs. CA0) on early embryo development, and embryo ploidy. Study design, size, duration This retrospective study included 83 couples who underwent ICSI egg donation cycles at Chachava Clinic, Tbilisi, Georgia. Thirty-three semen specimens were processed using conventional swim-up (SU) and 427 Mii were inseminated. Forty-four semen samples were separated by CA0 and 519 Mii were inseminated. Clinical outcome measures included fertilization rate (number of 2PN over number of inseminated oocyte), good embryo rate (embryo number with more than 8 cells over inseminated oocyte), and euploidy rate using PGT-A. Participants/materials, setting, methods Male age and basic semen parameters via CASA (CEROS II, Hamilton-Thorne) were recorded. SU involved layering 1 mL neat semen to 1.2 mL HTF medium, inclining the tube at 45° for 60 min at 37 °C, collecting 1 mL uppermost, adding 2 mL HTF for centrifugation at 500×g for 5 min, and resuspending sperm pellet with 0.5 mL HTF. Semen samples were sorted using LensHooke® CA0 device following the manufacturer’s instructions. Main results and the role of chance In comparing demographic characteristics between SU and CA0 arms, CA0 exhibited higher median total motility (SU: 40% vs. CA0: 54%, p = 0.02), with no significant differences in female age and Mii oocyte count. This study assessed the impact of sperm separation methods on early embryo development, finding no significant differences in fertilization rate (SU: 79% vs. CA0: 77%) and good embryo rate (SU: 64% vs. CA0: 62%). However, CA0 demonstrated a significantly higher euploidy rate than SU (SU: 59% vs. CA0: 69%, p = 0.03). Further analysis within subpopulations, including normozoospermic and asthenozoospermic (total motility <40%), revealed equivalent euploidy rates between SU and CA0 for normospermic specimens (SU: 66% vs. CA0: 68%). However, a significant differentiation in euploidy rate emerged for asthenozoospermic cases (SU: 59% vs. CA0: 74%, p = 0.02). Specifically, comparing outcomes in normozoospermic, a significant reduction was observed in asthenozoospermic cases using SU (66% vs. 50%, p = 0.02), whereas CA0 provided improved euploidy rates regardless of semen quality (68% for normozoospermic and 74% for asthenozoospermic). In conclusion, this study suggests that the non-invasive live sperm sorting device enhances embryo euploidy rates, serving as a reliable sperm preparation method, ensuring selection consistency despite sample variations. Limitations, reasons for caution This study’s limitations include its retrospective nature and single-center trial design. To more effectively assess CA0 efficiency, a randomized controlled trial targeting diverse patient eligibility criteria is warranted. Wider implications of the findings Attributed to the improved embryo euploidy with CA0, we anticipate enhanced ART outcomes, such as increased embryo transfer rate and prevention of ART failures related to embryo aneuploidy. Additionally, CA0 offers practical benefits by streamlining and standardizing the sperm separation process with only three steps involved. Trial registration number not applicable

P-762 Discovery and quantification of microplastics in human cumulus granulosa cells

Abstract Study question The impact of microplastics (MPs) on human health has received increasing attention; their accumulation in female reproductive system and possible harms have not been quantified. Summary answer This study firstly reveals the contamination of human cumulus granulosa cells and follicular fluid by MPs and the effects of these contaminants on ART outcomes. What is known already MPs are ubiquitous in soil, sea, air, fresh water, and other environments. MPs are ingested by humans through the food chain, air and direct contact, resulting in various health problems, such as endocrine disruption, immune disturbance and reproductive toxicity. MPs exposure has been found to have a great impact on females, as MPs may accumulate in the ovaries and reduce the number of follicles, ovarian size, pregnancy rate, embryo production and fertility. To date, one study has found evidence of MPs in human placentas for the first time, but MPs contamination in other human reproductive systems has not been identified. Study design, size, duration The inclusion criteria comprised female patients of infertile couples excluding male factors who visited the outpatient department of the Reproductive and Genetic Hospital of CITIC-Xiangya between January 2019 and January 2021.We randomly selected 16 women who met the criteria and assessed their CCs and follicular fluid samples for the presence of MPs. Participants/materials, setting, methods In the present study, 16 cumulus granulosa cell samples and 2 follicular fluid samples were collected, and MPs were detected via pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). Main results and the role of chance MPs were detected in both cumulus granulosa cells and follicular fluid, with an average abundance of 313.14±246.69 mg/kg in cumulus granulosa cellsand 12.88±15.54 mg/kg in follicular fluid. Polyethylene terephthalate was the predominant polymer in cumulus granulosa cells. There were no significant differences in the IVF fertilization rates, ICSI fertilization rates, IVF good-quality embryo rates, or ICSI good-quality embryo rates between the high-MP group and low-MP group. However, embryos in the high-MP group exhibited a lower normal cleavage rate than did those in the low-MP group, while the embryos in the high-MP group exhibited a greater proportion of noncleavage than did those in the low-MP group. Limitations, reasons for caution It is not clear how long these particles may persist in the human body. The method we used can detect only the concentration of MPs but not the size of MP particles. Moreover, our method cannot be used to quantify smaller-diameter nanoplastics, which are considered to pose greater risks. Wider implications of the findings This is the first study in which MPs were identified and characterized in human ovarian granulosa cells, providing reference data for evaluating their toxicity to the human female reproductive system. Trial registration number Not applicable

P-768 Is it time for a consensus on key performance indicators for cycles involving donation and surrogacy?

Abstract Study question As there are no agreed standards for bench marking in cycles involving donation and surrogacy, how do we measure performance for these treatment types? Summary answer Until an international consensus is undertaken, we must define benchmarks using both in-house and published data from the Human Fertilisation and Embryology Authority (HFEA) register. What is known already Treatment cycles involving donor gametes or surrogacy have increased exponentially in recent years (Horsey, 2023; HFEA, 2020). Key performance indicators (KPIs) are critical for monitoring treatment effectiveness and safety. The Vienna Consensus (2017) is the accepted standard against which clinics should measure performance. However, the publication does not include data on cycles involving donation and surrogacy. In the absence of agreed standards, it is difficult for clinics to measure efficacy for these patient groups. Defining benchmarks for such cohorts is particularly challenging as the gametes used in donation and surrogacy treatment types may come from both fertile and sub-fertile populations. Study design, size, duration Retrospective analysis of anonymised United Kingdom (UK) data, published by the HFEA, from 2014 to 2018, was performed. Outcomes for 26290 donor intrauterine insemination (DIUI), 12262 in vitro fertilisation (IVF), 11341 intracytoplasmic sperm injection (ICSI) and 12453 frozen embryo transfer (FET) cycles, all using donor gametes, were included in this study. In cycles where surrogacy was indicated, gamete origin could not be determined, preventing the data from being included in this study. Participants/materials, setting, methods Using the complete HFEA data set, the average live birth rate, for each treatment type (excluding surrogacy), was calculated. This average was considered the target benchmark when comparing the average live birth rates at the clinic. Each quarter, a rolling 12 months of in-house retrospective data was used to assess clinic performance. Warning and action limits were set using standard deviations calculated using the 5 averages obtained from each year of HFEA data included. Main results and the role of chance The average live birth rates determined using the HFEA data, to be used as benchmarks for performance, were as follows; DIUI (13.7%), IVF (42%), ICSI (37%) and FET (36%). It is noteworthy that live birth rates reported by the HFEA were per embryo transfer (ET), rather than per embryo, meaning more than one live birth could be reported per ET. Discrepancies in outcome reporting meant that the national clinical pregnancy rate could not be determined for all treatment types, nor could fertilisation rate. Therefore, the clinic used in-house data to set benchmarks, action, and warning limits for these donor KPIs. Our findings whilst mining the national published data were that no cycle outcomes had been published beyond 2018, meaning these data may not be reflective of current national rates. Also, the national averages calculated will have been affected by individual clinic success rates. Knowing each clinic’s average would create a larger range, from which real world standard deviations could be determined, resulting in more meaningful warning and action limits. Such data was unavailable. The result being that, whilst UK national data is publicly available, it is flawed and insufficient for creating robust benchmarks that clinics can measure themselves against. Limitations, reasons for caution No national data, beyond 2018, is available for use in bench marking. Waiting for live birth data will delay any actions prompted by under-performance. Individual clinic success rate will have impacted the national average. Both fertile and sub-fertile populations will be included in both data sets. Wider implications of the findings The available UK data cannot be used to set robust benchmarks for donor or surrogacy KPIs. Small and new clinics are therefore unable to adequately measure their performance. With the increasing uptake of treatments involving donation and surrogacy, it is time for an international consensus on expected outcomes. Trial registration number not applicable

P-129 Transferring blastocysts derived from frozen-thawed cleavage embryos versus frozen-thawed blastocysts: A comparative analysis of pregnancy outcomes

Abstract Study question Is there an enhancement in pregnancy outcomes when cleavage-stage embryos are thawed, cultured for transfer as blastocysts, as opposed to transferring thawed blastocysts? Summary answer The strategy of culturing frozen-thawed cleavage embryos for 2 days and transferring them as blastocysts is not inferior to the transfer of frozen-thawed blastocysts. What is known already Embryo cryopreservation in IVF has many advantages, allowing couples to postpone pregnancy for personal reasons and reducing the risk of ovarian hyperstimulation syndrome. In the past, the common practice involved freezing and transferring cleavage embryos. However, a current trend favors single-embryo transfers at the blastocyst stage, promoting potential self-selection during extended culture. Several studies have raised concerns about potential damage during blastocyst vitrification, prompting investigation into whether cryopreserving cleavage-stage embryos for subsequent culturing to blastocysts yields superior outcomes compared to direct transfer of thawed blastocysts. Study design, size, duration This observational study was conducted at Hung Vuong Hospital from January 2022 to December 2023. We included 509 patients who underwent IVF followed by FET cycles. The D3-5 group (n = 167) comprised embryos that were frozen and thawed at the cleavage stage, subsequently extended two more days of culturing to the blastocyst stage for transfer. The D5 group (n = 342) involved frozen and thawed blastocysts. We excluded donor cycles, surrogacy, and cycles with preimplantation genetics testing. Participants/materials, setting, methods We used vitrification method for embryo cryopreservation. The freezing and thawing process were performed using either Kitazato or Cryotec medium kit, following manufacture’s instruction. Regarding the D3-5 group, thawed cleavage embryos were cultured for two more days until blastulation and transfer. Regarding the D5 group, blastocysts transfer was performed at least 2 hours after warming. The embryo transfer procedure was performed under abdominal ultrasonographical guidance. Pregnancy results were recorded and compared between two groups. Main results and the role of chance Regarding the D3-5 group, a noteworthy proportion of 65.3% cycles were unfortunately cancelled. The primary reason for cycle cancellations was the absence of developed blastocysts for transfer (85.3%). The remaining 14.7% of cancellations were attributed to other reasons. Fertilization rates were comparable between the two groups, with 80.8% in the D3-5 group and 81.5% in the D5 group (p = 0.464). However, the blastulation rate in the D3-5 group (40.1%) was lower than in the D5 group (46.4%), (p = 0.016). Patients in the D5 group demonstrated slightly better pregnancy outcomes compared to the D3-5 group: implantation rate (59.9% versus 51.7%, p = 0.302), clinical pregnancy rate (52.3% versus 44.8%, p = 0.36), ongoing pregnancy rate (37.7% versus 34.5%, p = 0746), live birth rate (33.9% versus 31%, p = 0.78), and miscarriage rate (26.3% versus 20.7%, p = 0.46). However, these differences were not statistical significance. Limitations, reasons for caution This is an observational study, we observed that thawing the cleavage-stage embryos then subsequently culture to the blastocyst stage may lead to the lower blastulation rate or even absence of developed blastocysts for transfer. This approach also increases the workload for embryologists. Wider implications of the findings Culturing frozen-thawed cleavage embryos for 2 days and transferring them as blastocysts increases workload for embryologists and poses a risk of cycle cancellation. We propose that the use of frozen-thawed blastocysts may be a more efficient and patient-friendly option. Trial registration number not applicable