ICSI Cycles Research Articles

Human 1PN and 3PN zygotes recapitulate all morphokinetic events of normal fertilization but reveal novel developmental errors

Does mono- (1PN) and tri-pronuclear (3PN) fertilization recapitulate the morphokinetic changes of normal bi-pronuclear (2PN) fertilization? Abnormal fertilization retraces the overall choreography of normal fertilization but reveals novel morphokinetic phenomena and raises scientifically and clinically relevant questions. ART has allowed the extracorporeal observation of early human development. Time-lapse technology (TLT) has revealed the complexity of the morphokinetic changes underpinning fertilization and the importance of this process for the genetic and cellular integrity of the embryo. Abnormal fertilization has remained neglected, despite its relevance to the physiology and pathology of early human development. This retrospective study involved TLT observation of normally (2PN, N = 2517) and abnormally (1PN, N = 41; 3PN, N = 27) fertilized oocytes generated in ICSI cycles performed between October 2019 and December 2020. Oocyte retrieval was carried out after clomiphene citrate-based minimal ovarian stimulation. Oocytes of patients with different diagnoses of infertility were included in the analysis, while cases involving cryopreserved gametes or surgically retrieved sperm were excluded. The study included 1231 couples treated for diverse infertility causes. The fraction of male factor cases was substantial (36.1%). Microinjected oocytes were assessed by a combined TLT-culture system. Oocytes not suitable for TLT assessment, owing to an excess of residual corona cells or inadequate orientation for correct observation, were not analysed. Phenomena relevant to meiotic resumption, pronuclear dynamics, cytoplasmic/cortical modifications, cleavage patterns and embryo quality were annotated and compared between groups. Extrusion of the second polar body (PBII) was observed in almost all 2PN/1PN (99.9% and 100.0%, respectively) and in a vast majority of 3PN zygotes (92.1%). Rates of PBII fusion with the ooplasm were much higher in 1PN and 3PN zygotes (P < 0.0001 versus 2PN). The cytoplasmic wave was observed not only in 2PN and 3PN but also in 1PN zygotes (positivity rates of 99.8% and 100% and 82.9%, respectively; P < 0.0001). More rarely, 2PN and 1PN zygotes emitted a third polar body (PBIII). The average times of this event were comparable. The presence and position of the cytoplasmic halo were comparable among the three classes of zygotes. In the 1PN group, the single PN was maternally or paternally derived in 17 and 24 zygotes, respectively, while in the vast majority of 3PN zygotes (121/127) the supernumerary PN was of maternal origin. Average times of maternal PN appearance were comparable, while average times of paternal PN appearance were delayed in 3PN zygotes (P = 0.0127). Compared with the control group, the area of the maternal PN was larger in 1PN zygotes, but smaller in 3PN zygotes (P < 0.0001). The paternal PNs displayed the same trend (P < 0.0001), although such values were consistently smaller than maternal PNs. The area of the third PN in the 3PN group was on average more than 50% smaller than those of maternal and paternal PNs. In maternal PNs of 3PN zygotes, nucleolus precursor bodies (NPBs) aligned along the area of PN juxtaposition at a lower rate compared with the 2PN group. The rate of NPB alignment was ∼50% smaller in 1PN zygotes (P = 0.0001). In paternal PNs, the rates of NPB alignment were not statistically different among the three groups. Asynchronous PN breakdown was increased in 3PN compared with 2PN zygotes (P = 0.0026). In 1PN zygotes, a developmental delay was observed starting from the disappearance of the cytoplasmic halo, reaching 9 h at the time of the first cleavage (P < 0.0001). Higher rates of abnormal cleavage patterns and blastomere fragmentation (P < 0.0001) were observed in 1PN compared to 2N and 3PN zygotes. Cleavage progression was increasingly affected after abnormal fertilization, especially 1PN, finally resulting in blastocyst formation rates of 70.2%, 12.2% and 53.5% in 2PN, 1PN and 3PN embryos, respectively (P < 0.0001). Both maternal and paternal ages were higher in cases involving 3PN fertilization. The study data were obtained from ICSI, but not standard IVF, treatments carried out in a single centre. The study findings therefore require independent verification. This study reports the first detailed morphokinetic map of human abnormal fertilization. Collectively, this evidence prompts new scientific hypotheses and raises clinical questions relevant to the aetiology and the treatment of abnormal fertilization. This study was supported by the participating institutions. The authors have no conflicts of interest to declare. N/A.

Free centrifuge sorting method for high-count sperm preparation improves biological characteristics of human spermatozoa and clinical outcome: A sibling oocytes study.

Sperm processing for assisted reproductive technologies (ART) aims to separate immotile and debris from the motile spermatozoa in the semen. The purpose of this study was to assess the effect of free centrifuge sorting (FCS) approach based on a combination of rheotaxis and swim-up on sperm biological characteristics and ICSI clinical outcomes. Each semen sample was splitted into two equal parts for 67 ICSI cycles with donation oocytes. Parts were processed with the Direct Swim Up (DSU) (control) and with the FCS method (experimental). Sperm quality was assessed in terms of motility, fine morphology, mitochondrial membrane potential, lipid peroxidation and sperm DNA fragmentation. Also Following ICSI, the clinical outcomes were compared between the groups. Sperm progressive motility (93.5± 4.1% vs. 78.6± 8.2%; p< 0.001), the fraction of Class I (good) morphology (30.2± 9.4% vs. 23.7± 8.5%; p< 0.0001) and the rate of mitochondrial membrane potential (77.4± 7.2% vs. 66.9± 5.7%; p< 0.0001) were significantly higher in the FCS compared to DSU groups. The level of lipid peroxidation (0.5± 0.05% vs. 0.6± 0.06%; p< 0.0001) and concentration of DNA fragmentation (DF) (7.4± 1.6% vs. 15.4± 2.6%; p< 0.0001) were lower in sperm from the FCS group compared to DSU group. There were higher rates of high-quality embryo formation (p< 0.001), implantation and clinical pregnancy rates (p= 0.03) in the FCS group compared to the control group. The processing of seminal samples using FCS collected spermatozoa with better biological quality and resulted in higher reproductive outcomes in ICSI cycles.

Sheath in Embryo Transfer. A Step Towards Improving Pregnancy Rate in ICSI Cycles

Sheath in Embryo Transfer. A Step Towards Improving Pregnancy Rate in ICSI Cycles

Pregnancy Rate in Women with Normal Uterine Cavity and those with Corrected Uterine Cavitary Lesions in ICSI Cycles

Pregnancy Rate in Women with Normal Uterine Cavity and those with Corrected Uterine Cavitary Lesions in ICSI Cycles

A propensity score-based, comparative study assessing humid and dry time-lapse incubation, with single-step medium, on embryo development and clinical outcomes.

Does culture in a high relative humidity atmosphere improve clinical outcomes when using a time-lapse integrated incubator and single-step culture medium? Using an integrated time-lapse system and single-step culture medium, culture in a high relative humidity atmosphere increases the likelihood of embryos, especially those subjected to preimplantation genetic testing for aneuploidies, to achieve a pregnancy compared to those cultured in dry conditions. The use of a humid atmosphere inside incubators can reduce changes in culture media osmolality, which has been reported to have a significant effect on embryo quality and morphokinetics. Studies assessing the effect of humid culture (HC) in clinical outcomes are, however, scarce and inconclusive, mostly due to a high variability in culture conditions and reduced sample size. Retrospective cohort study performed over 1627 ICSI cycles performed during 3 consecutive years in which embryo cohorts were cultured in a time-lapse incubator with three dry and three humidified chambers, and using single-step culture medium. Clinical outcomes were compared between treatments in which embryo cohorts were cultured in either humid (n = 833) or dry (n = 794) conditions. The study includes autologous treatments, with (N = 492) and without (N = 372) preimplantation genetic testing for aneuploidies (PGT-A) and ovum donation treatments (N = 763), performed in three university-affiliated private IVF centres. Stimulation, oocyte pickup and fertilization were performed according to the standard procedures of the clinic. All embryo cohorts were cultured in the same model of time-lapse incubator, distributed to either a dry or humidified chamber, while the rest of the culture variables remained equal. The population was weighted by the inverse probability of treatment to control for all measured confounders. The association between HC and the main outcome was assessed by logistic regression over the weighted population. The E-value was reported as a way of considering for unmeasured confounders. Differences in embryo development and other secondary outcomes between the study groups were assessed by Pearson Chi-squared test, ANOVA test and Kaplan-Meier survival analysis. An univariable logistic regression analysis, weighted by the inverse probability of treatment, determined that embryos cultured in humid conditions are more likely to achieve a clinical pregnancy than those cultured in dry conditions (odds ratio (OR) = 1.236 (95% CI 1.009-1.515), P = 0.041, E = 1.460). Through stratification, it was determined that said effect is dependent on the type of treatment: no improvement in clinical pregnancy was present in ovum donation or autologous treatments, but a statistically significant positive effect was present in treatments with preimplantation genetic testing (OR = 1.699 (95% CI 1.084-2.663), P = 0.021, E = 1.930). Said increase does not relate with an improvement in later outcomes. Differences were also found in variables related to embryo developmental morphokinetics. The retrospective nature of the study makes it susceptible to some bias linked to the characteristics of the treatments. To lessen the effect of possible biases, cases were weighted by the inverse probability of treatment prior to the evaluation of the outcome, as means to assess for measured confounders. In addition, the E-value of the weighted OR was calculated as a sensitivity analysis for unmeasured confounders. A randomized prospective study could be performed for further assessing the effect of humid conditions in clinical outcome. These results support that embryo culture under conditions of high relative humidity contributes to optimize clinical results in undisturbed culture in a time-lapse incubator with single-step medium. To our knowledge, this is the largest study on the matter and the first performing a propensity score-based analysis. This work was supported by the ''Centro para el Desarrollo Tecnologico Industrial'' from the Spanish Ministry of Science, Innovation, and Universities (CDTI-20170310) and Generalitat Valenciana and European Social Fund (ACIF/2019/264). None of the authors have any competing interest to declare. N/A.

P-525 PGT-A with embryo pooling: does it result in a higher yield of euploid embryos and better clinical pregnancy outcomes?

Abstract Study question Does PGT-A with embryo pooling result in a higher yield of euploid embryos and better clinical pregnancy outcomes compared to PGT-A done after one cycle? Summary answer There is no evidence to support that embryo pooling for PGT-A purposes is favorable in preference to PGT-A after one cycle. What is known already PGT-A has been the method of choice that is thought to optimize embryo selection and enhance clinical outcomes. However, the number of embryos available for biopsy can be a source of limitation, as the yield of euploid embryos suitable for transfer can be affected when lower number of embryos are available for screening. A protocol that is suggested to overcome this limitation is to undergo multiple IVF/ICSI cycles with blastocyst biopsy and embryo pooling, then proceed with PGT-A screening once a sufficient number of biopsied embryos is attained. Study design, size, duration This is a retrospective study that included ICSI cycles from June 2019 to December 2021, involving 157 patients (average maternal age 36.4) that underwent PGT-A after one ICSI cycle (group A), and 79 patients (average maternal age 35.6 ) that underwent PGT-A after embryo pooling from more than one ICSI cycle (Group B). Patients in group B underwent pooling due to cost implications on the number of embryos to screen. Participants/materials, setting, methods A total of 843 embryos were biopsied and screened for group A, and 536 were biopsied and screened for group B. Following PGT-A screening, a number of frozen embryo transfers (FET) were conducted (120, 67 respectively). Investigations included: number of embryos available for biopsy, number of euploid embryos and clinical pregnancy outcomes after FET. Statistical analysis was calculated (P &amp;lt; 0.05). Main results and the role of chance Patients in group A proceeded with PGT-A after only one ICSI cycle, while in group B patients underwent 2.3 cycles on average. The average number of embryos suitable for biopsy was 5.4, and 6.9 per patient respectively. The rate of euploid embryos was 42.1% and 43.8%, and the average number of euploid embryos per patient was 5.4 and 6.5 respectively. A total of 144 embryos were transferred in group A with an average of 1.2 embryos per patient. Clinical pregnancy rate (CPR) was 60%, implantation rate was 54.9% and live birth rate (LBR) was 35.8%. In Group B a total of 67 embryos were transferred with an average of 1.1 embryos per patient. Clinical pregnancy rate (CPR) was 67.7%, implantation rate was 68.7% and live birth rate (LBR) was 40%. No significant difference was observed between both groups for clinical outcomes (P &amp;gt; 0.05). Limitations, reasons for caution This study is retrospective and small in size. Data on live birth rate requires further follow up as a number of patients are still in ongoing pregnancies. A larger data set study is needed to support conclusion. Wider implications of the findings Results from this study does not support the adoption of a pooling strategy for patients undergoing PGT-A. Although the number of embryos available for biopsy may increase slightly, the yield of euploid embryos is not significantly increased and the clinical pregnancy rate is not enhanced. Trial registration number not applicable

P-107 A Novel Sperm Selection Technique for Embryos of the Desired Sex

Abstract Study question Can a novel sperm selection technique (GST) yield higher rates of embryos and offspring of the desired sex in couples undergoing ICSI with PGT-A? Summary answer GST consistently enriched spermatozoa, resulting in a higher proportion of embryos and offspring of the desired sex without impairing clinical outcomes or offspring health. What is known already Although various methods to select sex-specific spermatozoa have been proposed over the years, many of these techniques have been shown to have varying degrees of success in addition to being time-consuming and costly. Moreover, the use of fluorescence and electrical charges in some of these methods has raised concerns about their potential contribution to congenital malformations. Here, we tested a novel sperm selection method aimed at achieving a higher proportion of embryos of the desired sex, without compromising clinical outcome or offspring health. Study design, size, duration Over a 6-year period, ejaculates from male partners of couples (n = 109) undergoing ICSI with PGT-A were processed using GST to enrich spermatozoa for the couples’ preferred sex. Standard sperm processing was carried out for couples undergoing ICSI exclusively to assess conceptus aneuploidy, comprising the control group (n = 1,261). The proportion of male and female spermatozoa in the initial and selected specimens, PGT-A results, and ICSI outcomes were compared between the two groups. Participants/materials, setting, methods A total of 1,370 couples were treated in 2,483 ICSI cycles. Standard sperm processing was performed for 1,261 couples who did not have an offspring sex preference. For 109 consenting couples, GST was used to enrich spermatozoa for their desired sex (IRB 1306014043). To confirm sex enrichment, ≥1,000 sperm cells were screened by fluorescent in-situ hybridization (FISH) for 9 chromosomes. The couples’ PGT-A results and ICSI outcomes were compared between the control and GST cohorts. Main results and the role of chance For the control cohort (n = 1,261), ejaculates were processed in the standard fashion. Spermatozoa sex ratio was unaffected. These couples (maternal age, 37.1±4yrs; paternal age, 39.1±6yrs) underwent 2,356 ICSI cycles (1.2±1), yielding an 80.9% fertilization rate (14,830/18,321). PGT-A results confirmed that 46.6% (n = 760) of their embryos were female and 53.4% (n = 872) were male. They achieved a 76.3% (725/950) implantation rate and a 64.9% (617/950) clinical pregnancy rate resulting in 569 healthy deliveries (48% female, 52% male). From the study cohort (n = 109), 60 couples desired a female and 49 desired a male child. Those who desired female offspring (maternal age, 37.9±4yrs; paternal age, 40.8±6yrs) obtained an 81.6% sperm sex enrichment, per FISH. They underwent 74 ICSI cycles and achieved a 77.6% (592/763) fertilization rate resulting in 78.1% (235/301) female embryos that generated a 79.3% (23/29) implantation rate, yielding 16 singleton deliveries of healthy female offspring that are developing normally. The 49 couples (maternal age, 37.6±3yrs; paternal age, 40.8±5yrs) preferring male offspring obtained an 80.8% sperm sex enrichment. They underwent 53 ICSI cycles and achieved a 74.7% (481/644) fertilization rate with an equivalent proportion of male embryos (231/292, 79.1%). Their implantation rate was 90.9% (20/22), yielding 14 healthy male singletons, all developing normally. Limitations, reasons for caution Although our sperm sex selection method does not guarantee offspring of a specific sex, it allowed couples participating in the study to obtain a greater proportion of conceptuses of their desired genotype. This method does not aim to replace PGT-A, but rather reduce embryo wastage. Wider implications of the findings Semen specimens processed by GST yielded satisfactory fertilization and embryo development, comparable to those from the control cohort. Moreover, offspring health was not negatively affected. These encouraging findings indicate that our method is safe and can consistently enrich for the desired embryo sex in a reliable and ethically palatable manner. Trial registration number n/a

O-251 Defining the exclusive role of male genome integrity on conceptus development

Abstract Study question To determine the effect of sperm DNA fragmentation on embryo development by ruling out a female factor component. Summary answer By utilizing healthy donor oocytes, it is possible to quantify the sole deleterious effect of sperm DNA fragmentation and explore the ooplasmic repair mechanism. What is known already In approximately 50% of couples with unexplained ART failure, a subtle male factor is present that is missed in a conventional semen analysis assessing concentration, motility, and morphology. Additional information on male gamete competence can be acquired by assessing Sperm Chromatin Fragmentation (SCF). Indeed, a fragmented male genome can give rise to poorly developing embryos, leading to impaired implantation, lower pregnancy, and higher miscarriage rates. It has been previously seen that an oocyte, according to its age, can repair the damaged DNA contributed by the male gamete. Study design, size, duration In last decade, 316 couples, who have an elevated SCF, underwent ICSI cycles and resulted in disappointing clinical outcomes. To exclude an eventual confounding female factor, couples who utilized donor oocytes were identified and clinical outcome was compared to a control. To measure the role of an impaired sperm genome and a concurrent oocyte repair mechanism, we compared clinical outcomes between cycles in the same couples who used their own and subsequently donor oocytes. Participants/materials, setting, methods We included 381 couples screened for SCF; 65 underwent ICSI cycles with donor oocytes; of these, 46 underwent a previous ICSI cycle with their own oocytes. Fertilization, implantation, clinical pregnancy, and delivery rates were compared between above-mentioned groups. A TUNEL assay was used to measure SCF (≥500 spermatozoa were assessed/sample at a 15% threshold). Paired t and Chi-square tests were used to compare ages and clinical outcomes, respectively. Main results and the role of chance We included 381 couples (maternal age, 37.8 ± 4.2; paternal age, 41.8 ± 8.2), whose male partners had the following semen parameters: average volume of 2.5 mL, concentration of 25.4x106/mL, 33% motility, and normal morphology of 1.7. Of these, 316 had elevated average DNA fragmentation of 25.5% and utilized their own oocytes in 683 ICSI cycles, resulting in a 67.5% fertilization, 12.2% implantation, 27.9% clinical pregnancy, and a 22.5% ongoing/delivery. Of the 381 couples, 65 underwent ICSI utilizing donor oocytes (SCF of 24.3%) resulting in a significantly higher fertilization (78.4%) (P &amp;lt; 0.00001) and embryo implantation rates (34.2%) (P &amp;lt; 0.0001). Similarly, albeit not statistically significant, the clinical pregnancy and ongoing/delivery rates increased from 27.9% to 37.1% and from 22.5% to 30.0%, respectively. To further explore the exclusive role of the male gamete in embryo development, we identified a cohort of patients (n = 46), with an elevated average DNA fragmentation of 23.6%, who underwent an ICSI cycle using their own oocytes and a subsequent cycle using donor oocytes. Compared to cycles where couples used their own oocytes, fertilization, embryo implantation, clinical pregnancy, and ongoing/delivery rates rose from 67.5% to 76.6% (P &amp;lt; 0.0001), 2.8% to 24.7% (P &amp;lt; 0.00001), 8.4% to 38.8% (P &amp;lt; 0.0001), and 1.2% to 28.6% (P &amp;lt; 0.00001), respectively. Limitations, reasons for caution Although this study attempts to control for a concurrent confounding female factor, it cannot be completely excluded. It is difficult to assess the extent and contribution of the ooplasmic repair mechanisms on the male genome. Wider implications of the findings Ooplasmic repair mechanisms of healthy female gametes appear to repair or improve the deleterious impact of sperm DNA fragmentation on ART outcomes. Trial registration number not applicable

O-099 The use of cumulus cells (CCs) to select spermatozoa for intracytoplasmic sperm injection improves blastocyst development and quality

Abstract Study question Is there a role for sperm selection based on the capacity to pass through cumulus oophorous column in improving the laboratory outcomes in ICSI cycles? Summary answer A higher rate of blastocyst formation and quality are obtained with the use of CCs to select spermatozoa for ICSI compared to conventional density gradients. What is known already The sperm selection for ICSI is an operator-dependent procedure based on sperm morphology and motility. ICSI bypasses all natural barriers of sperm selection, which may impact embryo development. During natural fertilization, only the spermatozoa that pass-through cumulus cells will have the chance to fertilize the egg. Spermatozoa capable of traverse COCs have better morphology, higher chromatin integrity and greater acrosomal reaction. Hyaluronic acid is an important component of these cells and plays a key role in the selection of spermatozoa with intact DNA. However, there is scarce data regarding this selection method for ICSI on the development of the embryo. Study design, size, duration This is a prospective randomized sibling oocyte pilot study (February-December 2021). A total of 996 metaphase II (MII) oocytes were randomized into two groups. In the control group (Group 0, n = 489 MII), ICSI was performed with sperm selected by conventional density gradients. In the study group (Group 1, n = 507 MII) the oocytes were microinjected with sperm selected through CCs. The main outcome was blastocyst formation rate and quality. Reproductive results were analyzed as secondary outcomes. Participants/materials, setting, methods Ninety-eight couples undergoing ICSI (private center) with ≥6 fresh own MII obtained after ovarian stimulation, embryo culture until blastocyst stage and ejaculated sperm samples were included in the study. All seminal samples were processed using density gradients and those in the study group were additionally passed through CCs (the patient's own CCs were used in all cases). Fertilization and day 5 blastocyst formation rates were assessed. Embryo quality was scored according to Gardner's criteria. Main results and the role of chance Data was similar with regards to cause of infertility, maternal age (36.47 vs 36.63 y.o.), sperm count (60.53 vs 58.25 mill/ml) and progressive motility (57.45 vs 57.50%) between group 0 vs 1, respectively. Fertilization rate was similar between groups (74.0% vs 78.7%; p = 0.419. Group 0 vs 1, respectively). However, the blastocyst formation rate was significantly higher in the study group compared to control (70.9% vs 60.1%; p = 0.002). In addition, high-quality blastocyst rate was significantly higher in the CCs compared to the control group (49.6% vs 38.5%; p = 0.003). After the analysis of 145 treatments (131 frozen vs 14 fresh transfers) the cumulative clinical pregnancy rate in the study group was 47.2%, higher than in the control group (32.1%), but without reaching statistical difference (p = 0.07). Limitations, reasons for caution This is an exploratory study, aimed to evaluate the effectiveness of CCs as a more “physiologic” selection of spermatozoa for ICSI treatments in terms of laboratory outcomes (blastocyst formation rate and quality). Albeit not reaching statistical significance, the higher cumulative pregnancy rate in the study group seems clinically relevant. Wider implications of the findings Our results suggest that CCs can select more competent sperm for ICSI. This “physiologic-resembling”, operator-independent selection method has the potential of increasing pregnancy outcomes in ICSI cycles, pending of confirmation in larger studies including cumulative embryo-transfer cycles. Trial registration number NA

O-206 The impact of oocyte central granularity (CG) on the outcomes of ICSI cycles: live birth achievement is only compromised if CG-derived embryos are transferred

Abstract Study question Does oocyte central granularity (CG) compromise post-ICSI live birth achievement? Are morphologically normal oocytes obtained from CG cycles also affected? Summary answer Oocytes with CG, but not morphologically normal oocytes obtained from cycles providing CG oocytes, present lower competence to achieve a live birth. What is known already Ooplasm central granularity (CG) is associated with alterations in the actin cytoskeleton and meiotic spindle potentially affecting meiotic accuracy, fertilisation dynamics and embryo development. We have recently reported lower fertilisation rates and slower morphokinetics associated with CG. However, to clarify whether CG does affect oocyte developmental competence and if it is associated with endocrine alterations that may also impinge on the performance of morphologically normal oocytes obtained in the same cycle, adequately powered studies assessing the clinical outcomes of cycles transferring embryos derived from CG oocytes or from their morphologically normal companions are needed. Study design, size, duration Retrospective analysis including 866 control ICSI cycles/patients providing only morphologically normal oocytes (NO) and 211 CG cycles/patients providing NO and CG oocytes, from July 2014 to August 2021. Clinical outcomes were compared (1) between cycles providing CG oocytes (CG) or not (Control 1), and (2) among cycles achieving a fresh transfer in which only NO were obtained (Control-T), CG oocytes were obtained but NO-derived embryos were transferred (NOT), and CG-derived embryos were transferred (CGT). Participants/materials, setting, methods Patients undergoing their first ICSI cycle (n = 866; Control) or their first ICSI cycle providing CG oocytes (n = 211; Treatment-groups) were included. Cycle characteristics and outcomes were compared using the Fisher’s exact test and the Wilcoxon sum rank test. Multivariate analyses were performed to assess the potential interference of maternal age, infertility cause, number of embryos transferred and day of embryo transfer on the relationship between CG and live birth. Main results and the role of chance Patients providing CG oocytes (n = 211) presented higher maternal age (37.6± 4.5 vs. 36.5± 4.3; p= p &amp;lt; 0.001), higher basal FSH (8.5± 3.8 vs. 7.9± 4.1 IU/L; p = 0.03), lower AMH (2.2± 2.2 vs. 3.2± 3.5 ng/mL; p &amp;lt; 0.001), higher incidence of ovarian infertility (17.5% vs. 4.9%; p &amp;lt; 0.001) and higher FSH total dose (2255.3± 866.4 vs. 1825.6± 752.3 IU; p &amp;lt; 0.0001), compared to controls (n = 866). Implantation, abortion and live birth rates did not differ between cycles providing (CG) or not (Control) CG oocytes (22.7% vs. 24.9%, 17.1% vs. 27.7%, 25.8% vs. 22.9%, respectively). Nevertheless, implantation and live birth rates were lower in cycles in which CG-derived embryos were transferred (CGT; n = 87) compared to cycles in which CG oocytes were obtained but only NO-derived embryos were transferred (NOT; n = 57), or to cycles in which CG oocytes were not obtained (Control-T; n = 595). Implantation rates were 11.4%, 30.9% and 24.9%, while live birth rates were 10.5%, 30.2% and 22.9% for CGT, NOT and Control-T, respectively (p &amp;lt; 0.001 for implantation rates in CGT vs. NOT and CGT vs. Control-T; p &amp;lt; 0.04 for live birth rates in CGT vs. NOT and CGT vs. Control-T). No significant differences were observed between NOT and Control-T. Limitations, reasons for caution Our study is subjected to the limitations of a retrospective analysis and thus the data may be affected by other variables that were not controlled for. Moreover, the intrinsic difficulty in obtaining a higher number of cycles in which only CG-derived embryos are transferred, limits the power of the analysis. Wider implications of the findings Our findings shed light on a still unsolved question in ICSI practice. The present data indicate that CG is associated with markedly reduced oocyte developmental competence, while the presence of CG oocytes in a cycle does not affect the quality of the companion normal oocytes. Trial registration number not applicable

P-186 ART outcomes in ICSI cycles with normal and morphologically variant (MV) oocytes in the same cohort

Abstract Study question To evaluate ART outcomes of ICSI cycles from normal oocytes (A) Vs normal and MV oocytes (B) and all MV oocytes in the cohort (C). Summary answer Clinical pregnancy rate (CPR), Implantation rate (IR) and live birth rates (LBR) were clinically satisfactory even in group C What is known already In routine IVF laboratories, oocytes are subjected to a rapid evaluation using an inverted microscope to evaluate morphological variants in the cytoplasm, peri-vitelline space and zona pellucida, which is very superficial and subjective. Post ICSI, focus entirely shifts to embryo morphology studies and outcome of such morphologically variant oocytes are less explored. Transferring embryos selected by morphological assessment alone and by neglecting oocyte quality may result in compromised in vitro development, low pregnancy rates, miscarriage rates and further negative consequences Study design, size, duration A prospective observational study of 212 women who underwent ICSI cycles from September 2020 to December 2021 were taken up for the study of which, 106 subjects had all normal oocytes in the cohort (A), 67 subjects had both normal and abnormal oocytes (B) and 28 subjects had all morphologically variant oocytes (C) Participants/materials, setting, methods 212 consecutive women aged 24-38 undergoing ICSI cycles in a private fertility hospital were included. ICSI was performed for all oocytes, embryos cultured until Day 5. Primary outcome measure was to compare ART outcomes between three groups. Secondary outcome measure was to determine minimum threshold of normal oocytes required for higher live birth rate by comparing ART outcomes of patients with at least 7 normal oocytes (n = 51) Vs those with &amp;lt;7 normal oocytes (n = 55). Main results and the role of chance The study showed a highly significant difference in CPR (P &amp;lt; 0.0001), IR (P = 0.0001) and LBR (P = 0.01) when embryos with all morphologically normal oocytes were transferred compared to embryos with morphologically variant oocytes were transferred. There was no difference in ICSI outcomes including fertilization rate, ICSI degeneration rate, early embryo development rate and blastulation rate between the three groups. The study also revealed that a minimum of 7 normal oocytes in the cohort gave significant difference in CPR (P = 0.01), IR (P = 0.02) and LBR (P = 0.0005) when compared to the women who had less than 7 normal oocytes in the same cohort. The outcome of each of the normal and MV oocytes were evaluated by culturing the MV oocytes separately in culture until Day 5. The embryos formed from MV oocytes were vitrified separately and was transferred only when there were no embryos were available for transfer from the normal oocytes obtained from the same cohort. Limitations, reasons for caution The study is to be continued further, until &amp;gt;1000 oocytes are evaluated to confirm if more robust results are obtained, so that sample size in all the three groups are also higher. Wider implications of the findings This study implies that instead of discarding or not injecting oocytes with morphological variants, transferring those embryos with some morphologically variant oocytes also results in a clinically satisfactory outcome and live birth rates. Trial registration number Not applicable

P-261 Morphokinetics-oriented embryo selection for day 2 transfers: adapting transfer strategy to the times of Covid-19

Study questionCan embryo selection oriented by a morphokinetic algorithm developed for day 2 transfers provide pregnancy rates similar to those achieved by elective frozen blastocyst transfers?Summary answerTransfers on day 2 of embryos selected by a morphokinetic algorithm can provide pregnancy rates similar to those achieved with elective frozen blastocyst transfers.What is known alreadyThe comparative efficacy of cleavage stage fresh transfers in relation to blastocyst fresh transfers remains controversial. On the other hand, elective transfers of frozen blastocysts have been found to provide satisfactory implantation and delivery rates, which may result from favourable embryo quality and/or endometrial receptivity associated with freeze-all cycles. The covid-19 pandemic has imposed changes to the ICSI laboratorial routine, particularly pushing for a reduction in embryo culture time. In this context, we developed an algorithm based on fertilization and cleavage morphokinetic parameters, aiming to optimize embryo selection for day 2 transfers.Study design, size, durationThis study aimed to assess the performance of a morphokinetic algorithm to select cleavage stage embryos for day 2 transfers, using as a parameter the outcomes from elective frozen blastocyst transfers. A retrospective analysis of data generated in our clinic during pandemia was performed, comparing patient profiles and clinical outcomes of day 2 transfers following algorithm-oriented embryo selection (n = 128), with those of contemporary elective transfers of frozen blastocysts (n = 230) from freeze-all patients with OHSS risk.Participants/materials, setting, methodsPatients were in their first ICSI cycle, 128 of which underwent a day 2 transfer of a single embryo selected by an algorithm based on fertilization (tPNf) and cleavage (T2 e T4) parameters. The remaining 230 patients had a elective frozen blastocyst selected by morphology transferred after endometrial preparation. Cycle characteristics and outcomes were compared using Fisher’s and Wilcoxon sum rank tests. A multivariate analysis tested the association of treatment and cycle variables with pregnancy.Main results and the role of chancePatients providing embryos transferred on day 2 after algorithm-oriented selection presented lower maternal age (35.5 ± 4.1 vs. 36.4 ± 4.4; p = 0.02), higher basal FSH (8.3 ± 3.6 vs. 6.9 ± 2.1 IU/L; p < 0.001) lower AMH (2.3 ± 2.0 vs. 5.7 ± 6.3 ng/mL; p < 0.0001), different distribution of infertility factors (p < 0.001; male factor: 64.1% vs. 39.6%, tubal: 3.1% vs. 11.3%, unexplained: 15.6% vs. 29.1%, for day 2 and frozen blastocyst transfers, respectively) and lower oocyte yield (6.9 ± 4.0 vs. 17.2 ± 6.5) as compared to patients providing frozen blastocysts.Beta-hCG, clinical pregnancy, ongoing pregnancy and miscarriage rates (assessed until the 12th week of pregnancy) did not differ between algorithm-oriented day 2 and frozen blastocyst transfers (53.1% vs. 50.9%, 40.6% vs. 45.7%, 33.6% vs. 36.5% and 17.3% vs. 20.0%, respectively).The multivariate analysis indicated maternal age as the only variable significantly and independently associated with ongoing pregnancy achievement [OR = 0.89 (0.84-0.94); p,0.001)].Limitations, reasons for cautionOur study is subjected to the limitations of a retrospective analysis and the data may be affected by variables not controlled for that are heterogeneous between groups such as different endometrial conditions and the use embryo cryopreservation.Wider implications of the findingsOur findings indicate that morphokinetic algorithms can be successfully applied to improve outcomes of day 2 transfers to a level similar of that achieved with elective frozen blastocyst transfers. Our data represent a valuable parameter for the improvement or adaptation of ICSI practice in the face of logistic constraints.Trial registration numbernot applicable

P-780 Ten years electronic witnessing in the IVF laboratory

Abstract Study question What did we learn after 10 years of electronic witnessing? Summary answer Only when applied correctly, electronic witnessing can prevent sample mix-up. What is known already In the early days of IVF, manual double witnessing was used to reduce the misidentification of gametes for insemination. However, risk for human errors due to fatigue, automatism, distraction or work load make this a mistake-prone approach. Hence, there is increasing interest in using electronic witnessing systems for patient and sample identification. Although not always mandatory, such systems have been implemented in many fertility laboratories to avoid identification errors. A mismatch is generated when non-matching samples are simultaneously present in a single workstation. Yet, when the system is incorrectly used, corrective intervention by an administrator (administrator assign) may be required. Study design, size, duration This evaluation investigates the mismatch (MM) rate and administrator assigns (AA) over a 10-year period (March 2011- December 2021) with the use of RI Witness (RIW) (CooperSurgical). Radio frequency identification tags (RFID) were used for patient and sample identification. Conventional IVF and ICSI cycles, and frozen embryo cycles (FET) were included since 10 years, IUI cycles since 8 years. Participants/materials, setting, methods The total number of tags and witness points (WP) were recorded. Witness points represent all the actions in RIW that have been performed during the entire process. MM and AA were collected and stratified by procedure (sperm preparation, oocyte retrieval, IVF/ICSI, (embryo)biopsy, vitrification/warming, embryo transfer, medium changeover and IUI). Critical MM (such as mis-labeling or non-matching samples within one work area) and critical AA (such as non-RIW-identified samples and unconfirmed WP) were selected. Main results and the role of chance A total of 109,655 cycles were included: 53,023 IVF/ICSI, 36,347 FET, and 20,285 IUI cycles. The 724,096 used tags, led to a total of 849,650 WP. The overall MM rate per WP was 0.25% (2,132/849,650), 1.9% per cycle, of which 144 critical mismatches occurred in the different procedures: sperm preparation: 66, oocyte retrieval: 5, IVF/ICSI: 33, (embryo)biopsy: 1, vitrification/warming: 26, embryo transfer: 8, medium changeover: 3 and IUI: 2. The mean critical MM rate per WP per year was 0.017% ± 0.007% and 0.13 ± 0.05% per cycle. The overall AA rate per WP was 0.11% (940/849,650), 0.86% per cycle, including 320 critical assigns: sperm preparation: 83, oocyte retrieval: 34, IVF/ICSI: 173, (embryo)biopsy: 3, vitrification/warming: 6, embryo transfer: 0, medium changeover: 21. Critical AA rate per year was on average 0.041%/WP ± 0.01% and 0.30 ± 0.07% per cycle. All MM and AA rates remained stable during this 10-year period, except for sperm preparation, where the removal of a single witness point led to a huge increase in AA. RIW should still be used in combination with the manual labelling of bottom and lid of dishes and tubes to guarantee correct assignment in case of RFID malfunction or AA. Limitations, reasons for caution The procedures and method of integration of RIW may vary from one laboratory to another and result in differences in the potential risks. RIW cannot (yet) identify individual embryos, making double manual witnessing indispensable at certain critical steps, where potential errors are not recorded. Wider implications of the findings Using an electronic witnessing is considered to be the ultimate tool to safeguard correct identification of gametes and embryos. But this is only possible when used correctly and requires proper training and attention of the staff. It may also induce new risks, i.e. blind witnessing of samples by the operator. Trial registration number not applicable

P-112 Utilizing surgical sperm retrieval to improve clinical outcomes for men with high sperm chromatin fragmentation in their ejaculates

Abstract Study question In men with elevated sperm chromatin fragmentation (SCF), can surgical sperm retrieval from the proximal area of the male genital tract improve clinical outcomes? Summary answer Spermatozoa retrieved from the epididymis or testes demonstrate higher genomic integrity and are associated with enhanced embryo implantation, clinical pregnancy, and delivery rates. What is known already Sperm genomic integrity is a key factor in achieving a successful pregnancy. During spermiogenesis, alteration of DNA topology involving nuclease and ligase mechanisms is required for supercoiling and DNA compaction. If a proper DNA repair mechanism fails during this intricate process, spermatozoa with DNA fragmentation should be picked up by the epididymis and phagocytized. When this mechanism fails, along with the presence of superimposed reactive oxygen species in the male genital tract, spermatozoa with DNA fragmentation may reach the ejaculates. We have previously demonstrated that retrieving spermatozoa directly from the epididymis and testes yields gametes with higher conserved genomic integrity. Study design, size, duration In a nine-year timespan, we identified over 1,000 men with high SCF in their ejaculates; 144 patients consented to undergo surgical retrieval of spermatozoa. In a preliminary assessment, their clinical outcomes were compared to those from men with normal SCF (n = 539). Of these men, 53 consented to undergo subsequent ICSI cycles with their female partners using surgically retrieved spermatozoa from the epididymis (n = 15) or testes (n = 38). Fertilization and clinical outcomes were compared. Participants/materials, setting, methods Ejaculated, epididymal, and testicular specimens were screened by terminal deoxynucleotidyl dUTP nick-end labeling (TUNEL) to assess SCF using a commercially available kit. A total of 500 spermatozoa/sample were screened with a normal threshold of 15%. Surgical samples from the epididymis and testes were cryopreserved in multiple vials for subsequent ICSI cycles. Main results and the role of chance In the preliminary assessment, ICSI outcomes utilizing ejaculated spermatozoa were compared between men with normal (n = 539, 9.3±3%) and abnormal (n = 144, 28.2±13%) SCF. Although fertilization did not differ, implantation (24.1% vs. 7.8%) and clinical pregnancy rates (CPR, 35.8% vs. 14.6%) were significantly impaired (P &amp;lt; 0.001) in men with elevated SCF. SCF assessment on surgically retrieved spermatozoa revealed a decreasing trend from 28.2±13% in the ejaculate to 16.6±9.2% in the epididymis and 10.1±5.7% in the testes. A total of 53 men underwent 79 subsequent ICSI cycles using surgically retrieved spermatozoa. Compared to their historical cycles using ejaculates (n = 65), ICSI with surgically retrieved sperm substantially improved implantation (18.8% vs. 2.1%; P &amp;lt; 0.001), CPR (36.5% vs. 5.3%; P &amp;lt; 0.0001), and ongoing/delivery rates (33.8% vs. 4.1%; P &amp;lt; 0.0001). Among these couples, 15 underwent 21 ICSI cycles utilizing epididymal spermatozoa with a mean SCF of 16.6±9%. Implantation (26.5% vs. 3.7%; P &amp;lt; 0.05), CPR (68.4% vs. 4.2%; P &amp;lt; 0.0001), and ongoing/delivery rates (57.1% vs. 4.2%; P &amp;lt; 0.0001) were significantly improved compared to the historical cycles. The remaining 38 patients underwent ICSI with testicular spermatozoa with an average SCF of 10.1±6%. Implantation (15.0% vs. 2.6%; P &amp;lt; 0.01), CPR (25.5% vs. 6.1%; P &amp;lt; 0.01), and ongoing/delivery rates (23.6% vs. 3.0%; P &amp;lt; 0.05) were higher when compared to historical cycles. Limitations, reasons for caution Although surgically retrieved spermatozoa can be used to enhance clinical outcomes in couples with high SCF, epididymal spermatozoa yielded higher pregnancy rates despite the higher level of SCF in these specimens compared to testicular spermatozoa. This can be explained by the corrective action of the ICSI procedure itself. Wider implications of the findings Our study demonstrated that sperm DNA integrity progressively increases through the journey of the male genital tract. In couples with a compromised sperm genome, surgically retrieved spermatozoa can be beneficial. Therefore, assessing SCF might be used a routine tool to evaluate the male gamete. Trial registration number N/A

O-249 The impact of semen parameters on ICSI and pregnancy outcomes in egg recipient cycles with PGT-A

Abstract Study question To assess the effect of male factor on ICSI and pregnancy outcomes in egg recipient cycles combined with preimplantation aneuploidy testing Summary answer In egg donor cycles where sperm was obtained from azoospermic men there was a significant effect on ART and clinical outcomes. What is known already The increased incidence of chromosomal problems in men with azoospermia and its correlation with chromosomally abnormal embryos and with adverse pregnancy outcomes has been shown by multiple studies. Nevertheless, few studies in the literature outline the impact of non-obstructive azoospermia in the subgroup of couples who opt to use donated eggs. The egg donation model offers an opportunity to isolate the male factor and evaluate its impact on IVF-ICSI and pregnancy outcomes. The aim of our study was to study the effect of non-obstructive azoospermia(NOA) on ICSI outcomes compared with oligoasthenozoospermia(OATS) and moderate male factor(MMF) infertility, from ART parameters to clinical outcomes. Study design, size, duration This is a retrospective longitudinal cohort study involving 1,594 ICSI cycles using donor eggs performed between January 2016 and May 2020. The cohort was divided into three groups according to the male partner’s sperm parameters: couples with NOA accounting for 479 cycles (30%); couples with OAT-S (sperm number &amp;lt;5 x 106/ml), accounting for 442 cycles (27.8%); couples with moderate male factor, with sperm number &amp;gt;_5 x 106/ml and &amp;lt;15 x 106/ml, accounting for 673 cycles (42.2%). Participants/materials, setting, methods Participants: Subfertile couples who opted to use donor eggs. Donor eggs were utilized, due to history of severe female infertility, including low ovarian reserve, poor response to ovarian stimulation, repeated IVF failures or premature ovarian failure. Setting: Private IVF Unit Intervention: ICSI with ejaculated/surgically retrieved sperm, blastocyst culture, PGT-A and frozen-thawed euploid embryo transfer. Outcomes: The primary outcomes were live birth, fertilization, blastocyst development and euploidy rates; the secondary outcomes were clinical pregnancy, miscarriage rates, preterm labour and SGA. Main results and the role of chance The fertilization rate was significantly reduced in the NOA group as compared to the OATS and the MMF group: 30.3% versus 63% and 77.3%. Logistic regression analysis adjusted for confounders highlighted NOA as a negative predictor of obtaining an euploid blastocyst per inseminated oocyte. When the analysis was performed per obtained blastocyst, no correlation between male factor and euploidy rate was observed. The clinical pregnancy rates were similar in the MMF and OATS group, but significantly lower in the NOA group (38.2% versus 36.3% and 29.5% respectively). The miscarriage rate in the NOA group was 11.8%; higher than the OATS and MMF group (7% and 2.7%). The live birth rate per ET was significantly lower in the NOA group compared to the OATS and the MMF group (20.4% vs 30.3% and 35.4%,p&amp;lt;0.05). The live birth rate was significantly lower in the OATS group compared to the MMF group(p &amp;lt; 0.05). The risk of preterm labour was significantly higher in the NOA group, compared to the OATS and MMF group (55.1% versus 46.8 and 16.1%,p&amp;lt;0.001) and a higher number of babies born with a birth weight &amp;lt;2.5kg came from the NOA group, compared to the OATS and MMF group (34% versus 19.8% and 12.2%, p &amp;lt; 0.001). Limitations, reasons for caution The present study is limited by its retrospective design and the fact that the confounding factors of advanced maternal age, endometrial ageing and uterine factors cannot be fully evaluated. However, the study was controlled for female and male partner's age. Neonatal outcomes were not available. Wider implications of the findings Non-obstructive azoospermia can impair early embryonic potential and reduce live birth rates. Our study also shows an association between NOA and preterm birth. Future research should focus on prospective collection of data and long-term follow up of babies born by fathers with non-obstructive azoospermia. Trial registration number Not applicable

O-311 Evaluating the Reproductive Potential of Azoospermic Men by Profiling the Genome of Surgically Retrieved Spermatozoa

Abstract Study question Can whole exome sequencing of surgically retrieved spermatozoa from azoospermic men pinpoint mutations related to the etiology of their infertility and ability to support pregnancy? Summary answer Identifying key germline mutations involved in spermatogenesis helps explain reproductive failure, regardless of the etiology of the azoospermia. What is known already Azoospermia accounts for approximately 15% of male factor infertility cases. Although it can be caused by pre-testicular factors, the most recognized forms are testicular and post-testicular. Post-testicular azoospermia is mainly attributed to a mechanical obstruction, whereas testicular azoospermia, the most challenging form, is characterized by scattered functional germinal epithelia that may fail to support the meiotic process during sperm development. To elucidate the etiology of this condition, genetic studies on somatic cells have been carried out. Here, we perform whole exome sequencing (WES) on surgically retrieved spermatozoa to preferentially detect germline mutations that may be passed on to offspring. Study design, size, duration Over 3 years, we recruited patients undergoing epididymal sperm aspiration for acquired obstructive azoospermia (OA; n = 19) or testicular biopsy for nonobstructive azoospermia (NOA; n = 11). Eight men were included as fertile controls. Copy number variants (CNVs) and gene mutation profiles were obtained through WES and compared between the OA and NOA cohorts, followed by sub-analyses within those two categories according to whether they generated a clinical pregnancy (fertile) or not (infertile), while controlling for maternal age. Participants/materials, setting, methods Spermatozoal DNA was extracted and amplified from the surgically retrieved specimens (concentration, 742±520 ng/ul; quality, 1.7±0.1 nm). CNVs and gene mutations were detected using CLC Genomic Server 9.0. Genes were considered duplicated or deleted when the read depth was &amp;gt;1.5 or &amp;lt; 0.5 times the median read depth in the control. Common mutations were compared between the OA and NOA cohorts, as well as according to the couples’ clinical outcomes. Female partners had negative infertility workups. Main results and the role of chance Of 30 men (paternal age, 42.3±7yrs), 19 OA men underwent epididymal sperm retrievals (concentration, 1.1±4x106/ml, 9±12% motility), while 11 NOA men underwent testicular biopsies (concentration, 0.03±0.4x106/ml, 0.5±1% motility). WES did not indicate a significant difference in sperm aneuploidy between the two etiologies (OA, 1.7%; NOA, 1.8%) compared to the control (1.1%). In OA patients, only 3 housekeeping-related genes were deleted, while in the NOA cohort, 5 genes involved in RNA transcription (POLR2L), apoptosis (AP5M1), and basic spermiogenic functions (AP1S2, AP1G2, APOE) were deleted. OA patients and their partners (maternal age, 36.8±4yrs) underwent 19 ICSI cycles resulting in a delivery rate of 47.4% (9/19). Those able to reproduce (n = 9) shared a mutation in ZNF749, specifically affecting sperm production. The infertile men (n = 10) all shared a PRB1 deletion, controlling essential DNA replication. NOA men and their partners (maternal age, 38.2±2yrs) underwent 11 ICSI cycles, yielding a delivery rate of 72.7% (8/11). The fertile men (n = 8) all shared a MPIG6B deletion, involved in stem cell lineage differentiation. All of their infertile counterparts (n = 3) presented gene deletions not only involved in spermato/spermio-genesis (n = 4) but, most importantly, also superimposed with those encoding early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC8, REPIN1, MAPRE3, ARL4C). Limitations, reasons for caution Although maternal age was controlled for, confounding factors related to the female partner cannot entirely be excluded. While men unable to reproduce shared common gene mutations providing information about their condition, these findings still need to be confirmed in larger observations. Wider implications of the findings By performing WES, we were able to identify specific mutations associated with compromised embryo developmental competence of surgically retrieved spermatozoa. DNA sequencing technologies help identify gametes capable of sustaining a pregnancy even in the most severe form of male infertility, laying the groundwork for precision medicine in this field. Trial registration number N/A

P-164 COVID-19 vaccination does not impair fertilisation outcomes in IVF or ICSI cycles. A retrospective cohort study of patients’ cycle outcomes before and after vaccination

Study questionDoes vaccination against Covid-19 affect fertilisation outcomes for patients returning for further IVF/ICSI cycle following vaccination?Summary answerFertilisation outcome and selection of fertilisation method based on semen parameters are not altered after COVID-19 vaccination of both partners.What is known alreadyThere has been much speculation regarding the impact of the COVID-19 vaccine on fertility in both men and women. While vaccine hesitancy has been high amongst people of reproductive age, there is no current evidence that COVID-19 vaccines impact fertility. However, infection with SARS-CoV-2 virus can cause severe complications in pregnancy. Fertility may also be impacted as suggested by observation of decreased sperm count in some men, presence of SARS-CoV-2 virus in the testicles as well as invasion of SARS-CoV-2 in ovaries, uterus, vagina and placenta.Study design, size, durationThe study included 89 returning, consenting couples undergoing IVF/ICSI in 2021 at Westmead Fertility Centre soon after receiving their COVID-19 vaccine. The fertilisation method and outcomes were compared to their most recent cycle preceding vaccination (2017-2021).Participants/materials, setting, methodsFertilisation method was decided based on semen parameters. Semen ejaculates on the day of oocyte retrieval were assessed according to WHO criteria (5th edition). Conventional IVF was performed for patients with normozoospermic samples above lower reference limits for concentration and motility and ICSI was done in patients falling below these criteria. Fertilisation method and outcomes for IVF and ICSI were compared to the most recent cycle performed prior to either partner receiving their COVID-19 vaccine.Main results and the role of chance89 couples returned for an IVF/ICSI cycle following vaccination against Covid-19. Fertilisation outcomes were not significantly different following COVID-19 vaccination (NS, p > 0.05, c2). Average fertilisation rate (2PN/MII oocyte) per couple undergoing conventional IVF post-vaccination was 71.9±1.1% compared to 68.1±1.1% in the previous cycle (NS), while average fertilisation rate per couple undergoing ICSI post-vaccination was 51.7±1.2% compared to 47.6±1.2% pre-vaccination (NS). The average age of women/men at the time of OPU post-vaccination was 36.1±0.6/38.3±0.7 years, compared to 35.3±0.6/37.5±0.7 years in the treatment prior vaccination. In the post-vaccination cycle, 55 patients had normozoospermic samples on the day and underwent conventional IVF (61.8%), while 34 patients underwent ICSI (38.2%). Vaccination against COVID-19 did not affect fertilisation method selection with 82/89 (92.1%) of couples having the same method in pre- and post-vaccination cycles. Fertilisation method was different for seven couples (7.9%), with two choosing ICSI post-vaccination, and five being driven by change in semen parameters. Four couples had ICSI in the pre-vaccination cycle and IVF in the post-vaccination cycle, while one couple had IVF in the pre-vaccination cycle and ICSI in the post-vaccination cycle, all resulting in a non-significantly different fertilisation rate per couple (70.5±3.8% post-vaccination compared to 66.7±3.6% in the pre-vaccination cycle, NS).Limitations, reasons for cautionThe results represent the experience gained from current practice and not of a prospective controlled study. The developmental potential of embryos generated and clinical outcomes were not explored.Wider implications of the findingsIVF/ICSI fertilisation outcomes are likely not affected by both partners being vaccinated against COVID-19 when considering fertilisation rates, semen characteristics and fertilisation method. This information may assist vaccine hesitant couples of reproductive age in making their choice regarding vaccination.Trial registration number2101-08 QA

P-619 A prospective RCT comparing luteal phase treatment with estradiol and follicular antagonist for ovarian follicular synchronization in ICSI cycle in women with low ovarian reserve

Study questionIn women with low reserve for ovarian follicular synchronization, luteal phase treatment with estradiol (LE) or follicular antagonist(FA) would offer better outcomes?Summary answerData concludes that priming protocols with FA seem to offer statistically significant blastulation rate with similar outcome for metaphase 11 and fertilization rate.What is known alreadyThe heterogenecity of follicular growth, particularly in women with low reserve, may cause decreased retrieval of mature oocytes and reduced blastocysts. Luteal estradiol and follicular anatagonist have been used as priming protocols for synchronizing follicles by suppressing endogenous FSH. This RCT intends to compare these two protocols, in terms of clinical outcome.Study design, size, durationThis is a Prospective RCT conducted between September 2020-December 2021 with prior approval from Institutional Ethical committee (ECR/1312/INST/TG/2019). Total number of subjects were 100. Women with poor response, defined based on POSEIDON criteria were randomized into LE (n = 50) and FA (n = 50) and recruited into the study protocol based on a computer generated randomization sheet.Exclusion criteria: Age>40Participants/materials, setting, methodsAfter randomization, all patients were started on Inj rFSH & Inj HP-HMG1. Estradiol valerate pre treatment armEstradiol dosage of 4mg/day, started 7 to 10 days before day 1 of the stimulation cycle.2. Follicular Antagonist armUltrasound performed on cycle day 1/2 to identify the absence of ovarian cyst or follicle >10mm. Pretreatment with Inj Cetrotide 0.25mg sc once day for 5-7 days,thereafter stimulation was started.All the subjects underwent Frozen Embryo replacement cycle .Main results and the role of chanceBaseline variables of patients were comparable between both arms. There was 1-dropout in FA arm due to covid -19 infection and no dropouts in LE arm. Hence a total of 49 patients in FA arm and 50 patients in LE arm were available for analysis.Total days of stimulation, total dose of FSH used ,number of oocytes retrieved, M11 rate, fertilization rate, blastulation rate, FSG/oocyte used were the primary outcomes.Implantation rate, ongoing pregnancy rate ,live birth rate were the secondary outcomesOutcomes between FA Vs LE were as followsDays of stimulation – 10.65 vs 10.10 (p = 0 .289)FSH dosage- 2654 vs 2360 (p = 0.730) Fertilization rate – 91% vs 80% (p = 0.48) Blastulation rate – 67% vs 40% (p = 0.006) All outcomes compared between the two arms seemed to be similar except follicular antagonist group that showed to have a higher blastulation rate compared to luteal estradiol group which was statistically significant.Limitations, reasons for cautionThe RCT was performed on a small sample size and should be confirmed on a larger cohort.Wider implications of the findingsThis study provides assuring data on using the two agents, with statistically significant blastulation rate in FA group. Furthur research on larger sample size is required .Trial registration number12

P-209 Overweight and obese women have slower developing embryos and a lower rate of morphokinetic top-quality blastocysts compared to underweight and normal weight women

Abstract Study question Does maternal body mass index affect embryo morphokinetics and the rate of top-quality blastocysts Summary answer Overweight and obese women had slower developing embryos and a lower rate of top-quality blastocysts compared to underweight and normal weight women What is known already Time-lapse monitoring (TLM) allows for a detailed collection of embryo morphologic and kinetic changes compared to traditional periodic daily assessments. TLM allows for continuous observation of the developing embryo in time intervals for dynamic assessment of embryo morphokinetics. This may overcome any potential and unnecessary environmental fluctuations in comparison to the removal of embryos from the incubator for traditional static morphologic evaluations. There is a continuing strive to assess what factors are involved in embryo morphokinetics. Previous studies found maternal weight (either being underweight or overweight) to possibly be a contributing factor to embryo kinetic development, with conflicting results. Study design, size, duration Embryos derived from GnRH antagonist ICSI cycles between July 2013-December 2020 were included. Embryo division timings and morphokinetic top-quality embryo rates were compared between underweight (&amp;lt;18 kg/m2) normal weight (8.5–25 kg/m2) overweight (25–30 kg/m2 ) and obese (≥30 kg/m2) women. Top-quality embryos with the highest chance to implant were defined: tPNf &amp;lt;24.08, t2&amp;lt;26.6, S2&amp;lt;0.9, t8&amp;lt;56 hours post insemination (hPi) for cleavage embryos and t2&amp;lt;26.6, S2&amp;lt;0.9, t8&amp;lt;56 and tSB&amp;lt;96.6 hPi for blastocysts. Participants/materials, setting, methods Optimal time durations: time to polar body extrusion (tPB2), time to pronucleus fading (tPNf), cleavage timings (t2-t8), synchrony of the second and third cycles (S2 and S3), duration of the second cycle (CC2) time to start blastulation (tSB) were based on an in-house model including embryos with known implantation data (KID). Multivariate logistic regression analysis: age, type of gonadotrophins, number of oocytes aspirated and peak estradiol levels was performed. Pregnancy rates were calculated from transfers. Main results and the role of chance We analyzed 1,953 embryos derived from 705 GnRH-antagonist ICSI cycles; 123 embryos from 43 cycles of underweight women, 888 embryos from 319 cycles of normal weight women, 517 embryos from 186 cycles of overweight women and 425 embryos from 157 cycles of obese women. Mean BMI was 17.3 kg/m2for underweight women, 21.8 kg/m2 for normal weight, 26.6 kg/m2 for overweight and 33.8 kg/m2 for obese women. Maternal age, gravidity and parity were similar between the groups. Pregnancy rates were similar between the groups. Overweight and obese women had a statistically significant longer mean time across cell stages t5-8 and tSB compared to underweight and normal weight women. Overweight and obese patients had the lowest rate of top-quality blastocysts (10.9% and 13.4% respectively) compared with underweight and normal weight women versus (33.9% and 20.1%, respectively), p = 0.0003. Top-quality blastocyst rates remained lower in overweight and obese women after adjusting for maternal age, type of gonadotrophins, number of oocytes aspirated and peak estradiol levels. Limitations, reasons for caution The strengths are a single center study with embryos cultured under the same standardized laboratory conditions and a relatively large number of embryos in each group. Only GnRH-antagonist ICSI cycles were included to standardize the protocol and control for fertilization time. The retrospective nature of the study is a limitation. Wider implications of the findings Developing an in-house model for morphokinetic parameters is an important additive tool for improving embryo selection and assessment of embryo quality. Finding patient characteristics that influence morphokinetic embryo quality are important in further improving IVF success rates. Trial registration number None

P-157 Embryo culture under high humidity conditions in time lapse system does not improve ongoing pregnancy rates: a retrospective propensity score model study 299 ICSI cycles

Abstract Study question Does culture in high relative humidity conditions (HC) improve ongoing pregnancy rates when using a time-lapse system (TLS) for embryo culture? Summary answer Using The use of a TLS under HC does not increase the likelihood a pregnancy in comparison with cultured in dry condition (DC). What is known already Factors potentially affecting embryo development demand precise control in the IVF laboratory environment, especially for correct incubator usage. TLS provide stability during embryo culture, which is a well-known key factor for undisturbed embryo development. The humidity content of embryo culture atmosphere is especially relevant to prevention of oscillations in culture media osmolality. Previous data suggest that culture under HC has a significant effect on embryo quality and morphokinetics. However, studies assessing the effect of HC on clinical outcome are rare and inconclusive. Study design, size, duration A retrospective study of 299 women undergoing their first ICSI cycle from April 2021 to December 2021, of whom patients assigned to dry or humid conditions were 184 and 115, respectively. Participants/materials, setting, methods We used a GERI incubator, with 6 separated chambers for individual patients (3 HC, 3 DC). The effect of HC on ongoing pregnancy rate was assessed by the propensity matched sample, to reduce potential differences between women undergoing either HC or DC and to obtain unbiased estimation of treatment effect. Variables included in the propensity score model were female age, AMH, FSH, BMI, partner’s age, sperm quality and number of embryos transferred. Main results and the role of chance Patient demographics were homogeneously distributed in the two study groups. After adjusting for several confounding variables and applying the Propensity Score, no statistically differences were observed for embryo quality on day 3 [51,64 ± 34,33 (DC) Vs 53,83 ± 30,71 (HC)], blastulation rate [45,5% ± 22,60 (DC) Vs 45,9 ± 22,80 (HC)] and ongoing pregnancy rate. A trend was observed of increased ORs for ongoing pregnancy in HC conditions [OR = 1.56, 95% CI (0,77-3,15), p=0.21]. Limitations, reasons for caution The study is limited by its retrospective design, although a propensity score model may mitigate such a limitation. The lack of data on live birth rate is an additional limitation. Wider implications of the findings These results suggest that HC conditions does not increases the likelihood of embryos to achieve an ongoing pregnancy with respect to those cultured in dry conditions. Further data collection and analysis are ongoing to investigate more thoroughly and extended clinical outcome results. Trial registration number N.A.