ICSI Outcome Research Articles

#267 : The Spindle Localization ICSI (SL-ICSI) Improves the Fertilization Rate Decreased by Aging

Background and Aims: The position of meiotic spindle in metaphase II oocyte can be different from the underneath of the first polar body. In order to prevent the meiotic spindle damage during ICSI, confirming the position of the spindle is effective. In this study, we analyzed the meiotic spindle deviation and outcomes of ICSI with the patient age. Method: From 2017 to 2022, 5620 oocytes with the first polar body underwent SL-ICSI were analyzed. SL-ICSI was performed 4-5 hours after oocyte retrieval. Furthermore, the meiotic spindle deviation from the polar body was classified according to the angle; Group A: 0-5∘, Group B: 6-45∘, Group C: 46-90∘, Group D: 90∘<. The spindle invisible oocytes were regarded as group E. The average age, the fertilization rate and good blastocyst rate in each group were analyzed. Statistical analysis was performed by one-way ANOVA and Tukey’s post-hoc test. Results: The proportions of spindle-visible oocyteswere 98.7%; 41.2%, 36.1%, 14.6%, 6.8%, and 1.3% respectively, in group A to E. The mean agesin each group were 39.0, 39.6, 40.1, 40.6, and 40.7, respectively. In addition, the correlation between the age and the location of spindle was revealed (A-BCDE: [Formula: see text] < 0.001). The fertilization rates were 84.8%, 84.9%, 84.7%, 83.5%, 50.7%, respectively, and significantly decreased in group E ([Formula: see text] < 0.001). Abnormal fertilization rates were 4.3%, 5.1%, 6.4%, 7.3%, 20.5%, respectively, and significantly increased in group E ([Formula: see text] < 0.001). Good blastocyst rates were 49.8%, 52.4%, 47.6%, 50.5%, 3.3%, respectively, and significantly decreased in group E ([Formula: see text] < 0.001). Conclusion: The introduction of SL-ICSI can be effective for elderly patients. The meiotic spindle of oocytes is highly likely to deviate from the first polar body by aging. Furthermore, if the spindle-invisible oocyte is observed, ICSI should be carefully carried out.

Novel Method to Improve IVF and ICSI Outcomes

Novel Method to Improve IVF and ICSI Outcomes

#305 : Preparation of Epididymal/Testicular Spermatozoa; Technical Aspects and Its Impact on Success

Background and Aims: The introduction of ICSI 30 years ago paved the way for epididymal/testicular spermatozoa to be used to fertilise oocytes in couples undergoing ART. Currently, spermatozoa is recovered from the epididymis or testis using varying approaches, including micro-surgery and fine-needle aspiration, mostly in men with obstructive and nonobstructive azoospermia. The samples delivered to the laboratory for processing are of varying quality with regards to amount of material/tissue, presence of spermatozoa, and other cells. The success of processing the tissue can have a direct impact on the outcome of the subsequent ICSI and our aim for this review was to identify ways to improve the preparation techniques for epididymal/testicular samples. Ethics approval was obtained from WSLHD HREC Ref 5583QA. Methods: The review included 527 ICSI cycles for 311 patients (2016-2022) where epididymal/testicular spermatozoa had been used. Approaches for sample preparation included mini-gradient centrifugation and removal of red blood cells using erythrocyte lysis buffer (ELB). In cases with severe oligoasthenozoospermia (105 cycles, 79 patients), a phosphodiesterase inhibitor (PI) was added to induce motility. Results: Employing mini-gradient centrifugation or adding ELB did not result in lost spermatozoa for any cases, but vastly reduced sample debris allowing for easier identification of spermatozoa. Fertilisation rate after ICSI was 57.9±1.2%. Motile spermatozoa were found and injected in all 105 samples after addition of PI and the fertilisation rate after ICSI was 55.3±2.7. To date cycles with PI have resulted in 26 live births with no abnormalities and all infants were of appropriate weight for gestational age. Conclusions: Employing precise preparation techniques for epididymal/testicular samples is paramount to allow for timely identification of motile spermatozoa for ICSI and assure optimal overall outcome. Sharing and discussing successful approaches with the wider embryology community allows for discussion and refining of techniques which can benefit all.

Mitofusin 1 as Marker of Oocyte Maturation in Relevance to ICSI Outcome in Infertile Females

Pro-fusion proteins as mitofusins-1 are required for controlling mitochondrial shape which determined by a dynamic balance between organelle fusion and fission and also supports oocyte development. When compare with somatic cell, mitochondria of oocyte are tiny and circular in presence. The aim is to study the mitofusin-1 in the follicular fluid as a marker for evaluating of oocyte maturation in women undergoing ICSI cycles. Fifty infertile females were included in cross-section study was undergoing ICSI procedure with age 20 to 42 years. After retrieval of oocyte and the number of oocytes was recorded by embryologist and follicular fluid Samples were used for the measurement of Mitofusin 1. Mitofusin 1 levels by ELISA kit (Mybiosource /USA).The results showed considerable higher mitofusin1levels in patients with good oocytes quality (3.71 ± 1.35 vs. 2.45 ± 1.12 & p=0.001). There were much positive correlations between follicular fluids mitofusin-1 with both total oocytes count (r= 0.374 & p= 0.007) and MII oocytes (r=0.383 & p=0.006); and there was no much correlation between mitofusin-1 levels with MI oocyte (r= - 0.100 & p= 0.490), germinal vesical oocyte (r=0.103 & p= 0.475) and fertilization rate (r= 0.054 & p= 0.711). High follicular fluid levels of Mitofusin 1 may positively impact oocyte development and pregnancy rate.

The Prevalence of Bacterial Vaginosis among Infertile Iraqi Women and Subsequent ICSI outcome

The vaginal microbial makeup has a substantial impact on women's health. The typical vaginal microbial profile in healthy women of reproductive age comprises aerobic, facultatively anaerobic, as well as obligately anaerobic organisms. However, the optimal vaginal microbiota is dominated by lactobacilli and is thought to defend against pathogenic microbial colonization and infection by producing lactic acid, antimicrobial metabolites, and lowering immune system activation. Recent research has come up with the term "molecular bacterial vaginosis (BV)" to describe non-optimal vaginal microbiota that is low in Lactobacillus species (spp.) and has a lot of facultative and absolute anaerobic bacteria specifically Gardnerella vaginalis. In the context of IVF, BV has been linked to infertility. The study comprised forty-six women to explore the vaginal and follicular microbiota of asymptomatic infertile Iraqi women starting an ICSI trial. At the time of ova pick-up, a high vaginal swab and follicular fluid were acquired from each instance for DNA extraction to test for the presence of microorganisms by the use of a real-time PCR special commercial kit. In conclusion, molecular technologies have offered a more thorough view into the vaginal microbiota, which may explain why vaginal dysbiosis shifts during the ICSI-ET treatment. Even though the majority of women have no clinically visible infection, the fact is that they have an aberrant vaginal micro-ecology profile. Nevertheless, isolating microorganisms from follicular fluid did not reduce the likelihood of fertilization or the pregnancy rate during ICSI cycles.

The outcome of ICSI with in vitro matured Metaphase I oocytes

The outcome of ICSI with in vitro matured Metaphase I oocytes

Effect of Micronutrients and L-Carnitine as Antioxidant on Sperm Parameters, Genome Integrity, and ICSI Outcomes: Randomized, Double-Blind, and Placebo-Controlled Clinical Trial.

The evaluation of sperm DNA integrity is recommended in the sixth edition of the 2021 World Health Organization guidelines. Oxidative stress has been identified as a crucial factor leading to genome decay, lipid peroxidation, and nucleoprotein oxidation. This double-blind, placebo-controlled clinical trial aimed to assess the effect of oral antioxidant treatment (Fertilis), which contains L-carnitine and some micronutrients, in the improvement of conventional sperm parameters, sperm DNA integrity and in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcomes. A total of 263 participants were enrolled and randomly divided into two groups: 131 participants received the antioxidant treatment, while 132 participants received a placebo. The male partners in both groups underwent the antioxidant treatment or the placebo for a duration of three months. For each participant, we performed a hormonal test, an infectious test, a spermogram, a TUNEL assay for sperm DNA fragmentation, a toluidine blue staining for sperm DNA decondensation, and an IVF/ICSI procedure. Sperm characteristics analysis (volume, count, motility, and vitality), sperm DNA fragmentation, and sperm DNA decondensation were assessed and compared to the results preceding the antioxidant treatment. The study outcome revealed a significant decrease in the DNA fragmentation index and a significant increase in sperm motility after 3 months of treatment (p = 0.01 and p = 0.02, respectively). Additionally, a significant improvement in clinical pregnancy rate (p = 0.01) and life birth rate (p = 0.031) was observed. No significant changes were observed in conventional sperm parameters (volume, count, and vitality) or sperm DNA decondensation (SDI). Antioxidant therapy has a beneficial impact on achieving pregnancy, whether through spontaneous conception or assisted reproductive procedures (ART).

The impact of serum and follicular fluid secreted frizzled-related protein-5 on ICSI outcome in Iraqi infertile women with different body mass index

Introduction and Aim: Adipose tissue adipokines and cytokines impede oocyte maturation and endometrial epithelial receptivity. Secreted frizzled-related protein 5 (Sfrp5), a novel antagonist adipokine secreted by adipocytes, may have anti-inflammatory and insulin-sensitizing actions, but data is conflicting. This study examined serum and follicular fluid Sfrp5 levels in Iraqi infertile women with varying BMIs undergoing IVF/ICSI, as well as ICSI outcomes. Materials and Methods: This prospective cross-sectional study comprised 90 infertile females aged 18–45 who underwent IVF/ICSI at the High Institute of Infertility Diagnosis and Assisted Reproductive Technologies Centre in Baghdad, Iraq. Participants received antagonist ovarian hyperstimulation. All individuals' blood fasting sugar, serum fasting insulin, serum, and follicular fluid SFRP5 were tested on oocyte retrieval day. HOMA-IR was computed. Oocyte maturity and embryo morphology were categorized. Serum B-hCG was measured 14 days after embryo transfer. Results: A notable disparity was observed in the levels of serum and follicular Sfrp5, total oocyte count, mean metaphase II (MII), grade I, and pregnancy rate (p<0.05) among the participants categorized as normal weight, overweight, and obese. A notable disparity was observed in the average germinal vesicles (GV), grade III (GIII) embryos, and HOMA-IR (p < 0.05) among patients with varying body mass index (BMI) values. The study found a positive correlation between the levels of Sfrp5 in both serum and follicular fluid and the total count of oocytes, MII oocytes, grade I embryos, and pregnancy rate. However, there was a negative correlation between Sfrp5 levels and HOMA-IR, with statistical significance at p<0.05. Conclusion: Increased BMI is associated with lower serum and follicular fluid Sfrp5, reduced total oocyte count, reduced metaphase II oocyte count, more germinal vesicle oocytes, reduced embryo quality and lower pregnancy rate. Serum and follicular fluid Sfrp5 levels are negatively correlated with HOMA-IR but positively associated with ICSI outcome.

Interaction of Vitamin D and Tumor Necrosis Factor-α can modulate the Outcomes of ICSI

Interaction of Vitamin D and Tumor Necrosis Factor-α can modulate the Outcomes of ICSI

Can Vitamin D Supplemental Therapy improve the Disturbed Follicular Fluid Milieu and affect the Outcome of ICSI?

Can Vitamin D Supplemental Therapy improve the Disturbed Follicular Fluid Milieu and affect the Outcome of ICSI?

O-147 Factors influencing ICSI outcome after sperm retrieval in nonobstructive azoospermia patients with different types of etiologies: a retrospective study of 1157 patients

Abstract Study question What are predictors of fertilization, clinical pregnancy, miscarriage and livebirth delivery outcomes after microdissection testicular sperm extraction (mTESE)-ICSI in NOA patients with different etiologies? Summary answer Predictors for ICSI outcome were different in patients with various etiologies and type of NOA are associated with ICSI outcome in these patients. What is known already NOA patients due to spermatogenic dysfunction, accounting for about 60 percent of the total azoospermia cases, may have opportunities to be fathers with their own biological children by mTESE combined with ICSI. For them, there were some of predictive factors for ICSI outcome including sperm motility, sperm morphology, testis volume and levels of reproductive hormone including follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T), while the predictive factors of ICSI outcome remains underappreciated, especially about the relationship between ICSI outcome and male factors after reviewing the large scale clinical data. Study design, size, duration This retrospective study involved 1157 NOA patients with treated 1534 complete ICSI cycles at the Reproductive Medical Centre of Peking University Third Hospital from March 2012 to October 2021. Participants/materials, setting, methods 1534 ICSI cycles included 558, 359, 243, 155, 128, 56 and 35 cycles performed in males with idiopathic NOA (iNOA), Klinefelter syndrome (KS), AZFc microdeletions, cryptorchidism, a history of mumps orchitis, cryptozoospermia and with other causes separately. Primary outcome was livebirth delivery per ICSI cycle (LBR) and secondary outcomes were clinical pregnancy rate per ICSI cycle (PR) and fertilization rate (FR). Multi-variable logistic and linear regression was used to analyze ICSI outcome. Main results and the role of chance Overall LBR, PR and FR were 37.35%, 44.98%, 49.34% separately. Patients with mumps orchitis showed the highest LBR, PR and FR of 53.13%, 60.94% and 59.52% respectively, and AZFc-deleted patients displayed the lowest LBR, PR and FR of 25.93%, 30.04% and 32.87% separately. Lower female age (0.947 [0.904;0.993], p = 0.024), more transferred embryos (1.345 [1.225;1.477], p＜0.001), higher AFC (1.033[1.011;1.056], p = 0.003) were related with higher LBR, and higher male BMI (0.974[0.950; 0.999], p = 0.046) and lower AFC (1.057[1.034; 1.080], p＜0.001) were related with lower PR. Comparing outcome of mumps orchitis group, patients with cryptorchidism were associate with lower LBR(0.467[0.281; 0.778], p = 0.003), PR (0.440[0.268;0.721], p = 0.001) and FR(-0.169 [-0.226; -0.11], p＜0.001) separately. AZFc-deleted patients had a relationship with lower LBR (0.426[0.260; 0.697], p = 0.001), (0.273[0.170; 0.437], p＜0.001) and FR (-0.282 [-0.336; -0.228], p＜0.001) respectively. KS patients were related with lower LBR (0.455[0.288; 0.720], p = 0.001), PR (0.477 [0.306; 0.745], p = 0.001) and FR (-0.073 [-0.126; -0.020], p = 0.007) separately. iNOA patients were associated with lower PR (0.616 [0.409; 0.928], p = 0.020) and FR (-0.076 [-0.124; -0.029], p = 0.002) respectively. Furthermore, patients with cryptozoospermia were related with lower FR (-0.141 [-0.220; -0.061], p = 0.001). Limitations, reasons for caution Sperm characteristics maybe predictors for ICSI outcome which is a potential selection bias. Pituitary prolactin and Anti-Müllerian hormone weren’t included as covariates due to limited information in our database. We couldn’t verify the conclusion in more population from multicenter despite the current study with the large sample size so far. Wider implications of the findings Our results provide valuable information for NOA patients who want to counsel surgeons about their ICSI outcome to help patients and surgeons to perform a shared decision-making for optimal therapy methods, especially for AZFc-deleted patients with worse ICSI outcome who may consider donor semen to get better ICSI outcome. Trial registration number not applicable

P-590 Similar mean follitropin delta daily/total doses yield satisfactory ICSI outcomes in different subgroups of maternal age and body mass index: a “real-world” experience with Rekovelle®

Abstract Study question Can similar mean follitropin delta daily/total doses yield satisfactory ICSI outcomes in different subgroups of maternal age and body mass index (BMI)? Summary answer Similar mean follitropin delta daily/total doses yield satisfactory number of oocytes and maturity rates, and pregnancy rates in different subgroups of maternal age and BMI. What is known already Rekovelle® (follitropin delta) is a novel recombinant human FSH (rFSH) expressed from a host cell line of human fetal retinal origin (PER.C6®) and is the first commercially available rFSH product derived from human cell lines. Rekovelle® allows the individualization of the initial dose of gonadotropin using predictive response factors to COS, such as anti-müllerian hormone (AMH) levels and body weight. In clinical practice different approaches can be adopted to obtain the most satisfactory response to controlled ovarian stimulation (COS). The objective of this study was to describe data on “real-world” Rekovelle® administration, regarding the response to COS and ICSI outcomes. Study design, size, duration This non-interventional study based on secondary use of data included patients undergoing ICSI in a private university-affiliated IVF center from Jan/2018 to Dec/2021. Each patient or cycle had to meet the following inclusion criteria: pre-menopausal women, undergoing COS with Rekovelle® (16 ug) daily for ICSI; diagnosed with infertility, or with partners diagnosed with male infertility factors, eligible for ICSI using fresh sperm from partner ejaculation; presenting with both ovaries. Participants/materials, setting, methods Ovarian response to stimulation and ICSI outcomes were described for 362 ICSI cycles. The primary outcome measures were the numbers of retrieved oocytes and maturity rates, and the secondary outcome were the ongoing pregnancy rates per fresh and per fresh and/or frozen-thawed embryo transfer. The results obtained with the population enrolled in the ESTHER-1 trial (extern), and a population stimulated with follitropin alpha 300 IU (on site) were recorded (data not shown, to be presented). Main results and the role of chance The mean total dose of follitropin delta administered in subgroups of age and BMI were as follows: age ≤35 y-old 156µg, 36-39 y-old 158 µg, and ≥40 y-old 157µg, and BMI: <18.5 165µg, 18.5-24.9 156µg, 25.0-24.9 159µg, and ≥30 171µg. Patients in the Follitropin delta group showed acceptable outcomes in different age and BMI groups (Table1). Adequate embryo morphokinetic development and euploidy rates were also observed, suggesting that the modified protocol does not interfere with oocyte/embryo competences and embryo implantation potential. This was further corroborated by satisfactory cumulative pregnancy rates. Limitations, reasons for caution Given its descriptive nature, we have focused on the number and maturity stage of retrieved oocytes instead of clinical ICSI outcomes. The use of historical cohort groups is another drawback. Despite the eligibility criteria for inclusion in the analysis, potential differences in the baseline characteristics cannot be ruled out. Wider implications of the findings Patients in different subgroups of age and BMI stimulated with similar mean follitropin delta daily and total doses showed satisfactory number of retrieved oocytes and maturity rates, and cumulative pregnancy rates without increasing OHSS, providing a rationale for conducting clinical trials to confirm the benefits of using this modified protocol. Trial registration number N/A

O-300 Human sperm selection using cumulus oophorus complexes compared with conventional sperm preparation method on sperm quality and ICSI outcomes: A pilot study

Abstract Study question We compared a sperm selection protocol using cumulus oophorus complexes with a swim up preparation, evaluating the effects on sperm parameters and ICSI outcomes Summary answer Spermatozoa selected using cumulus oophorus complexes showed higher rates of normal morphology, good motility compared with basal semen and the traditional pellet swim up protocol. What is known already It is known that the head of a mature spermatozoa has a hyaluronan receptor that allows them to bind with hyaluronan acid. Cumulus oophorus complexs (COCs) extracellular matrix is made mainly by hyaluronic acid (HA). Various studies have confirmed the importance of COCs in fertilization, demonstrating that spermatozoa that are able penetrate through a layer of COCs are more likely to have a normal morphology and be more effective in producing acrosome reactions than spermatozoa that are not able to do. Moreover, these spermatozoa have been shown to have higher chromatin integrity and zona-binding capacity, resulting in increased fertilization competence. Study design, size, duration A prospective study was conducted between July and December 2022. A total of 50 participants were recruited for the study, randomly selected between patients attending ICSI. The patients were classified as group A when the sperms were selected using pellet swim up and COC selection and as group B when it was used pellet swim up treatment alone. Patients older than 39 years, with endometriosis or repeated IVF failure or severe male factor were excluded. Participants/materials, setting, methods 3 drops of 30 mlt aligned and connected each other were prepared, covered with mineral oil. In group A, in the middle drop, was transferred COC fragments, while the central drop was empty in group B. In the left drop were added 20 mlt of sperm suspension after swim up, and from the right drops were recovered the sperms after passing the middle drop with or without COC, that were used for ICSI treatment. Main results and the role of chance The aim of the study was to verify the effectiveness of COCs, collected after ovarian stimulation, in selecting the best sperms to be used in ICSI procedure. No statistical difference was found between group A and B in the median age of female patients, FSH/LH units administred, E2 and endometrium thickness at triggering using hCG, in the sperm concentration, motility and normal morphology in the baseline seminal samples. We found a statistical difference in the traditional seminal parameters comparing the two different treatments. In particular, we found an higher rates of sperms with rapid and progressive motility and normal morphology in group A between basal semen sample and after sperm selection using COC filter (86.7 vs 31.3%; 8.2 vs 4.4 %). We found a lower sperm concentration in group A vs group B after sperm treatment (2.3 vs 4.7 M/ml), and an higher rates of rapid progressive motility and normal morphology in group A vs group B (86.7 vs 38.6 %; 8.2 vs 4.6%). We found an higher but not statistically significative pregnancy and implantation rates in group A compared with group B probably because of the limited number of patients analyzed (25 vs 19.2 %; 25 vs 20.6%) Limitations, reasons for caution The study was conducted on a small participant cohort. We analyzed the traditional sperm parameters, but it is necessary to evaluate the chromatine integrity of the sperms after COC selection to confirm that the higher sperm competence in fertilization and embryo development could be based on a better genetic pattern Wider implications of the findings Data seem to demonstrate that the use of COC allows to select higher competent sperms if compared with traditional pellet swim up treatment. The clinical and biological ICSI outcomes seem to be improved by COC treatment but it is necessary to increase the sample size in future experimental trial Trial registration number not applicable

P-312 Automated sperm selection software (SiD) is as good as human selection for ICSI

Abstract Study question Automated sperm selection or human eye-based sperm selection, does it make a difference on IVF lab outcomes? Summary answer Single sperm selection by the AI SiD was as proficient as highly experienced embryologists and embryo development may have improved for inexperienced embryologists. What is known already The implementation of artificial intelligence in reproductive medicine has proven effective for a variety of applications. One application for this technology is single sperm selection during ICSI. The computer assisted program SiD, was designed to grade sperm, in real-time, based on motility parameters transforming qualitative sperm assessments into a quantitative score. SiD’s assistance can be used to select the most optimal sperm for ICSI. The application of sperm selection by AI is expected to bypass user bias, fatigue, limited training, while also optimising time. However, there are limited studies correlating the usage of AI for sperm selection with ICSI outcomes. Study design, size, duration Between August and November 2022, sibling MII oocytes were randomly divided in two groups; 1-ICSIgroup: ICSI performed with sperm selected by the embryologist based on motility and 2- SID-ICSIgroup: ICSI performed with sperm selected using SiD software. Priority was given to sperm classified as “Best” by SID software for ICSI. “good” sperm were considered as second choice. Morphology selection was performed before ICSI in both groups. Embryos were cultured during 6 days in same conditions. Participants/materials, setting, methods Sperm preparation was performed using a microfluidic chamber (Zymot™). Sperm selection was performed as previously described, and ICSI was performed following routine procedures. Biological outcomes were analysed in both groups and compared including fertilization rate, cleavage rate, embryo and blastocyst development rate, top quality embryo rate and blastocyst development rate. Biological outcomes in both groups were also compared based on embryologist seniority. Finally, embryo morphokinetics parameters were analysed in both groups through manual TLT annotations. Main results and the role of chance A total of 226 MII oocytes were included in “ICSI group” and 227 in “SID-ICSI group. A non significant trend towards better outcomes was observed in the SID-ICSI group for all biological outcomes including fertilization rate (81,5% vs 80,5%), cleavage rate (98,4% vs 97,3%), day 3 embryo development rate (77,8% vs 73,1%), top quality development rate at day 3 (50,8% vs 50,0%), blastocyst development rate at day 5 (49,7% vs 45,1%), good quality blastocyst development rate at day 5 (48,1% vs 46,2%), top quality blastocyst development rate at day 5 (25,4% vs 23,1%). Similarly, a non-significant increase was observed in all these outcomes when comparing both groups when sperm selection is performed by a junior embryologist. Conversely, no difference was seen in both groups when sperm selection is performed by a senior embryologist. Finally, no significant difference was observed in the SID-ICSI group for all fertilization events (tPB2, cytoplasmic wave, tPN1, tPN2, presence of cytoplasmic halo, tPNf, and disappearance of cytoplasmic halo) other cleavage timings (t2->t10, tM, tSC, tEC, tSB) and embryonic cell cycles (ECC1, ECC2, ECC3 s2, s3). Limitations, reasons for caution The ZymotTM microfluidic chamber was used for sperm preparation. This device optimises sperm selection, giving rise to an already optimised population of spermatozoa. Future investigations evaluating the effect of SiD automated sperm selection software on biological outcomes in samples prepared with a less effective technique would potentially yield conversing results. Wider implications of the findings This pilot study demonstrated that the usage of the automated sperm selection software SiD gives rise to similar biological outcomes and embryo morphokinetics, indicating its effectiveness in sperm selection. We expect this program would be useful in the presence of less experienced laboratory members helping by standardizing sperm selection. Trial registration number NA

P-041 Enhancing Embryos of the Desired Sex in Couples Undergoing PGT-A while Minimizing Embryo Wastage

Abstract Study question Can we enhance the proportion of euploid embryos of the desired sex for couples undergoing ICSI and PGT-A with sex selection while reducing embryo wastage? Summary answer Using a novel sperm selection technique, we generated higher proportions of embryos and offspring of the desired sex without impairing clinical outcomes or offspring health. What is known already Although controversial, the wish for offspring of a specific sex is common in the U.S. Many couples, particularly those requiring IVF with PGT-A, request a euploid embryo of a specific sex. This is often associated with the generation of more conceptuses of the unwanted sex, resulting in embryo wastage. We propose utilizing a simple and safe sex selection technique (SST) to skew spermatozoa toward the desired sex so that concurrent embryos can be generated in higher proportions. Study design, size, duration Over a 7-year period, ejaculates from male partners of couples (n = 144) undergoing ICSI with PGT-A were processed using SST to enrich spermatozoa for their preferred sex. Standard sperm processing was carried out for couples undergoing ICSI exclusively to assess conceptus aneuploidy, comprising the control group (n = 1,440). The proportion of male and female spermatozoa in the initial and selected specimens, PGT-A results, and ICSI outcomes following frozen embryo transfer (FET) were compared between the two groups. Participants/materials, setting, methods A total of 1,584 couples were treated in 2,723 ICSI cycles. Standard sperm processing was performed for 1,440 couples who did not have offspring sex preferences, comprising the control. For 144 consenting couples, SST was used to enrich spermatozoa for either sex (IRB 1306014043). To confirm sex enrichment and ploidy, ≥1,000 sperm cells were screened by fluorescent in-situ hybridization (FISH) for nine chromosomes. Embryology and PGT-A outcomes were compared between the control and SST cohorts. Main results and the role of chance Couples (n = 1,440) from the control cohort (maternal age, 37.1±4yrs; paternal age, 39.1±6yrs) underwent 2,541 ICSI cycles, yielding an 80.1% fertilization rate (15,901/19,859). PGT-A confirmed that 46.6%(n = 4,479) of conceptuses were female and 53.4%(n = 5,132) were male. These couples achieved 76.2%(866/1136) implantation and 64.9%(737/1136) clinical pregnancy rates, resulting in 624 healthy deliveries (48% female, 52% male). From the study cohort (n = 144), 79 desiring female offspring (maternal age, 37.9±4yrs; paternal age, 40.8±6yrs) obtained an 81.6% sperm sex enrichment. They underwent 98 ICSI cycles, achieving a 76.7%(792/1032) fertilization rate, resulting in a greater proportion of female embryos (79.1%, 351/444) than the control (P<0.05), of which 78.6% (276/351) were euploid. Following FET, 39 couples obtained a 79.5% (31/39) implantation rate, yielding 27 clinical pregnancies with deliveries, thus far, of 16 female singletons, all developing normally. The remaining 65 couples (maternal age, 37.6±3yrs; paternal age, 40.8±5yrs) preferring male offspring obtained 80.8% male sperm enrichment and underwent 84 ICSI cycles, achieving a 74.9%(723/965) fertilization rate with a greater proportion of 79.3%(249/314) male embryos compared with the control (P<0.05), of which 66.3%(165/249) were euploid. Following FET, their implantation rate was 90.0%(36/40), yielding 25 clinical pregnancies that resulted in the deliveries, thus far, of 20 male singletons, all developing normally. Limitations, reasons for caution Although SST does not absolutely grant offspring of a specific sex, it allowed couples to obtain a greater proportion of conceptuses of their preferred genotype. This method does not replace PGT-A for sex selection, but rather enhances the generation of embryos of the desired sex, thereby reducing embryo wastage. Wider implications of the findings We confirmed a significantly higher proportion of conceptuses of the desired sex. Thus far, embryo developmental competence, aneuploidy rates, delivery rates, and offspring health were not impaired by SST-processed spermatozoa. This supports the safety of SST, rendering it effective and ethically palatable. Trial registration number n/a

O-138 Factors influencing sperm retrieval rate of microdissection testicular sperm extraction in nonobstructive azoospermia patients with different etiologies: a retrospective study of 3104 patients

Abstract Study question What are predictors of successful sperm retrieval (SRR) with microdissection testicular sperm extraction (mTESE) in men with non-obstructive azoospermia (NOA) according to different etiologies? Summary answer Predictors for SRR were different in patients with various etiologies and effects of age on SRR in idiopathic NOA and KS patients were opposite. What is known already NOA patients due to spermatogenic dysfunction, accounting for about 60 percent of the total azoospermia cases, may have opportunities to obtain sperm by mTESE and overall SRR of mTESE in NOA patients is about 50%. There were some of predictive factors for SRR at mTESE including age, testis volume, serum follicle stimulating hormone (FSH), testosterone (T), inhibin B, anti-Mullerian hormone and testicular histopathology, while the predictive factors of SRR remains underappreciated, especially in accordance with different type of etiology. Most notably, there are some controversies about the relationship between SSR and age at mTESE surgery. Study design, size, duration This retrospective study involved 3104 NOA patients with different etiologies treated with the first mTESE at the Reproductive Medical Centre of Peking University Third Hospital from March 2012 to December 2022. Participants/materials, setting, methods 3104 NOA patients were classified into seven groups according to etiologies including 1530 males with idiopathic NOA (iNOA), 763 males with Klinefelter syndrome (KS), 345 males with microdeletion of the AZFc, 177 cases with the history of cryptorchidism, 131 with a history of mumps orchitis, 89 males with cryptozoospermia and 69 cases with other causes. The end-point was the presence of one or more sperm. Multi-variable logistic regression was used to analyze sperm retrieval outcome. Main results and the role of chance Overall SRR was 43.46%, and there were different SRR in NOA patients with various etiologies with highest SRR of 89.31% in patients with a history of mumps orchitis and lowest SRR of 29.35% in iNOA patients. For patients with a history of mumps orchitis, there was a negative relationship between T and SRR (0.805 [0.654, 0.991] p = 0.041). For cryptozoospermia patients, shorter infertility duration was predictive for successful SRR (0.837 [0.704, 0.995], p = 0.044). Males with bigger testes had more likelihood of positive SRR (1.077 [1.005; 1.153], p = 0.035) in patients with cryptorchidism. BMI was an independent factor of SRR in AZFc-deleted patients (1.084 [1.013, 1.161], p = 0.020). For KS patients, lower male age (0.938 [0.898, 0.979], p = 0.004) and bigger testes (1.214[1.099, 1.342], p＜0.001) were predictive for successful SRR. Lower levels of FSH (0.984[0.970, 0.998] p = 0.025) and higher male age(1.079[1.050, 1.110], p＜0.001) were predictive for successful sperm retrieval in iNOA patients. So, the optimum age range of iNOA male undergoing mTESE should be 30 to 35 years old with the SRR of 29.13% when considering low SRR of iNOA males aged＜30 years (23.96%) and the negative effect of female age on ICSI outcome. Limitations, reasons for caution Surgeon performing mTESE maybe another predictor for SRR which is a potential selection bias. Inhibin B and Anti-Müllerian hormone weren’t included due to limited information in our database. We couldn’t verify the conclusion in more population from multicenter despite the current study with the large sample size so far. Wider implications of the findings Our results provide valuable information for NOA patients who want to counsel surgeons about their treatments to help patients and surgeons to perform a shared decision-making for optimal therapy methods, especially about optimal age treated with mTESE in iNOA and KS patients. Trial registration number not applicable

P-200 Tailoring IVF laboratory key performance indicators of the Vienna consensus in testicular sperm aspiration cases

Abstract Study question Are the IVF laboratory KPIs of the Vienna consensus equally applicable to ICSI cycles performed with ejaculated or testicular sperm aspiration (TESA) samples? Summary answer In addition to standard ICSI cycles, the Vienna KPIs are largely applicable to TESA cases, except for 2PN fertilization and day 3 development rates. What is known already The ESHRE SIG Embryology and Alpha Scientists in Reproductive Medicine produced the first international consensus on a systematic set of IVF laboratory KPIs. These efficiency measures can reveal the possible impact of extrinsic factors, such as operator skills or culture media, on the IVF process. However, to control for intrinsic factors – i.e., specific gamete characteristics – the Vienna Consensus focused on a “reference population” as defined by female age ≤39years, exclusion of PGT cases and use of own fresh oocytes and ejaculated sperm. This leaves the relevance of the Vienna KPIs to other patient populations, including TESA cases, uncertain. Study design, size, duration This was a retrospective, single-center cohort analysis of 1678 ART ICSI cycles carried out between 2010 and 2022. Treatments involving TESA and ejaculated sperm were 105 and 1573, respectively. Participants/materials, setting, methods Inclusion criteria were indication for IVF/ICSI with own ejaculated or TESA spermatozoa and blastocyst culture of all embryos formed in each cohort. Oocyte donation and PGT cycles were not included. Ovarian stimulation was performed with either recombinant-FSH or hMG, combined with GnRH to prevent spontaneous LH surge. Fertilization of collected oocytes was achieved by ICSI. Main results and the role of chance Both maternal and paternal age were comparable between the two groups. In the TESA cases, 2PN rate was lower (N = 593/915, 64.8% versus N = 7276/10350, 70.3%; p < 0.01), while 1PN rate was higher (N = 32/915, 3.5% versus N = 246/10350, 2.4%; p = 0.04). Other fertilization and developmental rates were comparable: 3PN (N = 24/915, 2.6% versus N = 341/10350, 3.3%); microinjection damage (N = 52/915, 5.7% versus 632/10350, 6.1%); failed fertilization (N = 4/105, 3.8% versus N = 61/1573, 3.9%); cleavage (N = 579/593, 97.6% versus N = 7105/7276, 97.6%); day 2 development (N = 318/593, 53.6% versus N = 3887/7276, 53.4%); day 3 development (N = 248/593, 41.8% versus N = 3279/7276, 45.1%); total good blastocyst development rate (N = 147/361, 40.7% versus N = 1728/3944, 43.8%). Compared with the Vienna Consensus, all ICSI outcomes were within normal ranges as defined by competency and benchmark values, while in TESA cycles 2PN fertilization and day 3 development rates were slightly below the competency thresholds. Limitations, reasons for caution The study, especially because retrospective and with small sample size, requires external independent validation Wider implications of the findings The study confirms the robustness of the Vienna Consensus recommendations for the monitoring of the IVF laboratory performance, while highlighting the need for fine-tuning individual indicators in specific patient populations. Trial registration number not applicable

P-227 ICSI outcomes after using in-situ microfluidics of fluidic walls versus DGC: a prospective non-inferiority comparative pilot study in sibling oocytes

Abstract Study question Does the novel strategy in-situ microfluidics (isM) yield comparable ICSI outcomes to the control sperm selection methodology density gradient centrifugation (DGC)? Summary answer ICSI outcomes show that handmade in-situ microfluidics of fluidic walls are as effective as DGC to achieve fertilization and usable day 5 blastocyst formation rate. What is known already Microfluidics technologies are proving their capability to select suitable sperm for ICSI. Increasingly, publications show novel and ingenious microfluidics strategies aimed to integrate sperm biomimicry during in vitro sperm selection while simplifying the IVF-workflow. We recently designed a microfluidics system which allows selecting sperm for ICSI in the same ICSI-dish, only using microfluidics and disregarding centrifugation, washing and plasticware. A previous proof-of-concept study showed that this methodology was efficient to select suitable sperm for ICSI as it could separate at least 20 progressive spermatozoa in less than 15 minutes in a clean microdroplet free of any remaining seminal plasma. Study design, size, duration The present pilot study included a total of 280 fresh MII-oocytes allocated in a 1:1 ratio to the study (isM-ICSI) and control (DGC-ICSI) groups. The statistical power was established at 80% at a CI 95%. For comparison we relied in the KPIs: ICSI normal fertilization rate (INFR) and Day 5 usable blastocyst rate (D5UBR). The non-inferiority margin (Δ) was established using 2-sigma warning limit and our historical control mean values (ΔINFR = 7% and ΔD5UBR = 10%). Participants/materials, setting, methods An informed consent was signed by all participants. The comparative study included 29 consecutive ICSI cases performed in fresh oocytes from women under 40 years old and with an oocyte yield ≥6 MII. All semen samples had ≥1 × 106/ml progressive spermatozoa and were split into three aliquots: 1-seminogram (100 µl), 2-isM (10 µl), 3-DGC (surplus). Embryos were cultured in bench-top Time-lapse incubators, using GTL-media under 6,4% CO2 and 5% O2 conditions up to blastocyst stage. Main results and the role of chance Sperm characteristics showed the following total mean values for total concentration (42,83 ± 31,1 × 106/ml), total motility (51,28 ± 14,16 %) and progressive sperm concentration (17 ± 12 × 106/ml). The mean number of MII per patient was 9,66. From the total number of oocytes (n = 280), 139 were allocated to isM (study group) and 141 to DGC (control group) groups. INFR was 72,0 % vs 76,6 %, respectively. Non-significant differences were observed (p = 0,54). We observed that the mean total number of usable blastocysts (day 5/6) per patient was 1,83 (53/29) in isM vs 1,66 (48/29) in DGC. Moreover, D5UBR was 53,0% vs 44,4 % (p = 0,26) with a difference between the study and the control of within the non-inferiority margin. We observed a lower rate of arrested embryos (up to day 6) in the study group (19,0% vs 30,0%), although results were not statistically significant (p = 0,08). Limitations, reasons for caution This pilot study includes a limited number of oocytes. The present isM protocol depends on a free-hand preparation. Although the use of templates and the training reduces inter and intra-operator variability, a ready-to-use surface would benefit the operability. Further investigation is required to evaluate clinical pregnancy and live birth outcomes. Wider implications of the findings Sperm DNA fragmentation is cited among the causes of embryo arrest. As the use of isM resulted in a lower proportion of arrested embryos, exploring its capacity to select non-fragmented sperm warrants further interest. The isM protocol streamlines sperm selection for ICSI, reduces costs and risks along the procedure. Trial registration number na

O-301 Can advanced sperm selection techniques improve blastocyst euploidy rate of abnormal sperm DNA fragmentation cases to the level of normal ones; prospective randomized controlled trial

Abstract Study question To compare PGT-A outcomes of Physiological ICSI (PICSI) and magnetic activated cell sorting (MACS) sperm selection techniques to a control of normal sperm DNA fragmentation. Summary answer PGT-A outcomes did not differ between using normal sperm DNA fragmentation semen to the semen processed by PICSI or MACS. What is known already Sperm DNA fragmentation can negatively affects pre-implantation embryo development in all stages after ICSI. Advanced sperm selection techniques like PICSI and MACS are thought to be efficient to improve fertilization, cleavage, and blastocyst formation rates in cases with abnormal SDF. It has become evident that a blastocyst's aneuploidy is not only contributed by the oocyte,but can also be caused by the sperm.The relation between sperm selection techniques such as PICSI or MACS and blastocyst euploidy is not investigated yet. Study design, size, duration A prospective randomized controlled trial of 351 ICSI cycles in a private fertility center, from October 2019 to December 2022. Cases were included in the study if: female had ≥5 MII oocytes, male had abnormal sperm DNA fragmentation (SDF) index (>20% by TUNEL assay), all blastocysts (≥4BB) had trophoectoderm biopsy. Participants/materials, setting, methods Included cases were randomized on the day of ICSI to PICSI or MACS group, along with a control arm of cases with normal SDF needed for comparison. PGT-A was done using Illumina platform (Miseq), and the results were reviewed by artificial intelligence (Cooper Surgical). Cases were categorized according to the female age into 2 subgroups(<35, and > 35 years). Statistical analysis of all pre-implantation embryo development parameters and PGT-A results were done using SPSS. Main results and the role of chance We found no significant differences between the female age, male age, count, motility, SDF, no. mature oocytes between the studied groups. Limitations, reasons for caution The results included all pre-implantation embryo parameters but, subsequent post-implantation events like implantation, pregnancy and miscarriage rates will add more data about the competency of the resulted embryos. Wider implications of the findings The usage of advanced sperm selection techniques like PICSI or MACS has showed comparable results to that of normal SDF cases, through eliminating the adverse effects of SDF on ICSI and PGT-A outcomes. Trial registration number NCT05494216

P-196 PIEZO-intracytoplasmic sperm injection (P-ICSI) can improve the clinical outcomes of ICSI

Abstract Study question Can P-ICSI improve the clinical outcomes of ICSI, such as degeneration, fertilization and development to blastocyst, compared to conventional ICSI (C-ICSI)? Summary answer P-ICSI can improve the clinical outcomes of ICSI by increasing the number of fertilized oocytes and decreasing the number of degenerated oocytes after ICSI. What is known already In human IVF-ET, some of oocytes are damaged after ICSI when retrieved oocytes are inseminated by ICSI. The application of P-ICSI has been limited although it was developed over 30 years ago. Recently, it has been reported that P-ICSI can decrease damage after ICSI and improve fertilization and embryonic development compared to C-ICSI. Study design, size, duration This study included 50 subjects undergoing IVF and was performed between April and October 2022. Sibling oocytes were randomly assigned to C-ICSI and P-ICSI. Participants/materials, setting, methods A total of 1,207 oocytes were retrieved from 50 patients. Among them, 852 oocytes with visible spindle were inseminated by C-ICSI or P-ICSI in sibling oocytes. Four hundred twenty-five oocytes were inseminated by C-ICSI and 427 oocytes were by P-ICSI. Fertilization, degeneration after ICSI and blastocyst development on day 5 or day 6 were compared between the P-ICSI and C-ICSI. The differences between C-ICSI and P-ICSI were statistically analyzed using Mann-Whitney U test. Main results and the role of chance Normal fertilization rate of P-ICSI (78.2 ± 16.5%) were higher than that of C-ICSI (71.2 ± 18.9%). The difference was statistically significant (P = 0.0447). The oocyte degeneration rates after ICSI were 9.3 ± 11.8% (C-ICSI) and 4.8 ± 9.4% (P-ICSI). The degeneration rate of C-ICSI was significantly higher than that of P-ICSI (P = 0.04338). Blastocyst formation rate of C-ICSI was 58.6 ± 25.0% and that of P-ICSI was 64.3 ± 26.3%. The blastocyst formation rate of P-ICSI was higher than that of C-ICSI but the difference was not statistically significant (P = 0.2113). The rates of blastocysts that were available for embryo replacement or cryopreservation were higher in P-ICSI (56.0 ± 25.4%) than in C-ICSI (50.4 ± 24.7%) although the difference was not significant (P = 0.26272). Limitations, reasons for caution Pregnancy outcomes data were not available. The data of this study were obtained from single fertility center. More large-scale multicenter studies will be needed to confirm the effectiveness of P-ICSI. Wider implications of the findings Consistent with other studies, these results show that P-ICSI reduces the damage rate of oocytes after ICSI. The implementation of P-ICSI has the potential to improve clinical outcomes of human IVF although it is more complicated and takes longer than that of C-ICSI. Trial registration number not applicable