Intracytoplasmic Sperm Injection Cycles Research Articles

SPERM FACTORS AND EGG ACTIVATION: Fertilization failure after human ICSI and the clinical potential of PLCZ1.

Two decades have passed since the discovery of phospholipase C zeta (PLCZ1) as the sperm oocyte-activating factor. At present, there is a general consensus that PLCZ1 is responsible for triggering the calcium (Ca2+) oscillations necessary to start the oocyte activation process in mammals. One proof is that abnormal, reduced, or absent PLCZ1 in human spermatozoa leads to fertilization failure (FF) after intracytoplasmic sperm injection (ICSI). ICSI is the most effective assisted reproduction technique and enables overcoming almost all male infertility conditions. Despite fertilization rates of up to 80%, FF does occur in 1-3% of ICSI cycles, which leaves these patients with few options for obtaining genetically related offspring. Assisted oocyte activation (AOA) using Ca+2 ionophores has emerged as a useful treatment option for these patients. While AOA has been proven very beneficial for the treatment of sperm-related FF, some cases of female-related FF cannot be overcome by AOA. Therefore, the development of appropriate diagnostic tests that predict the prognosis of AOA treatment would be advantageous to improve the clinical management of these patients and shorten the time to pregnancy. The aim of this review is to provide an up-to-date overview of the genetic causes of FF after ICSI and to discuss the advantages and disadvantages of using PLCZ1 as a diagnostic marker or therapeutic molecule in comparison with currently available diagnostic tests and treatments.

The Application of Artificial Intelligence in Predicting Embryo Transfer Outcome of Recurrent Implantation Failure.

Background: Recurrent implantation failure (RIF) refers to that infertile patients have undergone multiple in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycles and transferred multiple embryos without embryo implantation or clinical pregnancy. Due to the lack of clear evidence-based medical guidelines for the number of embryos to be transferred in RIF patients, how to obtain the highest single cycle pregnancy success rate with as few embryos transferred as possible while avoiding multiple pregnancy as much as possible, that is, how to balance the pregnancy success rate and multiple pregnancy rate, is a great challenge for clinicians and RIF patients. We urgently need an effective and reliable assisted decision-making method to help clinicians find this balance, and an artificial intelligence (AI) system will provide an efficient solution. Design and Methods: In this research, we filtered out the RIF data set (n = 45,921) from the Human Fertilisation and Embryology Authority (HFEA) database from 2005 to 2016. The data set was divided into two groups according to the number of embryos transferred, Group A and B. Group A included 34,175 cycles with two embryos transferred, while Group B included 11,746 cycles with only one embryo transferred, each containing 44 features and a prediction label (pregnancy). Four machine learning algorithms (RF, GBDT, AdaBoost, and MLP) were used to train Group A and Group B data set respectively and 10-folder cross validation method was used to validate the models. Results: The results revealed that the AdaBoost model of Group A obtained the best performance, while the GBDT model in Group B was proved to be the best model. Both models had been proved to provide accurate prediction of transfer outcome. Conclusion: Our research provided a new approach for targeted and personalized treatment of RIF patients to help them achieve efficient and reliable pregnancy. And an AI-assisted decision-making system will be designed to help clinicians and RIF patients develop personalized transfer strategies, which not only guarantees efficient and reliable pregnancy, but also avoids the risk of multiple pregnancy as much as possible.

O-134 Predictive value of seminal oxidation-reduction potential (ORP) and sperm DNA fragmentation (SDF) analysis for reproductive outcomes of intracytoplasmic sperm injection (ICSI) cycles

Abstract Study question Do seminal oxidation-reduction potential (ORP) and sperm DNA fragmentation (SDF) predict ICSI cycle outcomes? Summary answer Both SDF and seminal ORP have strong prognostic value in predicting good fertilization (≥80%), blastocyst development (≥60%), clinical pregnancy and live birth. What is known already Seminal oxidative stress (OS) has been found to play an important role in various aetiologies of male infertility and is induced by the imbalance between reactive oxygen species (ROS) and antioxidants. OS is a major cause of sperm DNA fragmentation (SDF). It is well established that high levels of seminal OS and SDF are negatively associated with ART outcomes. Previous studies have associated high levels of SDF with an increase risk of miscarriage and a decrease in live birth rate. Study design, size, duration This prospective study included 144 couples who had a fresh autologous ICSI cycle between June 2018 and December 2020. There was no restriction to patients with severe male factor infertility. All cryopreservation and third party assisted reproductive techniques were excluded. Participants/materials, setting, methods This multi-centre study was performed at three ART Clinics in Cape Town, South Africa. All couples included in the study had fresh autologous ICSI cycles with freshly ejaculated semen. Seminal ORP testing was performed using the MiOXSYS system and SDF by means of a fluorescence microscopic TUNEL assay on freshly ejaculated semen samples that were used for ICSI. The ART outcomes evaluated were fertilization rate, blastocyst development rate, clinical pregnancy rate and live birth rate. Main results and the role of chance The study shows that seminal ORP significantly negatively correlates with fertilization rate (r=-0.267; P = 0.0012), blastocyst development rate (r=-0.389; P < 0.0001), pregnancy (r=-0.296; P = 0.0003) and live birth (r=-0.353; P < 0.0001). ROC curve analysis shows significant predictive power for ORP for fertilization (≥80%; AUC=0.652; P = 0.0012), blastocyst development rate (≥60%; AUC=0.762; P < 0.0001), pregnancy (AUC=0.677; P = 0.0002) and live birth (AUC=0.729; P < 0.0001). Comparable results were obtained for SDF. An average cut-off value for ORP of 0.55 mV/106 sperm/mL was calculated. The AUCs between the SDF and ORP results for all reproductive outcome parameters did not differ. For all reproductive outcome parameters, normal sperm morphology shows the lowest predictive power. ORP with male age as confounding factor had significant effects on odds ratios (OR) of fertilization (OR = 0.1446; CI: 0.048-0.430; P = 0.0001) and blastocyst development (OR = 0.2468; CI: 0.095-0.635; P = 0.0029), while it was not significant for pregnancy and life birth. Multivariate logistic regression with fertilization, blastocyst development, pregnancy and live birth as dependent parameters and high/low seminal ORP, primary and secondary infertility, unexplained infertility, polycystic ovaries, endometriosis, tubal factor myomectomy, ovarian insufficiency and advanced maternal age as independent variables showed overall model fits (P < 0.0002) with a significant (P < 0.0084) influence of ORP on all reproductive outcome parameters. Limitations, reasons for caution Since only ICSI patients were included in this study, this might have contributed to the fact the cut-off value of ORP is lower than the published cut-off. A further limitation is the use of a fluorescence microscopic TUNEL assay which drastically limited the number of sperm evaluated. Wider implications of the findings This is the first study showing predictive value of seminal ORP for reproductive outcome including pregnancy and live birth. The method is easy and much quicker than current methods for the determination of SDF. Hence, it can be used a point of care method. Trial registration number Not applicable

O-186 Paternal age is significantly related with the type of delivery and the sex of the newborn in IVF or ICSI cycles with donated oocytes

Abstract Study question Does paternal age have any effect on obstetric and perinatal outcomes in in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycles with donated oocytes? Summary answer Newborn sex and type of delivery, but not other obstetric and perinatal outcomes measured, were significantly different when comparing our paternal age groups. What is known already Currently there is a delay in fatherhood that has led to an increase of the average male age at which the first child is conceived. Studies relating paternal age with reproductive risks are increasing and results are controversial leading to a need of more research about it, which this study tries to address. Studies considering treatments with donated oocytes (controlled female factor) allow for a better understanding of male factor and reproductive risks. These risks for the mother and offspring need to be properly assessed and therefore it is important to study whether they can be affected by paternal age. Study design, size, duration This retrospective observational multicentric cohort study has considered pregnancies and children conceived from donor IVF-ICSI performed to couples in Spain IVIRMA clinics between January 2008 and March 2020 using patients’ own sperm. Paternal age ranged from 21 to 54 years. The study population was divided in three groups: <30(A, reference), 30-40(B) and >40(C) years. The data available and included consisted of 16382 patients who had a delivery with a live birth and 17988 singleton newborns. Participants/materials, setting, methods We evaluated pregnancies and children from donor IVF-ICSI with own semen, known age and resulting in a delivery. Data were obtained from the patient’s clinical charts to build the database to analyze the main outcomes of the study. P < 0.05 was considered statistically significant. We measured several obstetric and perinatal outcomes that were adjusted according to selected patients’, cycles’ and semen samples’ characteristics, as well as donor age, gestational age, type of delivery and newborn sex. Main results and the role of chance The analysis included: 233 newborns for A, 6539 for B and 11216 for C. Results are expressed as rate with 95%CI. There were no statistically significant differences for the incidence of gestational diabetes, anemia, pre-eclampsia or preterm birth; newborn weight, length, cranial circumference, Apgar score (1, 5 and 10 min) and NICU admission between the reference younger group (A) and the older paternal age groups (B and C). However, there were statistically significant differences for the incidence of hypertension between A 18.1%(10.5-28.1) and B 9.8%(8.8-10.9) with OR = 0.5(0.3-0.9)(p = 0.015); for delivery type between A[cesarean 44.0%(37.2-51.0)] and C[cesarean 64.0%(63.1-65.0)] with OR = 2.3(1.7-3.0)(p < 0.001); and for newborn sex between A[female 55.1%(48.2-61.8)] and B[female 48.2%(46.9-49.4)] with OR = 0.8(0.6-1.0)(p = 0.047). Therefore, these results suggest an increased risk of cesarean delivery in the older age group compared to the younger one and of having a male birth in the 30-40 group compared to the younger group. There was also a decreased risk of hypertension in 30-40 group compared to < 30 group, which should be clarified in further studies. After adjusted analysis, there were still significant differences for newborn sex between A and B (p = 0.048) and for delivery type between A and C (p = 0.043), but no significant differences for hypertension were observed. Limitations, reasons for caution Due to the retrospective nature of this study there are some biases derived from the clinical practice. Although there is some missing data limiting sample size, this is still large. Moreover, it considers donated oocytes (controlling female factors) limiting the generalization of our results to a population of young women. Wider implications of the findings Studies focusing on paternal age are increasing due to fatherhood delay, being results controversial. Paternal age comparison showed significant differences for hypertension, delivery type and newborn sex, however adjusted analysis only found an increased risk of cesarean delivery in C vs. A and of male newborn in B vs. A. Trial registration number NA

O-296 Paternal age does not affect obstetric and perinatal outcomes in IVF or ICSI cycles with autologous oocytes

Abstract Study question Does paternal age have any effect on obstetric and perinatal outcomes in in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycles with autologous oocytes? Summary answer Gestational diabetes, delivery type, cranial circumference and preterm birth were significantly different between our age groups, although after adjusted analysis no significant differences were seen. What is known already In recent years, there has been an increase of the average male age at which the first child is conceived. Therefore, studies addressing paternal age and reproductive risks are increasing and results are controversial, so there is a need of more research about it, which this study tries to address. Although several studies suggest the threshold to consider paternal age ‘advanced’ at 40 years, this limit is not well established. The assessment of risks concerning the mother and offspring is important and, thus, so is the study of its association with paternal age. Study design, size, duration This retrospective observational multicentric cohort study has considered pregnancies and children conceived from IVF-ICSI performed to couples in Spain IVIRMA clinics between January 2008 and March 2020 using patients’ own sperm and autologous oocytes. Paternal age ranged from 18 to 51 years. The study population was categorized in three groups: <30(A, reference), 30-40(B) and >40(C) years. The data available included 14402 patients who had a delivery with a live birth and 16118 singleton newborns. Participants/materials, setting, methods We evaluated pregnancies and children from autologous IVF-ICSI with own semen, known age and resulting in delivery. Data was exported to obtain the clinical database followed by the statistical analysis of the study main outcomes. P < 0.05 was considered statistically significant. Obstetric and perinatal outcomes were measured including delivery type, incidence of preterm birth, gestational diabetes, anemia, hypertension and pre-eclampsia; and newborn sex, weight, length, cranial circumference, Apgar score and neonatal intensive care unit (NICU) admission. Main results and the role of chance Results are expressed as rate or mean with 95%CI. There were no statistically significant differences for the incidence of anemia, hypertension or pre-eclampsia; newborn sex, weight, length, Apgar score (1, 5 and 10 min) and NICU admission between the reference younger group (A) vs. the older paternal age groups (B and C). However, there were statistically significant differences for the incidence of gestational diabetes when comparing A 5.1%(3.5-7.2) with B 7.7%(7.1-8.3) with OR = 1.5(1.1-2.2)(p = 0.021) and with C 9.1%(7.1-11.4) with OR = 1.9(1.2-2.9)(p = 0.005); for delivery type when comparing A[cesarean 33.8%(31.3-36.4)] with B[cesarean 37.8%(37.0-38.7)] with OR = 1.2(1.1-1.3) (p = 0.004) and with C[cesarean 49.3%(46.7-52.0)] with OR = 1.9(1.6-2.2) (p < 0.001); for cranial circumference when comparing A 34.5(34.3-34.7) with C 34.8(34.6-35.0) with p = 0.03; and for the incidence of preterm birth when comparing A 7.9%(6.6-9.5) with C 5.9%(4.7-7.2) with OR = 0.7(0.5-1.0)(p = 0.034). These results suggest an increased risk of gestational diabetes and cesarean delivery as paternal age increases. Moreover, group C showed a larger cranial perimeter and a lower risk of preterm birth when compared to group A. However, after adjusted analysis (according to selected patients’, cycles’ and semen samples’ characteristics, gestational age, newborn sex and type of delivery) these differences were no longer statistically significant, so probably other clinical variables are involved in the differences seen. Limitations, reasons for caution Despite the retrospective nature of this study that leads to biases derived from the clinical practice and to the presence of missing data limiting sample size, the study includes a huge sample size. Wider implications of the findings Currently, fatherhood delay has led to a growing interest in studies addressing paternal age and reproductive risks. Our paternal age comparison showed significant differences for gestational diabetes, delivery type, cranial circumference and preterm, no longer significant after adjusted analysis, so probably other clinical variables were involved. Further studies are needed. Trial registration number Not applicable

O-208 Use of epididymal spermatozoa in in vitro fertilization cycles impacts the morphokinetics of embryos cultured in a time-lapse imaging incubator system

Abstract Study question Does sperm origin impact embryo morphokinetics (the moments at which an embryo reaches a developmental milestone) and clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles? Summary answer The use of epididymal sperm correlates with late cell cleavages, and higher multinucleation and abnormal cleavage rates compared to those from men without infertility factor. What is known already ICSI with sperm surgically retrieved from the epididymis (PESA) dramatically altered the pregnancy prognosis in couples with severe male factor infertility, including obstructive azoospermia. The integration of time-lapse imaging (TLI) into the in vitro fertilization (IVF) laboratory allowed for an in-depth analysis of embryonic development. However, the impact of the use of epididymal sperm on embryonic morphokinetics has been poorly studied. We hypothesized that factors related to sperm origin may interfere with the speed and pattern of cell divisions. Study design, size, duration Women undergoing ICSI with epididymal sperm between Jan/2019 and Dec/2020 in a private university-affiliated IVF center were included (PESA group, n = 32 cycles/276 embryos). A matching was performed to form two groups of women undergoing ICSI using ejaculated sperm from partners with idiopathic male factor infertility (IMF group, n = 32 cycles/284 embryos) or without male factor infertility (Control group, n = 32 cycles/246 embryos). Generalized linear models followed by Bonferroni post hoc were used to compare morphokinetics between groups. Participants/materials, setting, methods Embryos were cultured in the EmbryoScope incubator, which recorded the following kinetic markers: timing to pronuclei appearance and fading (tPNa and tPNf), two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), morulae (tM), start of blastulation (tSB) and blastulation (tB). Durations of second and third cell cycles (cc2 and cc3) and timing to complete synchronous divisions s1, s2, and s3 were calculated. The KIDScore ranking was recorded. Main results and the role of chance Embryos derived from epididymal sperm showed significantly slower divisions compared to those from ejaculated sperm (Table 1). The KIDScore rank was significantly lower for embryos deriving from epididymal sperm (3,1 ± 0.2) compared to those deriving from ejaculated sperm from IMF and control groups (5.4 ± 0.1 and 5.6 ± 0.2, p < 0.001, respectively). The incidence of multinucleation was significantly higher in embryos deriving from epididymal sperm (23.2%) compared to those deriving from ejaculated sperm from IMF and control groups (2.8% and 3.7%, p < 0.001, respectively). The incidence of abnormal cleavage patterns (direct or reverse) was significantly higher in embryos deriving from epididymal sperm (11.1%) compared to those deriving from ejaculated sperm from control group (4.3%, p = 0.001). Similar clinical outcomes were observed between the groups. Limitations, reasons for caution Retrospective nature of this study and the small sample size may be a reason for caution. Wider implications of the findings The use of epididymal sperm for ICSI correlates with delayed cell cleavage, and increased incidences of multinucleation and abnormal cleavage patterns. This finding highlights the importance of TLI for the identification and de-selection of slow-growing embryos for transfer, in ICSI cycles with surgical sperm retrieval. Trial registration number not applicable

P-099 The effect of sperm DNA quality on blastocyst and euploid rates

Abstract Study question Do sperm DNA quality measurements influence the blastocyst rate and/or euploid rates? Summary answer Sperm DNA fragmentation has a negative influence on the rate of blastulation. It does not impact on euploidy rates assessed by pre-implantation genetic testing (PGT-A). What is known already Some studies show that sperm DNA has no influence on embryo development and that its negative influence begins following implantation. Other studies show that every step of the fertility process from fertilization onwards is impacted by the male gamete as well as the female gamete. To date, data has been confounded by different sperm DNA tests and different clinical practices and parameters measured. Study design, size, duration This was a retrospective study using the sensitive Comet assay to analyse whether it’s three sperm DNA fragmentation scores influence embryo development or euploidy. Participants/materials, setting, methods Data from 110 cycles of intracytoplasmic sperm injection (ICSI) where the blastocyst rate (number of blastocysts/number of mature oocytes injected) was obtained from the patient database of a UK single centre. Inclusion criteria: Only outcomes from the first assisted reproductive cycle after a Comet assay had been performed were included. Included cycles were within one year of the test. Main results and the role of chance Blastocysts were ranked in 4 groups- 0.25-0.49, 0.5-0.74, 0.75-1.00. Three Comet scores were analysed: Average Comet Score (damage across the semen sample; ACS), Low Comet Score (proportion of sperm with good DNA; LCS) and High Comet Score ( proportion of sperm with high DNA damage; HCS). Clinical thresholds were calculated by comparison with fertile donor sperm. High ACS and LCS and low HCS were all associated with higher blastocyst rates (ANOVA, p = 0.013; Kruskal Wallis, p = 0.027, Post Hoc pairwise comparisons between blastocyst groups (p = 0.016-0.024). No relationship was found between euploid rate (number of euploid embryos/total embryo number) and sperm DNA damage p = 0.761). Limitations, reasons for caution Retrospective design requiring more data to clarify these associations coupled with the lack of randomisation and low power analysis. Wider implications of the findings This study reports the negative impact of sperm DNA fragmentation on blastocyst formation rates. Male partner diagnostic assessments such as sperm DNA quality testing are useful adjuncts to semen analysis. Further research to determine if there is a relationship between sperm DNA damage and euploid embryo outcomes is ongoing. Trial registration number IRB-001C06-01-22

P-003 Sperm retrieved by microdissection testicular sperm extraction (MD-TESE) is associated with altered sex ratio of offspring after intracytoplasmic sperm injection in men with non-obstructive azoospermia

Abstract Study question Does the sperm retrieved surgically by MD-TESE of men with Non-Obstructive Azoospermia affect the live birth sex ratio? Summary answer Our data indicates that sperm retrieved in men with non-obstructive azoospermia alters the sex ratio in favor of female offspring. What is known already Non-Obstructive Azoospermia is the most severe form of male infertility and is the most common cause of Azoospermia. Among the different surgical techniques, MD-TESE gives the highest rate of sperm retrieval and when combined with intracytoplasmic sperm injection (ICSI), live births had been achieved with variable success, Sex ratio of offspring can vary substantially due to several variables, however no studies have investigated the resultant birth sex ratio in this setting. Study design, size, duration Retrospective Cohort study. Data were collected between May 2009 and December 2019. A total of 100 consecutive men with non-obstructive Azoospermia underwent MD-TESE for sperm retrieval. 46 couples underwent ICSI and live birth rate, cumulative live birth rate and sex ratio of offspring analysed. Participants/materials, setting, methods One-hundred men underwent MD-TESE out of which 46 proceeded with ICSI. Demographic data including male and female age, testicular volumes and serum hormone values are given as mean ± SD with a range (minimum and maximum). Fertility outcomes including sperm retrieval, fertilization rate, number of embryos transferred, and live birth rates and cumulative live birth rates were recorded. Chi-square test was performed to compare the proportions. Main results and the role of chance Sperm retrieval was successful in 58%(58/100). Testicular volume and hormonal parameters did not vary among patients with positive or negative sperm retrieval. Histology gave best correlation with sperm retrieval. Hypospermatogenesis yielded the highest sperm retrieval 93%(26/29), followed by Maturation arrest 78%(7/9), then Hyalinosis 46% (6/13) and lastly Sertoli cell only 38%(19/50). 46 couples underwent ICSI cycles where the mean age of patients and their wives were 36.4 ± 3.7y and 33.0 ± 4.3y. Fertilization rate and mean number of transferred embryos were 51.4 % and 1.7. Live birth rate and Cumulative Live Birth rate per Embryo transfer were 60.5% (26/43) and 74.4% (32/43) and per started ICSI cycle were 56.5% (26/46) and 69.6% (32/46), with a twin rate of 15.3%(4/26). Mean gestational length and mean body weight at birth were 39.0 ± 1.4w and 3228.5 ± 5.5 g. Number of live offspring were 36 (Female: Male = 26:10) giving rise to sex ratio of 2.6 to1 in favor of female offspring (P < 0.05). There were no neonatal death, and one baby had phenylketonuria. Limitations, reasons for caution This is the first study to test the hypothesis of sex ratio variation related to the source of sperm; the strength of our study is that all procedures were performed by the same surgeon, so there are no operator-dependent differences. This finding needs to be confirmed in larger cohort studies. Wider implications of the findings It has been demonstrated that different stressors may alter the sex of the offspring. While in the present study the stressor is the parental hypogonadism associated with non-obstructive azoospermia, We hypothesise that testicular environment may direct spermatogenesis in favor of X carrying sperm around the time of sperm retrieval. Trial registration number N/A

P-138 Transcriptomic signatures in trophectoderm and inner cell mass of human blastocysts with expected pregnancy rates

Abstract Study question Is it possible to identify the molecular factors that contribute to the implantation potential of blastocysts? Summary answer Genes correlated with expected pregnancy rate in trophectoderm (TE) and inner cell mass (ICM) respectively were identified, and aneuploidy alone couldn’t predict the pregnancy expectation. What is known already The selection of suitable embryos for transfer is critical for achieving successful pregnancy outcomes in assisted reproductive technology (ART). Although pre-implantation genetic testing for aneuploidy (PGT-A) as well as morphological and chronological evaluation of embryos, have been conducted in clinical practice, they do not fully guarantee successful pregnancy. Recently, transcriptional events in early human embryonic development have been analyzed using RNA-sequencing (RNA-seq) and researchers are attempting to apply this information to ART. Study design, size, duration To determine the correlation between blastocyst evaluation and pregnancy rate, we retrospectively analyzed 1,890 cases underwent frozen-thawed blastocyst transfer from March 2018 to December 2020. A total of 13 blastocysts that were cryopreserved for clinical use between February 2011 and September 2018, then scheduled for disposal and with consented for research, were subjected to RNA-seq without distinguishing between conventional in vitro fertilization (c-IVF) and intracytoplasmic sperm injection (ICSI). Participants/materials, setting, methods Blastocysts were donated by infertile couples undergoing c-IVF or ICSI cycles at the Yamashita Shonan Yume Clinic with informed consent under ethical approval. TE and ICM cells were collected from blastocysts by using a micromanipulator and then subjected to RNA-seq. Gene expression analysis and digital karyotyping using RNA-seq were performed simultaneously for TE and ICM cells, respectively. One-way analysis of variance, chi-square test and Tukey's multiple comparison test were used for this study. Main results and the role of chance Blastocysts were classified into three groups to correlate with pregnancy rates based on the diameter of the blastocyst and the time to reach this size: those taking less than 130 h to reach a diameter of > 170 μm (Group 1, n = 676), those taking more than 140 h to reach a diameter of < 180 μm (Group 2, n = 158), and the rest (Group 3, n = 1,056). The pregnancy rates of Groups 1, 2 and 3 were 59.0%, 16.5%, and 34.2%, respectively (p < 0.01). Assessing the differences in overall transcripts correlated between Group 1 (n = 5), Group 2 (n = 4), and Group 3 (n = 4), 26 and 67 differentially expressed genes (DEGs) were identified in ICM and TE cells, respectively. Importantly, downregulated genes in TE of blastocysts with lower expectation of pregnancy included tight junction-related genes, such as CXADR, CLDN10, and ATP1B1, which were implicated in peri-implantation development. Digital karyotyping revealed karyotypic abnormalities and mosaicism in all groups with no common abnormalities observed, suggesting that aneuploidy alone cannot predict the pregnancy expectation. Limitations, reasons for caution Although 93 genes potentially related to implantation have been identified, it is still unclear how these genes are involved in implantation. In vitro implantation models using human embryos and artificial embryos currently under development are expected to contribute to the elucidation of the functions of these genes. Wider implications of the findings Our results provide reliable candidates for genes that could allow for non-invasive selection of high-quality blastocysts for ART and add to the knowledge base of transcriptional events in human peri-implantation development. Trial registration number not applicable

P-270 Assisted gamete treatment to pinpoint acquired meiotic maturity and overcome oocyte activation deficiency contributed by both gametes

Abstract Study question How can we treat couples with complete and persistent fertilization failure with ICSI linked to a combination of oocyte- and sperm-related oocyte activation deficiency (OAD)? Summary answer By targeting spindle presence, we optimized oocyte response to chemical activation and enhanced fertilization. Genomic assessment confirmed gamete contribution. What is known already Total fertilization failure occurs in 1-3% of all intracytoplasmic sperm injection (ICSI) cases. In sperm-factor OAD, the lack of phospholipase C zeta (PLCζ) prevents the spermatozoon from initiating downstream calcium oscillation in the oocyte. In these cases, assisted gamete treatment (AGT), which exposes gametes to calcium ionophore, has been adopted to artificially trigger the influx of calcium ions and has been shown to effectively improve fertilization. However, AGT is limited to triggering an intracytoplasmic calcium influx and still requires optimal ooplasmic maturity. Study design, size, duration Over the past 17 months, we identified couples with compromised PLCζ and reported persistent fertilization failure with ICSI despite AGT treatment. We then devised a treatment plan comprising an extended in vitro culture (IVC) to pinpoint meiotic oocyte maturity confirmed by the presence of a meiotic II spindle and followed by AGT post-ICSI. Genomic assessment was also carried out. Participants/materials, setting, methods Two couples with recurrent and total fertilization failure even after AGT were included. PLCζ expression was assessed using immunofluorescence on ≥ 200 cells/specimen with a 30% threshold. In the follow-up cycles, IVC was extended for at least 8 hours between retrieval and ICSI. Metaphase II spindles were visualized by Oosight®. AGT was performed by exposing both spermatozoa and oocytes to calcium ionophore. NGS was performed on spermatozoa to identify gene mutations involved in fertilization. Main results and the role of chance We identified 2 couples (couple A: 37-year-old female, 39-year-old male; couple B: 32-year-old female, 33-year-old male) with the following semen parameters: average volume of 2.6 ml, concentration of 82.0x106/ml, 44% motility, and normal morphology of 3%. The oocyte maturation rate was 76.3% (45/59) but resulted in zero fertilized out of a total of 45 MII oocytes injected. In-house PLCζ assessment revealed a deficiency of oocyte activation factor at 12.9%. AGT treatment alone failed to enhance fertilization on a subsequent cycle, resulting in 0% (0/8) and 5.6% (1/18) fertilization rates for couples A and B, respectively. Couple A then underwent 3 ICSI cycles with extended IVC and AGT; upon examination of nuclear maturity, 91.4% (32/35) of oocytes displayed normal metaphase II spindle and achieved an overall fertilization rate of 43.8% (14/32). To date, 12 blastocysts were cryopreserved. In couple B, 27 oocytes out of 34 retrieved presented normal metaphase II spindles after extended IVC; ICSI with AGT yielded a fertilization rate of 63.0% (17/27). All 17 zygotes were cryopreserved. Overall, our treatment improved fertilization to an overall rate of 52.5% (31/59, P <0.00001). Genomic assessment of spermatozoa identified gene mutations involved in fertilization (ADAM15, ADAM30) and calcium channel activity (CATSPER1). Limitations, reasons for caution Assisted gamete treatment can enhance fertilization in cases of deficiency in PLCζ. However, chemical activation requires a responsive ooplasm that has reached meiotic maturity. These rare cases require precise diagnoses and tailored treatment techniques to address each aspect of sperm- and/or oocyte-factor OAD. Wider implications of the findings Our study has demonstrated the usefulness of extended IVC by targeting spindle presence to enhance chemical responses to AGT. Our findings show that although calcium ionophore can trigger the release of intracellular calcium and allow fertilization, a fully mature ooplasm is required. Trial registration number N/A

P-320 The Effects of Endometriosis on Oocyte Morphology and Embryo Development

Abstract Study question What is the effect of the endometriosis on oocytes morphological abnormalities in intracytoplasmic sperm injection cycles.? Summary answer An increase in the detection rate of refractile body and polar body fragmentation was observed in the morphology of oocytes obtained from endometriosis patient. What is known already The quality of the oocyte is suspected for the lower clinical success in endometriosis patients compared to other patient groups. The oocyte quality is determined by its morphological, cellular, and molecular evaluations. However, there are limited studies on the association between endometriosis and oocyte morphology. Study design, size, duration This was a retrospective cohort study, conducted between January 2020 and August 2021, at a tertiary care hospital. The women aged 24-35 years and BMI 19-30 kg/m2 ranges who underwent fresh GnRH antagonist cycles and stimulated with 150-300 IU dose of recombinant FSH (rFSH) and then trigerred with 250 micrograms/0.5 ml of rhCG were included. Participants/materials, setting, methods The patients who were cycled with the etiology of endometriosis (n = 46 cycles, Group 1) and those who were cycled with the etiology of male infertility (n = 64 cycles, Group 2) were included in this study. In addition to the comparison of intracytoplasmic and extracytoplasmic (polar body, perivitelline space, zona pellisuda) anomalies observed in oocyte morphology between groups, morphology anomalies that affect fertilization and blastocyst development within the group were evaluated. Main results and the role of chance The patient age, BMI, antral follicle counts, duration of infertility, and basal endocrine parameters among patients who were cycled with the etiology of endometriosis (n = 46 cycles, Group 1) and those who were cycled with the etiology of male infertility (n = 64 cycles, Group 2) were not different (p>0.05). As a result of the comparison of intracytoplasmic anomalies of 800 oocytes in total (n = 263 Group 1; n = 537 Group 2) between the groups; the refractile body determination (OR:1.23; 95% CI 1.08 -1.4, p <0.00) was higher in the endometriosis group compared to the other group. As a result of the comparison of extracytoplasmic anomalies between the groups; the polar body fragmentation (OR:1.3; 95% CI 1.01 - 1.85, p> =0.03) determination was higher in the endometriosis group compared to the other group. When the effect of oocyte morphology anomalies on fertilization and blastocyst development within the groups was evaluated by logistic regression analysis, it was found that granulation of the perivitelline space had a negative effect on blastocyst development only in the endometriosis group (β = -0.69; p> =0.31). Limitations, reasons for caution The main limitation of our study was its retrospective design, which precludes us from reaching a more definitive conclusion on the relation between oocyte morphology of endometriosis an fertility outcomes. Wider implications of the findings The previous studies indicate that PVS and PBI abnormalities may be ascribed to over-mature oocytes and also be negatively correlated with fertilization rate and embryo quality. Our study suggest that endometriosis seems to negatively affect oocyte quality and maturation process. Trial registration number not applicable

P-032 Sperm parameters can predict the success of assisted reproductive technology. Single-center and retrospective analysis of assisted reproductive technology cycles from 1992 to 2020

Abstract Study question Male parameters are largely underestimated in assisted reproductive technology (ART) success, so it was evaluated whether conventional semen parameters could predict assisted reproductive technology success Summary answer Sperm motility plays a role in predicting in vitro fertilization (IVF) success, while sperm morphology is the relevant parameter in intracytoplasmic sperm injection (ICSI) cycles. What is known already The detection of predictive markers would be very useful to choose the ART type and usually the male counterpart was relegated to a secondary role. Obviously, no convincing evidence that ART outcomes may be dependent on sperm parameters exists. The variation over the years of the reference values in semen analysis, together with the poor inter-laboratory standardization of sperm assessment, contributes to this very complex scenario. It remains undeniable that identifying thresholds of seminal parameters with prognostic significance in terms of pregnancy rate would have a considerable clinical impact in the assisted reproduction field. Study design, size, duration A single-center, retrospective, observational study was carried out including couples attending the Santa Maria Nuova Hospital-IRCCS of Reggio Emilia for infertility between January 1992 and December 2020. Only fresh ART cycles performed in a single third-level fertility center were considered for this study with a total of 22,013 cycles. Fertilization rate was the primary end-point, representing a parameter immediately dependent on male contribution. Pregnancy and live birth rates were considered in relation to semen variables. Participants/materials, setting, methods Both partners were aged over 18 years and satisfied national criteria to access ART procedures. The studies considering common sperm parameters obtained by conventional semen analysis, that is, sperm number, motility, and morphology. Semen analyses were performed according to the World Health Organization (WHO) manual available at the time of the ART cycle. Ovarian stimulation was performed applying different hormone stimulating protocols and ART approach used are IVF or the ICSI. Main results and the role of chance Considering the entire ART cohort, 5819 cycles were IVF (26.4%) and 16,194 were ICSI (73.6%). Considering strong ART outcomes, the overall pregnancy rate was 20.4% (4,368 cycles) for biochemical and 20.2% (4,314 cycles) for clinical pregnancies, respectively. Among the latter, the overall live birth rate was 63.3%. Interestingly, both biochemical (20.8% vs. 19.1%, p < 0.001) and clinical (20.7% vs. 18.5%, p < 0.001) pregnancy rates were significantly higher in ICSI than IVF cycles. On the contrary, the live birth rate was not significantly different between ART methodologies (64.0% in ICSI vs. 60.9% in IVF, p = 0.074). Only 610 cycles (2.8%) were interrupted since no oocytes were retrieved after gonadotropins stimulation. Considering only cycles with oocytes retrieved, the fertilization rate was significantly higher in ICSI (42.4±39.7%) compared to IVF cycles (38.4±49.2%) (F = 27.343, p < 0.001). In IVF setting, both progressive motility (p = 0.012) and motility after capacitation (p = 0.002) significantly predicted the fertilization rate (statistical accuracy = 71.1%). Sperm motilities also predicted pregnancy (p < 0.001) and live birth (p = 0.001) rates. In ICSI, sperm morphology predicted fertilization rate (p = 0.001, statistical accuracy = 90.3%). Limitations, reasons for caution These results, although statistically significant, show confidence intervals at the limits of significance. Therefore, this data must be carefully considered. From entering the ART path to its outcome, the variables encountered are numerous. The identification of a single parameter that influences, slightly, the final outcome, indicates a considerable clinical weight. Wider implications of the findings Interestingly, sperm motility plays a role in predicting in vitro fertilization success, while sperm morphology is the relevant parameter in ICSI. These parameters may be considered reliable tools to measure the male role on ART outcomes, potentially impacting the clinical management of infertile couples. Trial registration number not applicable

P-761 Perinatal outcomes following blastocyst versus cleavage-stage transfer of fresh embryos: an updated analysis of UK national data

Abstract Study question Are there differences in perinatal outcomes in singleton live births following transfer of fresh embryos at blastocyst versus cleavage-stage? Summary answer In singleton live births arising from fresh embryos, blastocyst transfer is not associated with higher perinatal risks than cleavage-stage transfer. What is known already Extended culture involves prolonged exposure to culture media. Although blastocyst transfer is currently the default strategy for many fertility clinics, there is a paucity of perinatal data from randomised trials. Previous observational research, including an earlier analysis of United Kingdom (UK) national data (up to 2012) by our group, has been suggestive of increased perinatal risks following blastocyst transfer. As laboratory practices have continued to evolve over time, we felt it was important to address this question again using more contemporary data (up to 2018) which have been recently made available by the UK Human Fertilisation and Embryology Authority (HFEA). Study design, size, duration A retrospective cohort study was undertaken using anonymised HFEA data on all fresh in-vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) cycles resulting in a singleton live birth (127,632) between 1999 and 2018. Perinatal outcomes were compared in babies conceived from the transfer of fresh embryos which were either at cleavage-stage (2-3 days of culture) or blastocyst-stage (5-6 days). Participants/materials, setting, methods All IVF/ICSI cycles resulting in singleton live births following the transfer of fresh blastocysts or cleavage-stage embryos were included. Logistic regression was used to calculate adjusted risk ratios (aRRs) with 95% confidence intervals (CIs) for perinatal outcomes including a healthy baby (term delivery, normal birth weight, no congenital anomalies) following either blastocyst or cleavage-stage transfer. Outcomes were separately compared in women who had a singleton live birth after their first fresh cycle of IVF/ICSI. Main results and the role of chance Of 127,632 singleton live births, 54,688 occurred after blastocyst transfer while 72,944 resulted from cleavage-stage transfer. The corresponding numbers in 64,812 women who underwent their first cycle of IVF/ICSI were 25,452 (blastocyst) and 39,360 (cleavage-stage). Outcomes following blastocyst and cleavage-stage transfer were comparable in terms of low birth weight (7.2% vs. 7.5%, aRR 0.98, 95% CI 0.93-1.05), very low birth weight (1.7% vs. 1.8%, aRR 0.95, 95% CI 0.82-1.10), high birth weight (6.4% vs. 7.3%, aRR 0.98, 95% CI 0.92-1.04), very high birth weight (1.2% vs. 1.4%, aRR 0.99, 95% CI 0.87-1.14), very preterm birth (1.7% vs. 1.7%, aRR 1.04, 95% CI 0.92-1.17) and congenital anomalies (0.6% vs. 2.5%, aRR 0.93, 95% CI 0.80-1.08). These findings were confirmed in the sensitivity analysis involving women undergoing their first fresh embryo transfer. In the general population, the risk of preterm birth was higher following blastocyst transfer (7.7% vs. 7.3%, aRR 1.07, 95% CI 1.01-1.13) but this difference was not maintained in the sensitivity analysis (aRR 1.05, 95% CI 0.96-1.14). The chance to deliver a healthy singleton was similar across both groups in both primary cycle-based analysis (aRR 1.00, 95% CI 0.98-1.02) and sensitivity analysis (aRR 1.00, 95% CI 0.97-1.02). Limitations, reasons for caution The primary analysis was unable to discriminate between cycles and women or to adjust for clustering of women within cycles. Data were unavailable for some key confounders (body mass index, smoking status, previous medical history and complications during pregnancy). Wider implications of the findings While our findings provide some reassurance to patients undergoing blastocyst transfer, the limitations of the current study mean that further research is needed using women based rather than cycle based data. Trial registration number not applicable

P-031 The extent of late paternal effect: does the blastocyst matter? A retrospective analysis of 703 single, frozen-thawed embryo-transfers

Abstract Study question Do the blastocysts derived from severe male infertility show different ongoing pregnancy rates(OPR) compared to blastocysts of couples requiring assisted reproduction techniques(ART) for other indications? Summary answer First, single, frozen-thawed embryo transfers of blastocysts derived from severe male infertility did not affect OPR in-vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI) cycles. What is known already Several studies reported a negative impact of severe male infertility on fertilization rate, embryo morphology and embryo cleavage speed, due to the so-called early paternal effect, caused by sperm cytoplasm deficiencies. Nuclear sperm defects are responsible for the late paternal effect, causing lower blastocyst formation rates and, theoretically, reducing implantation potential of the obtained blastocysts. However, the effects of male infertility on OPR and live birth rates (LBR) are still debated. Additionally, the heterogeneity in the definition and estimation of male infertility makes the existing data difficult to interpret. Study design, size, duration Retrospective analysis of first IVF/ICSI cycles performed between January 2019 and December 2021 in the Reproductive Sciences Unit of Gynaecology/Obstetrics Department of San Raffaele Hospital in Milan, Italy. Participants/materials, setting, methods First, single, frozen-thawed embryo transfers of infertile couples first IVF/ICSI cycles were included. Embryo transfers obtained by frozen semen were excluded. Couples characteristics, semen parameters and controlled ovarian stimulation (COS) data were collected. Semen quality was assessed using total motile count (TMC) and grouped into quartiles of TMC for the analyses. The effect of severe male factor infertility evaluated through TMC over OPR was the primary outcome of the study. Main results and the role of chance N = 703 transfers were analysed. Performing a logistic regression analysis adjusted for confounding factors (maternal age, COS protocols, number of oocytes retrieved and quality of transferred blastocysts), OPR was not influenced by TMC values [odds ratio (OR) = 0.999; confidence interval (CI) = 0.986-1.013; p = 0.932]. After grouping male infertility population into quartiles of TMC, no significant differences in OPR were found between extreme quartiles (TMC ≤2.55 million and TMC ≥20 million respectively), even when adjusted for confounders [OR = 1.007; CI 0.609-1.663; p = 0.98]. Therefore, the implantation potential of the obtained blastocysts, once formed, seems to be independent from sperm quality. Limitations, reasons for caution The major limitations of the present study are the retrospective design and sample size. Additionally, the estimation of male infertility as well as the routinary use of TMC in clinical practice are not yet standardized. However, having analysed only the first transfers of the included couples makes our results reliable. Wider implications of the findings Further prospective studies with larger sample size are needed to confirm our results and to validate the use TMC as a standard tool to classify male factor and to guide clinical choices. Pregnancy and obstetrical outcomes are also necessary to better understand the role of male infertility in human reproduction. Trial registration number not applicable

P-082 Microfluidic-based device selects sperm with less DNA damage and higher motility, what else?

Abstract Study question Does the microfluidic-based sperm selection device ZyMōt improve sperm parameters and other laboratory key performance indicator (KPI) values compared to the conventional swim-up method? Summary answer The microfluidic-based sperm selection device ZyMōt selects sperm with lower DNA fragmentation and higher motility than conventional swim-up method in intracytoplasmic sperm injection (ICSI) cycles. What is known already Elevated levels of sperm DNA fragmentation (SDF) in semen samples have been associated with poor embryo development and low pregnancy rates. SDF refers to breaks in the sperm's genetic material, mainly due to defects during spermatogenesis and other factors such as reactive oxygen species that are favored by centrifugation. Conventional sperm selection methods, by integrating centrifugation into their protocols, have become ineffective in selecting sperm with low SDF. In order to solve this problem and improve reproductive outcomes, microfluidic-based devices such as ZyMōt have been designed to avoid centrifugation and select spermatozoa with low SDF. Study design, size, duration Prospective, experimental, single-center study conducted from June to December 2021. A total of 14 couples with ≥ 10 retrieved oocytes were recruited for an intra-patient comparison. Semen sample was split and processed for ICSI by the conventional swim-up method or by the microfluidic-based device ZyMōt. Each fraction was used to fertilize half of the total number of oocytes retrieved. SDF index, semen parameters, useful blastocyst rate, fertilization rate and morphokinetic variables were observed. Participants/materials, setting, methods Oocytes retrieved were from own (n = 96) and donation cycles (n = 93). From each patient, the cohort of oocytes was divided into two groups: 1) inseminated with spermatozoa selected by swim-up and 2) inseminated with spermatozoa selected by ZyMōt 850 µL device. Embryo evaluation and development were then followed by time-lapse monitoring using EmbryoScope. Sperm chromatin dispersion (SCD) assay was used to measure SDF, analyzed by ImageJ. Each treatment followed routine protocol established in the clinical practice. Main results and the role of chance SDF index was significantly lower in ZyMōt group in comparison with swim-up group (10% vs 20%), indicating a better selection by the ZyMōt of sperm with less DNA breaks. Additionally, ZyMōt group also presented a significantly greater number of spermatozoa with progressive motility (96.9% vs 95.4%). In contrast, useful blastocyst rate showed a slightly, but not significantly, increment in ZyMōt group compared to swim-up group (53.2% vs 46.6%). No significant differences in fertilization rate or sperm recovery rate were observed between groups. Regarding morphokinetic parameters, timing variables from first cell division to blastocyst stage (t2-tB) showed no significant correlation with ZyMōt group contrasted with swim-up. Blastocysts were evaluated and a value was assigned with respect to their quality (A to D). There was a higher number of embryos with A grade in ZyMōt group and a higher number of embryos with D grade in swim-up group. The annotated variables were assessed using paired t-test and P value <0.05 was considered statistically significant. Limitations, reasons for caution The impact on reproductive outcomes may vary depending on whether the breakage is single- or double-stranded, however, SCD is not able to distinguish between them. These, together with the oocyte's ability to repair sperm damage, lead us to the explanation for the non-significance on embryo quality. Wider implications of the findings This study provides further insights into the use of ZyMōt for the selection of low SDF spermatozoa in a simple manner. Its use could be indicated in patients with a high SDF value in their semen samples. Benefits on reproductive outcome will be confirmed with larger sample size. Trial registration number 2102-VLC-007-MD

P-121 Azoospermia: are there neonatal differences regarding the source of sperm for ICSI?

Abstract Study question In couples with azoospermia undergoing intracytoplasmic sperm injection (ICSI), are neonatal outcomes different depending on sperm origin (epididymal-PESA, testicular-TESA or donor sperm)? Summary answer Newborns from PESA or TESA had similar neonatal outcomes. Comparing to donor cases, PESA/TESA cycles didn’t present increased levels of malformation or stillbirth. What is known already Since the introduction of ICSI and the demonstration that the spermatozoa derived either from the epididymis or the testis were capable of normal fertilization and pregnancy, it was possible for azoospermic men to father their own children. However, it raised concerns that the quality of spermatozoa in terms of DNA damage or maturation when collected from non-ejaculated semen could differ from that collected from ejaculated and whether sperm of different origins will affect the neonatal outcome and safety of ICSI. Study design, size, duration Retrospective cohort study performed at a reproductive medicine center, using data from 359 deliveries from 945 cycles of ICSI of couples with infertility due to azoospermia, performed between 1995 and 2021. Data were collected from electronic records. Participants/materials, setting, methods A total of 359 deliveries were divided in 3 groups, according to the sperm source: PESA (n = 138), TESA (n = 139) and DONOR (n = 82). A total of 402 newborns were evaluated: PESA (n = 162); TESA (n = 156); DONOR (n = 84). Neonatal outcomes and congenital malformations were analyzed for singletons, twins and triplets separately. Statistical analysis: Anova, chi-square and Fischer test, considering p < 0.05. Main results and the role of chance Comparing the 3 groups (PESA, TESA, and donor sperm), the following results were found: The mean birth weight (BW), length and Apgar score > 7 for singletons and twins did not differ between groups; neither did prematurity or extreme prematurity. According to birth percentile, there was a lower rate of small for gestational age (SGA) newborn in the epididymal group (PESA, 1.5% vs. TESA, 8.5% vs. sperm donor, 5.5%, p = 0.004). Also, in 158 twin births, the number of newborns with low birth weight (<2500g) was significantly smaller in the epididymal group (PESA, 32.9% vs. TESA, 62.5%; donor sperm, 73.1%, p = 0.001). There were three stillbirths in triplets (PESA group). We also found six cases of neonatal mortality (one in singleton pregnancy in TESA group, 3 in twins (two in PESA group and one donor sperm group), and two in triplets (one in TESA and one in PESA groups). The total number of congenital malformations was 8 (2.48%) (PESA n = 1, 0.62%; TESA, n = 2, 1.28%, and donor sperm, n = 5, 5.95%, p = 0.02). Limitations, reasons for caution The possibility of incomplete medical records is an important limitation to be considered in retrospective studies. Larger studies are important to better assess the risk of malformation in this population. Wider implications of the findings In this study, there were no differences on neonatal outcomes regarding the source of sperm used for ICSI, with PESA, TESA and donor groups, as well as congenital malformation. It showed equally safe options with testis, epididymal or ejaculated sperm for azoospermia treatments. Trial registration number not applicable

P-231 Embryo morphokinetics in male factor infertility

Abstract Study question Are embryo morphokinetics and division pattern susceptible to sperm origin? Summary answer Surgical retrieved sperm and Oligo-astheno-teratozoospermia have significant effect on morphokinetics and pattern of embryo development in IVF. What is known already Among couples seeking primary infertility treatment, approximately one third of them have male factor indication. Intracytoplasmic sperm injection (ICSI) has changed their prognosis to achieve successful pregnancy. Recently, there are many conflicting studies on the relation of sperm origin, embryo development and clinical outcome after fertilization burrier is passed by ICSI. Implementation of time-lapse monitoring system (TLM) allows us to analyze embryo development under stable conditions, and identify morphological, temporal, spatial parameters and cell cycle endpoints at which embryos with sperm of different origin exhibit distinct kinetics. The magnitude of variable morphokinetics in anticipating pregnancy outcome is not yet determined. Study design, size, duration This is a prospective cohort study that included 195 ICSI cycles stratified into three groups of different sperm origin (abnormal sperm (OAT), surgical sperm(SRS) and normosperm (CS)). Each group included around 400 zygotes for patients seeking IVF treatment in Fakih IVF/Dubai between December 2019 and December 2021 in Dubai / UAE. The mean age of patients is (22-40 years female/ 22-45 years male). Normospermia subset was limited to couples seeking gender selection and tubal factor. Participants/materials, setting, methods Embryo morphokinetics (time to polar body (tPB) until time to blastocyst (tB)) and cell cycle durations were analyzed. Embryos were examined for irregular cleavage phenotypes. The prevalence of each category of out of-range morphokinetics and abnormal cleavage among the different groups was compared. The portion of embryos displaying optimal ranges for the indicated morphokinetics was explored focusing on cell cycle duration. Finally, multi-variables influencing morphokinetics parameters were controlled. Chi2 test was performed in SPSS, P-value<0.05. Main results and the role of chance Embryos derived from SRS were later to reach tPNf and t2(26.66±9.16 and 29.03±8.52) than embryos derived from CS (25.15±4.20 and 27.84±4.98) and OAT (25.71±6.20 and 28.67±7.22). P < 0.05. Embryos derived from SRS reached t5(47.94±11.02) faster than both embryos derived from CS (49.61±8.89) and OAT (49.26±9.47). Embryos derived from SRS reached t6(51.64±10.44) faster than embryos from CS (53.10±8.96). P value=0.0. Embryos derived from SRS and OAT reached tSB (95.34±9.01 and 94.92 ±10.04) and tB(103.68±8.53 and 102.08±10.32) significantly later than embryos derived from CS (91.16±5.65 and 96.34±7.70). No correlation was found between sperm origin and irregular embryo phenotypes except for blastomere asymmetry and incomplete morulation. P = 0.0004. SRS and OAT had higher incidence of blastomere asymmetry at 2-, 3- and 4cell stage than CS. P = <0.05. For incomplete morulation, CS group had higher incidence of 1-3 fragments at morula stage(71.3%) than SRS and OAT (57.4% and 49%). However, OAT had higher incidence of 3-5 fragments at morula stage(35.9%) than SRS (224.1%) and CS (18.8%). Significantly, SRS displayed higher proportion of embryos out of range for most of the measured morphokinetics. P < 0.05. Accordingly, less proportion of embryos in SRS and OAT fall within optimal range for cell cycle duration P < 0.0. Antral follicle count, number of collected oocytes and male’s age had significantly affected morphokinetics and cleavage abnormality. Limitations, reasons for caution Selection of sperm by ICSI due to male- and severe male factor infertility may result in selecting live sperm with the best available normal morphology which may not reflect DNA fragmentation status of the overall sample where damage of DNA is more significant and lead to bias the results. Wider implications of the findings Embryo development potential with different sperm origin was compared based normal optimum morphokinetics or not. Further follow up study to investigate clinical outcome of implantation and clinical pregnancy rate, miscarriage rate and live birth rate is needed. Trial registration number DSREC-11/2021_07

P-691 Assessment of ovarian vascularity by three-dimensional vaginal power Doppler on day seven of menstrual cycle to predict the number of eggs collected in antagonist cycle

Abstract Study question Could ovarian vascularity indices on day 2 and 7 of stimulation, measured by 3-dimensional (3D) vaginal power Doppler, predict after controlled ovarian stimulation? Summary answer Ovarian vascularity index (VI) on day 7 may be an indicator of poor (<three mature eggs collected) ovarian responses to gonadotropins. What is known already Poor and/or hyper ovarian responses to gonadotropins may be related to cycle cancellation during controlled ovarian stimulation (COS). In this context, gonadotropin dose is often individualized using patient features that predict ovarian response (such as age, antral follicular count (AFC) and anti-Müllerian hormone (AMH)). In parallel, ovarian vascularity color doppler is a valuable evaluation method to predict the ovarian hyperstimulation syndrome and the growth/maturity of Graafian follicles. The aim of the present study is to estimate the utility of 3-dimensional vaginal power Doppler and ovarian vascular flow indices in the prediction of the number of mature occytes collected after COS. Study design, size, duration A prospective study was conducted on 296 couples undergoing intracytoplasmic sperm injection cycle at Al Hadi Laboratory and Medical center, Beirut, Lebanon. It was performed between January 2020 and dec 2021. Couples were categorized into poor responders group (3 or less metaphase II (MII) eggs collected) (36.1%), high responders group (16 or more MII eggs collected) group ( 6.7%), and normal responders group (more than 3 and less than 16 MII eggs collected) (57,2%). Participants/materials, setting, methods On the second and seventh day of the menstrual cycle, ovarian volume and vascularity parameters (vascularity index (VI), flow index (FI), and vascularity flow index (VFI)) were measured using the 3D power Doppler and the Virtual Organ Computer-Aided Analysis. On day 2 , the antral follicle count was evaluated and a blood sample for AMH testing was collected. Women included in the study have undergone COS using GnRH antagonist protocol. Main results and the role of chance Mean age was 35,88+/- 6 year , day2 VI and day2 VFI correlates with the number of egg collected (p = 0.014 and 0.045 respectively). 9 parameters were used to predict poor and high ovarian responses (Age, AMH, AFC, day2 VI, day2 FI , day2 VFI , day7 VI, day7 FI and day7 VFI) in a Receiver operator characteristics (ROC) curve model . Ovarian day7 FI significantly predicted poor ovarian response to gonadotropins (p = 0.038) with an area under the curve of 0.679. AFC predicted also poor ovarian response to gonadotropins (p = 0.006) . In parallel, AFC significantly predicted high ovarian response to gonadotropins (p = 0.002) and AUC (0.778) while all other 3D doppler parameters couldn’t predict high responders. Limitations, reasons for caution It will be necessary to perform a prospective analysis on a broad sample size to validate these findings. In addition, it will be interesting to assess the impact of ovarian vascularity on pregnancy outcomes. Wider implications of the findings Assessing ovarian vascularity during ovarian stimulation can help reduce the rate of cycle cancellation. In addition, more studies are welcomed in the field to unravel the mechanisms behind altered ovarian vascularity and to test the possibility of restoring normal ovarian physiology. Trial registration number Not Applicable

Should Intra Cytoplasmic Sperm Injection (ICSI) be the primary insemination method in women undergoing IVF cycles with donor sperm?

To compare efficacy of Intra Cytoplasmic Sperm Injection (ICSI) with conventional in vitro fertilization (IVF) on treatment outcome in women undergoing in vitro fertilization with donor sperm. We examined retrospectively the outcome data from 203 patients undergoing fresh cycles of conventional IVF (cIVF) or ICSI and an additional 77 frozen-thawed embryo transfer (FET) cycles during 2003-2014, all using donor sperm. Fertilization, cleavage, pregnancy and live birth rates and number of high-quality embryos were compared between cIVF and ICSI. Altogether 185 women underwent 479 transfer cycles of fresh embryos (237 cIVF vs. 224 ICSI and 18 "rescue ICSI" cycles). In addition, 77 FET cycles were compared (24 cIVF vs. 53 ICSI cycles). No differences were found between cIVF and ICSI in fertilization, cleavage, pregnancy and live birth rates (92.6% vs 92.2%, 73.4% vs 72.4%, 25.3% vs 27.2% and 13.1% vs 14.7%, respectively). Pregnancy and life birth rates remained similar even when FET cycles were included (25.8% vs 26.2% and 13.1% vs 13.7%, respectively). The use of ICSI was associated with lower rates of high-quality embryos (52.7% vs. 63.3%, P < 0.0001). A multivariate logistic regression analysis found that patients' age, number of transferred embryos and smoking were independently associated with the chance to conceive. Patient age correlated inversely with fertilization rate (r =-0.13, P < 0.006).Non-smokers were more likely to become pregnant (OR = 2.23, P < 0.012). Our results show that ICSI does not bypass the age-related decrease in oocyte quality in patients using donor sperm for IVF. Use of ICSI was associated with lower rates of high-quality embryos. The findings imply that ICSI should not be the primary method of insemination in patients undergoing IVF with donor sperm.

In vitro fertilization cycle and embryo transfer outcomes in oligoanovulatory patients with hypothalamic hypogonadism vs. polycystic ovary syndrome and compared with normo-ovulatory patients

ObjectiveTo study the difference in the live birth rates between anovulatory women with hypothalamic hypogonadism (HH) and those with polycystic ovary syndrome (PCOS) and normo-ovulatory women undergoing fresh embryo transfer or frozen embryo transfer (FET).DesignRetrospective cohort study.SettingAcademic medical center.Patient(s)Patients with oligoanovulation (HH, n = 47; PCOS, n = 533) and normo-ovulation (tubal factor infertility, n = 399) undergoing in vitro fertilization and intracytoplasmic sperm injection cycles from January 1, 2012, to June 30, 2019.Intervention(s)None.Main Outcome Measure(s)Live birth rate.Result(s)Patients with HH had longer stimulation durations than both patients with PCOS and tubal factor infertility. Patients with HH had fewer oocytes retrieved than patients with PCOS, but their numbers of blastocysts were similar. Patients with HH and tubal factor infertility had similar numbers of oocytes retrieved and blastocysts. In fresh embryo transfer cycles, the live birth rates were similar among patients with HH, PCOS, and tubal factor infertility (37.5% vs. 37.1% vs. 29.3%, respectively). When evaluating FET cycles, patients with HH had lower live birth rates than patients with PCOS (26.5% vs. 46.7%) and tubal factor infertility (42.6%).Conclusion(s)Live birth rates are similar among patients with HH, PCOS, and normo-ovulation undergoing fresh embryo transfer but are significantly lower in women with HH undergoing FET.