Spermatozoa For Intracytoplasmic Sperm Injection Research Articles

#49 : Does Percutaneous Epididymal Sperm Aspiration (PESA)-Sperm Attribute as the Game Changer in High-Risk Male Factor Fertility for Intracytoplasmic Sperm Injection (ICSI)?

Background and Aims: The surgically retrieved sperms either from PESA, TESA and microTESA are considered to contribute satisfactory outcome in ICSI cycles in male factor infertility especially in Azospermia. Although the different sources of sperm for ICSI don’t affect the fertilisation rates in ICSI cycles, but the sperm obtained from fresh PESA may become game changer with higher implantation rates, resulting in better clinical pregnancy and live birth outcomes. Method: This comparative study was based at single reproductive center and evaluated 344 PESA-ICSI patients with high-risk male factors from July 2021 to Dec 2022 henceforth were compared with ejaculated-ICSI. The following high-risk male infertility patients were included - 1. No blastocyst formation in two stimulation cycles with ejaculated sperm, 2. If >2 IVF failure (with/o > 4 blastocyst transfer), 3. Severe OAT/azoospermia, 4. Sperm morphology <2% (and/or DFI >25%) even after 6 week of lifestyle n antioxidant therapy and 5. Miscellaneous factor like morbid obesity BMI>40, age >45 with/o history of chronic smoking and alcoholic. The embryo quality was assessed from good blastocyst rate and pregnancy outcome was recorded as implantation and clinical pregnancy rate. Results: As per the inclusion criteria 344 high-risk infertile male (21%) underwent PESA-ICSI and compared with 1346 ejaculated-ICSI patients. The average no of M2 oocytes were takes for ICSI purpose is similar in both group (9.2 and 9.4 per oocyte retrieval). The blastocyst rate in PESA-ICSI group was 51% as compare 48% in ejaculated-ICSI but there was outstanding good-blastocyst rate (34% compared to 24%, P<0.01). Both the group showed nearly equal implantation rate (83% compare to 80.4%) but ongoing pregnancy up to 12 weeks was significantly high as compare to ejaculated-ICSI group (77% vs 62% P <0.01) Conclusion: The high-risk male infertility must be addressed with timely evaluation and decision. PESA offers safe, simple, daycare and promising results in terms of sperm yield and pregnancy outcome, must be offered as first line approach.

Outcomes comparison of testicular versus ejaculated sperm for intracytoplasmic sperm injection in infertile men with high DNA fragmentation: updated systematic review and meta-analysis.

The testicular sperm instead of ejaculated sperm for intracytoplasmic sperm injection (ICSI) in infertile men with high sperm DNA fragmentation (SDF) is a controversial topic. This updated systematic review and meta-analysis aims to evaluate whether couples with high level of SDF will benefit more from intracytoplasmic sperm injection with testicular sperm (Testi-ICSI) as compared to intracytoplasmic sperm injection with ejaculated sperm (Ejac-ICSI). A systematic search was conducted according to PRISMA guidelines, using PubMed, Embase, Web of Science and the Cochrane Central Register of Controlled Trials (CENTRAL), encompassing studies from the earliest record until May 2022. We included studies analyzing comparative pregnancy outcomes of testicular versus ejaculated sperm for ICSI in infertile men with high DNA fragmentation. The risks of bias and certainty of evidence were assessed using the Risk Of Bias In Non-randomized Studies of Interventions (ROBINS-I) and the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) framework, respectively. Eleven studies were included. Meta-analysis showed that SDF levels revealed a significant difference association [odds ratio (OR) =-25.81; 95% confidence interval (CI): -34.82, -16.81; I2=94%; P<0.00001] between testicular and ejaculated sperm. Compared with Ejac-ICSI, a non-significant tendency was observed for fertilization rates (FRs) in the Testi-ICSI group (OR =0.87; 95% CI: 0.67, 1.12; I2=81%; P=0.28). However, there was significant difference pointing to better outcomes for Testi-ICSI in clinical pregnancy rates (CPRs) (OR =2.36; 95% CI: 1.71, 3.24; I2=0%; P<0.00001), live birth rates (LBRs) (OR =3.10; 95% CI: 2.13, 4.51; I2=4%; P<0.00001) and miscarriage rates (MRs) (OR =0.28; 95% CI: 0.13, 0.60; I2=0%; P=0.001). Results of this updated meta-analysis reveal that SDF rates are lower in testicular sperm than in ejaculated sperm and that Testi-ICSI is correlated with better clinical outcomes, including higher CPRs, higher LBRs, and lower MRs in infertile males with high SDF levels. Nevertheless, with the overall low to moderate quality of the studies, further well-designed controlled studies are required.

Successful cryptozoospermia management with multiple semen specimen collection

To determine the prevalence of sperm suitable for intracytoplasmic sperm injection (ICSI) in fresh ejaculated semen samples provided by men scheduled for a microdissection testicular sperm extraction (mTESE) procedure. Secondary objectives included an evaluation of the effect of a short abstinence period on semen quality and ICSI outcomes for men with cryptozoospermia. Retrospective cohort study. Academic medical center. All men were scheduled to undergo a mTESE procedure by a single, high-volume surgeon at an academic center from September 1, 2015, to May 1,2021. Presence of sperm suitable for ICSI in the ejaculate on the day of scheduled mTESE. Prevalence of sperm suitable for ICSI in the ejaculate among previously diagnosed men with azoospermia. Secondary outcomes included changes in semen parameters, clinical pregnancy rate, and live birth rate. Of 727 planned mTESE procedures, 69 (9.5%) were canceled because sperm suitable for ICSI were identified in a fresh ejaculated sample produced on the day of scheduled surgery (typically one day before oocyte retrieval). Overall, 50 men (50/727, 6.9%) used these rare, ejaculated sperm for ICSI. Semen samples obtained with <24 hours of abstinence were more likely to have better motility than the sample initially provided on the day of the planned mTESE. The live birth rate per ICSI attempt using these rare, ejaculated sperm was 36% (19/53). Providing a fresh ejaculated semen sample on the day of mTESE allows nearly 10% of men with azoospermia to avoid surgery with satisfactory ICSI outcomes. Providing multiple ejaculated samples over a short period of time does not adversely affect sperm concentration and may enhance sperm motility in men with cryptozoospermia.

P-143 Will Use of Testicular sperm in ICSI cycles increase Embryo Aneuploidy?

Abstract Study question Will use of surgically retrieved Testicular Sperms (TESA) in Intra-Cytoplasmic Sperm Injection (ICSI) cycles increase embryo aneuploidy? Summary answer Use of TESA sperms for ICSI seems safe and doesn’t increase embryo aneuploidy. TESA performed for various indications also showed no variation with embryo aneuploidy. What is known already Invention of ICSI and improved surgical sperm retrieval techniques have helped men with severe male factor infertility to father their own genetic child. Sperms retrieved from seminiferous tubules of testes are considered immature as they haven’t completed the process of spermiogensis. It is hypothesized that use of TESA sperm for ICSI can increase embryo aneuploidy, which is still uncertain. Study design, size, duration This retrospective study conducted at a private fertility clinic from 2014–2022 (n = 265 patients; total of 860 embryos evaluated). Study populationTESA sperm used and PGT-A done (n = 66). TESA group was further sub-divided into 2 groups based on the indication for TESA,Non-Obstructive Azoospermia(n = 26) and Raised SDF(n = 40). Control Population Ejaculated sperm used and PGT-A done (n = 199) Only younger women(&amp;lt;37yrs) with self-gametes and RIFconsidered in this study. RIF was defined as women with 2 failed IVFcycles in thepast with at least 4 blastocystsbeing transferred. Participants/materials, setting, methods All fertilized oocytes inseminated by ICSI and subjected to extended blastocyst culture. TESA done as per our clinic’s SOP and for control group sperm preparation done by density gradient method. Trophectoderm biopsy was performed and sent for PGT-A to the genetic lab. Next-Generation sequencing (NGS) was the Comprehensive Chromosomal technique (CCS) used for assessing embryo ploidy status. Aneuploidy across all groups compared. Main results and the role of chance Following were the Aneuploidy rates of TESA sperm, Ejaculate sperm group and TESA done for NOA and SDF respectively. Ejaculate Sperm – Aneuploidy%- 42% TESA Sperm (irrespective of the indication)- Aneuploidy%- 44% (p = 0.6260) TESA Sperm (done for NOA)- Aneuploidy%- 44% (p = 0.7504) TESA Sperm (done for raised SDF) – Aneuploidy%- 32% (p = 0.05) Embryo aneuploidy was comparable between Ejaculate and TESA sperm group. Though TESA sperm is considered immature, it didn’t seem to increase embryo aneuploidy. Embryo Aneuploidy from TESA sperm ICSI cycles done for NOA or raised SDF also showed no altering trend. Data from this study is suggestive that TESA sperm doesn’t alter embryo ploidy status. Limitations, reasons for caution Data is retrospective with small sample size and unequal distribution. A well designed Randomized Trial might help test the inference of this study further. Wider implications of the findings Incidence of male infertility and use of ICSI is on a rise globally. Interventions to optimize embryo implantation and reproductive outcomes will help with better success rates and shorten duration to conception. Further research is warranted with severe male factor infertility to improve sperm selection techniques and reproductive outcomes. Trial registration number not applicable

A Machine Learning Approach for the Prediction of Testicular Sperm Extraction in Nonobstructive Azoospermia: Algorithm Development and Validation Study.

Testicular sperm extraction (TESE) is an essential therapeutic tool for the management of male infertility. However, it is an invasive procedure with a success rate up to 50%. To date, no model based on clinical and laboratory parameters is sufficiently powerful to accurately predict the success of sperm retrieval in TESE. The aim of this study is to compare a wide range of predictive models under similar conditions for TESE outcomes in patients with nonobstructive azoospermia (NOA) to identify the correct mathematical approach to apply, most appropriate study size, and relevance of the input biomarkers. We analyzed 201 patients who underwent TESE at Tenon Hospital (Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris), distributed in a retrospective training cohort of 175 patients (January 2012 to April 2021) and a prospective testing cohort (May 2021 to December 2021) of 26 patients. Preoperative data (according to the French standard exploration of male infertility, 16 variables) including urogenital history, hormonal data, genetic data, and TESE outcomes (representing the target variable) were collected. A TESE was considered positive if we obtained sufficient spermatozoa for intracytoplasmic sperm injection. After preprocessing the raw data, 8 machine learning (ML) models were trained and optimized on the retrospective training cohort data set: The hyperparameter tuning was performed by random search. Finally, the prospective testing cohort data set was used for the model evaluation. The metrics used to evaluate and compare the models were the following: sensitivity, specificity, area under the receiver operating characteristic curve (AUC-ROC), and accuracy. The importance of each variable in the model was assessed using the permutation feature importance technique, and the optimal number of patients to include in the study was assessed using the learning curve. The ensemble models, based on decision trees, showed the best performance, especially the random forest model, which yielded the following results: AUC=0.90, sensitivity=100%, and specificity=69.2%. Furthermore, a study size of 120 patients seemed sufficient to properly exploit the preoperative data in the modeling process, since increasing the number of patients beyond 120 during model training did not bring any performance improvement. Furthermore, inhibin B and a history of varicoceles exhibited the highest predictive capacity. An ML algorithm based on an appropriate approach can predict successful sperm retrieval in men with NOA undergoing TESE, with promising performance. However, although this study is consistent with the first step of this process, a subsequent formal prospective multicentric validation study should be undertaken before any clinical applications. As future work, we consider the use of recent and clinically relevant data sets (including seminal plasma biomarkers, especially noncoding RNAs, as markers of residual spermatogenesis in NOA patients) to improve our results even more.

Telomere Length in Human Spermatogenic Cells as a New Potential Predictor of Clinical Outcomes in ART Treatment with Intracytoplasmic Injection of Testicular Spermatozoa.

Predicting the clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles that use the testicular spermatozoa of azoospermic patients presents a challenge. Thus, the development of additional approaches to assessing the competence of a testicular-sperm-derived embryo without causing damage to gametes or the embryo is necessary. One of the key parameters in determining such developmental competence is telomere length (TL). We aimed to analyze TLs in spermatogenic cells from the testicular biopsy samples of azoospermic patients and determine how this parameter influences embryo competence for pre- and post-implantation development. Using Q-FISH, we studied the TL of the chromosomes in spermatogonia and spermatocytes I from the TESE biopsy samples of 30 azoospermic patients. An increase in TL was detected during the differentiation from spermatogonia to spermatocytes I. The patients' testicular spermatozoa were used in 37 ICSI cycles that resulted in 22 embryo transfers. Nine pregnancies resulted, of which, one was ectopic and eight ended in birth. The analysis of embryological outcomes revealed a dependence between embryo competence for development to the blastocyst stage and the TL in spermatogenic cells. The TLs in spermatogonia and spermatocytes I in the testicular biopsy samples were found to be higher in patients whose testicular sperm ICSI cycles resulted in a birth. Therefore, the length of telomeres in spermatogenic cells can be considered as a potential prognostic criterion in assessing the competence of testicular-sperm-derived embryos for pre- and post-implantation development. The results of this study provide the basis for the development of a laboratory test for the prediction of testicular sperm ICSI cycle outcomes.

Embryo development and live birth resulted from artificial oocyte activation after microdissection testicular sperm extraction with ICSI in patients with non-obstructive azoospermia.

The application of microdissection testicular sperm extraction (micro-TESE) to retrieve the sperm of patients with non-obstructive azoospermia (NOA) has greatly increased. Patients with NOA often have poor quality sperm. Unfortunately, there are few studies on artificial oocyte activation (AOA) performed on patients who successfully retrieved motile and immotile sperm by micro-TESE after intracytoplasmic sperm injection (ICSI). Therefore, this study sought to obtain more comprehensive evidence-based data and embryo development outcomes to aid consultation of patients with NOA who opted to receive assisted reproductive techniques and to determine whether AOA needs to be performed in different motile sperm after ICSI. This retrospective study involved 235 patients with NOA who underwent micro-TESE to retrieve adequate sperm for ICSI between January 2018 and December 2020.A total of 331 ICSI cycles were performed in the 235 couples. Embryological, clinical, and neonatal outcomes were demonstrated comprehensively between motile sperm and immotile sperm using AOA and non-AOA treatment. Motile sperm injection with AOA (group 1) showed significantly higher fertility rate (72.77% vs. 67.59%, p=0.005), 2 pronucleus (2PN) fertility rate (64.33% vs. 60.22%, p=0.036), and miscarriage rate (17.65% vs. 2.44%, p=0.018) compared with motile sperm injection with non-AOA (group 2). Group 1 had comparable available embryo rate (41.29% vs. 40.74%, p=0.817), good embryo rate (13.44% vs. 15.44%, p=0.265), and without an embryo for transfer rate (10.85% vs. 9.90%, p=0.815) compared with group 2. Immotile sperm injection with AOA (group 3) displayed significantly higher fertility rate (78.56% vs. 67.59%, p=0.000), 2PN fertility rate (67.36% vs. 60.22%, p=0.001), without an embryo for transfer rate (23.76% vs. 9.90%, p=0.008), and miscarriage rate (20.00% vs. 2.44%, p=0.014), but significantly lower available embryo rate (26.63% vs.40.74%, p=0.000) and good embryo rate (15.44% vs. 6.99%, p=0.000) compared with group 2. In groups 1, 2, and 3, the rates of implantation (34.87%, 31.85% and 28.00%, respectively; p=0.408), clinical pregnancy (43.87%, 41.00%, and 34.48%, respectively; p=0.360) and live birth (36.13%, 40.00%, and 27.59%, respectively; p=0.194) were similar. For those patients with NOA from whom adequate sperm were retrieved for ICSI, AOA could improve fertilization rate, but not embryo quality and live birth outcomes. For patients with NOA and only immotile sperm, AOA can help achieve acceptable fertilization rate and live birth outcomes. AOA is recommended for patients with NOA only when immotile sperm are injected.

Outcome analysis of ICSI assisted pregnancy using testicular sperm versus ejaculated sperm in man with severe oligozoospermia in the same ART cycle: A case report.

Intracytoplasmic sperm injection (ICSI) has become the most common method for couples with male factor infertility, and source of sperm for the procedure have evolved over time. but few have examined testicular sperm extraction vs. ejaculated sperm use for severe oligozoospermia in the same assisted reproductive technology (ART) cycle. Here, we evaluated the clinical outcomes after ICSI with testicular sperm or ejaculated in man with severe oligozoospermia in the same ART cycle. A couple who had failed the first ART cycle with ejaculated sperm, using the freshly ejaculated sperm and testicular sperm for ICSI during the second ART cycle by lack of enough sperm to fertilize in an ICSI attempt. The patient was diagnosed with severe oligozoospermia, and routine semen analysis revealed sperm concentration is less than 2 million/mL. The patient using testicular sperm versus ejaculated sperm with ICSI assisted pregnancy in the same ART cycle. We found that superior cleavage rate, number of embryos transferred and blastocyst rate with the use of testicular rather than ejaculated sperm-ICSI in the couple. The results described here suggest that use of testicular sperm may improve biologic outcomes, especially for couples with male-partner oligozoospermia who previous ICSI failures. Our case report supported the efficacy of testicular sperm preference over ejaculated sperm for ICSI in men with severe male factor infertility. It is a paradigm shift concerning the use of ejaculated sperm as the preferable source of sperm for ICSI, add to the small amount of literature on testicular sperm extraction vs. ejaculated sperm use for severe oligozoospermia in the same ART cycle.

Factors predicting the outcome of percutaneous epididymal sperm aspiration in men with obstructive azoospermia

To report on the live birth rates (LBRs) following percutaneous epididymal sperm aspiration (PESA) in men with obstructive azoospermia (OA) and factors affecting treatment outcome which is under reported in the literature. This is a multicenter study that was conducted in Egypt including all couples undergoing intra cytoplasmic sperm injection (ICSI) for OA using PESA-derived sperms. Men were subdivided according to aetiology into congenital, iatrogenic and idiopathic groups. Fertilization, pregnancy and LBRs were determined and compared in each group. The longitudinal LBR, crude and expected cumulative delivery rates (CCDR, ECDR) were calculated. Multiple logistic regression analysis was used to determine significant associations between maternal, paternal and ICSI factors with successful live births. Ninety couples were included in the study. Viable sperm for ICSI was retrieved in 89 men (98.9%). A total of 155 ICSI cycles with 17 frozen embryo transfers resulted in 81 pregnan-cies and 55 live births. After 5 cycles, the longitudinal LBR, CCDR and ECDR were 30%, 57.3% and 88.6% respectively. Maternal age and number of fertilized eggs were the only fac-tors significantly affecting LBRs. PESA is a minimally invasive procedure for secur-ing viable sperm for ICSI in OA men, with high cumulative delivery rates. Maternal age and number of fertilized eggs are the only factors that significantly affecting LBR. The contempo-rary longitudinal and cumulative LBRs provide objective out-come data to counsel OA patients undergoing fertility treat-ments.

Switching to testicular sperm after a previous ICSI failure with ejaculated sperm significantly improves blastocyst quality without increasing aneuploidy risk.

The use of testicular sperm is confined to patients with azoospermia, but there is evidence to support its use in males with poor semen parameters and/or previous intracytoplasmic sperm injection (ICSI) failures with ejaculated spermatozoa. We compared the aneuploidy rate and quality between embryos derived from ICSI cycles with ejaculated sperm (EJ-ICSI) and those from ICSI cycles using testicular spermatozoa (TT-ICSI) within the same couple. Retrospective study of 27 couples who first underwent an EJ-ICSI cycle that did not result in a livebirth and afterwards a TT-ICSI cycle. Only the two closer cycles of each couple were included. Preimplantation genetic test for aneuploidies (PGT-A) was performed in both ICSI cycles and classic parameters of embryo quality were assessed until blastocyst-stage. A total of 375 embryos from 54 ICSI cycles were evaluated. Aneuploidy rate was measured by two different parameters. Patients undergoing TT-ICSI presented a similar aneuploidy rate as EJ-ICSI group: 30.7% (23.4-38.0) vs 26.8% (18.1-35.5) per inseminated oocytes (P>0.05), and 76.2% (66.2-86.2) vs 72.1% (59.1-85.2) per the total number of biopsied embryos (P>0.05), respectively. Further, the good-quality blastocyst rate per correctly fertilized oocyte was significantly higher in TT-ICSI group (33.6% (30.4-36.9)) than EJ-ICSI group (24.2% (20.3-28.0)) (P<0.001). Switching to testicular sperm for ICSI yielded better-quality blastocysts without affecting the chromosomal load of the embryos in non-azoospermic couples with a previous unsuccessful ICSI using ejaculated sperm. This strategy is a good option for couples seeking a livebirth who do not want to use donor sperm.

P-013 Testicular spermatozoa for intra-cytoplasmic sperm injection after IVF failure with ejaculated spermatozoa: a propensity score-matched cohort study

Abstract Study question Does the use of testicular spermatozoa from non-azoospermic infertile men after failed IVF with ejaculated spermatozoa increases the pregnancy rate? Summary answer Depending on semen characteristics and obtained embryos number at the previous IVF failure with ejaculated spermatozoa, it seems licit to propose ICSI with testicular spermatozoa. What is known already Ejaculated spermatozoa are generally considered to possess better fertilisation potential than testicular spermatozoa as their maturation has been completed. However, it has been shown that in selected cases, the use of testicular spermatozoa from non-azoospermic infertile men resulted in a higher implantation and pregnancy rate than the use of ejaculated spermatozoa. One of the reasons for this result is a higher DNA fragmentation index (DFI) in ejaculated spermatozoa compared to testicular spermatozoa. This increase in DFI would negatively affect embryo development and implantation. Study design, size, duration A retrospective matched cohort study using propensity score matching (PSM) analysis was performed. After an IFV failure (cycle_1), IVF with ejaculated or testicular spermatozoa was performed (cycle_2). Female age, female BMI, IVF rank, stimulation protocol, gonadotropin total quantity, treatment duration, punctured oocytes number, and mature oocytes punctured were included in PSM. The matching was performed for IVF performed in cycle_1 for cases and controls identified in cycle_2. 26 couples were included between 01/01/2012 and 30/09/2021. Participants/materials, setting, methods Among the 126 couples, for 63 couples, the cycle_2 was performed with ejaculated spermatozoa (controls) and for 63 couples with testicular spermatozoa (cases). Mixed logistic regression was used to compare outcomes of cases versus controls groups. The cycle_1 have allowed finding the prognostic factors to propose a cycle_2 ICSI with testicular spermatozoa in case of cycle_1 IVF failure. The study outcomes were the pregnancy rate (PR) and the cumulative pregnancy rate (CPR). Main results and the role of chance In cycle_1, no difference was observed for the parameters included in PSM. The DFI was higher in case group (13.7% ± 10.5% versus 9.3% ± 4.4%, p &amp;lt; 0.05), no difference was observed for fertilization rate, blastulation rate and frozen embryo rate. However, the PR was higher in case group (22.2% versus 0.0%, p &amp;lt; 0.001), the same result was found for the CPR (25.4% versus 6.3%, p &amp;lt; 0.001). The main prognostics factors to propose a cycle_2 ICSI with testicular spermatozoa after cycle_1 IVF failure with ejaculated spermatozoa: teratozoospermia or cryptozoospermia in cycle_1 (OR = 6.0 [1.2 ; 31.1], p &amp;lt; 0.05), a number of obtained embryos greater than 7 in cycle1 (OR = 5.3 [1.5 ; 18.0], p &amp;lt; 0.01), independently of male age in cycle_1 (OR = 2.3 [0.8 ; 6.5], p &amp;gt; 0.05 Limitations, reasons for caution The main limitations of the current study is being retrospective rather than prospective randomized. However, the used of the propensity score to perform a case-control study allow this study to be more reliable and to obtain results corresponding to real life. Wider implications of the findings This study is in favour of using testicular spermatozoa after IVF failure with ejaculated spermatozoa when the semen morphology is altered or in case of cryptozoospermia, and when at least 8 embryos was obtained in the previous IVF procedure Trial registration number not applicable

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

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

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 &amp;lt; 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 &amp;gt; 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 (&amp;lt;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

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 &amp;lt; 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 &amp;lt; 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-085 Use of an artificial intelligence tool to assess single-sperm motility variables related to bias preference of ICSI sperm selection practice, normal fertilization, and blastocyst formation

Abstract Study question Which single-sperm WHO motility variables are associated with ICSI sperm selection preference, normal fertilization, and blastocyst formation when assessed by an artificial intelligence platform? Summary answer Injected sperm had higher VSL, VAP, and LIN compared with non-injected. Higher STR was associated with fertilization and higher WOB and less HMP with blastulation. What is known already Sperm selection for intracytoplasmic sperm injection (ICSI) is key to its success. Currently, the procedure may be subjective although the embryologist selects rapidly from a small population of sperm. There is no consensus on specific motility variables for single-sperm selection. SiD (IVF 2.0 Limited, UK) software is able to assess all sperm in a visual field and accurately compute the motility variables for each sperm in real-time. SiDTM could help to understand the association between motility variables related to ICSI success and to assess the differences in sperm selection practice between different clinics and operators. Study design, size, duration 473 ICSI videos and their corresponding outcome were retrospectively analyzed using SiD software and assessed against normal fertilization (2PN), and blastocyst formation (BF), from four fertility clinics in different countries between July 2021 to December 2021. Ethical approval was obtained. This study was non-interventional. The study parameters were defined as sperm that were selected or non-selected, 2PN, and BF. Participants/materials, setting, methods Motility variables were computed from ICSI videos for VSL, VCL, LIN, VAP, ALH, WOB, STR as defined by the WHO manual for the examination and processing of human semen; and HMP, a metric associated with sperm-head movements. Injected sperm were manually identified and the injection was visually verified by a senior embryologist. Extracted motility variables were standardized according to each clinic and PVP concentration. Study groups were tested with a two-sided Mann-Whitney U test. Main results and the role of chance Video analysis identified 304 selected and 9543 non-selected sperm. 2PN and BF status was known for 302 and 301 injected oocytes respectively (2PN rate: 79%, BF rate: 53%). Embryologists used VSL, VAP, and LIN as the preferred motility criteria to select sperm (p &amp;lt; 0.001). Using data from all clinics, we found a tendency for STR (p = 0.12) and LIN (p = 0.14) for 2PN and WOB (p = 0.11) and VAP (p = 0.18) for BF. There was partial consistency on sperm selection criteria amongst clinics: Clinic1 selected VSL, VAP, and LIN as the main variables to select sperm; Clinic2 VSL, WOB, and LIN; Clinic3 VSL, VAP, and VCL; and Clinic4 ALH, VCL, and VAP (p &amp;lt; 0.001). For Clinic1 higher HMP showed a correlation (p = 0.06, n = 21) to 2PN, while higher WOB (p = 0.12, n = 21) did for BF. Clinic2 showed a limited association to higher STR and 2PN (p = 0.12, n = 120), and a significant association between higher WOB and BF (p &amp;lt; 0.05, n = 120). Clinic3’s data suggested that higher STR was associated with 2PN (p &amp;lt; 0.05, n = 97) and low HMP with BF (p &amp;lt; 0.05, n = 97). Limitations, reasons for caution This retrospective study needs to be repeated prospectively with larger numbers in more locations using embryologists with different levels of ICSI experience. Clinical outcome data such as clinical pregnancy, miscarriage, and live birth impact is still required. Wider implications of the findings SiD was able to track and extract motility features from single sperm. SiDTM could assist embryologists in real-time to select sperm during ICSI and could be used as a training tool to reduce variability among embryologists. Trial registration number RPA-2021-03

Comparison of sperm quality after double slow freezing and double vitrification of stallion sperm

Intracytoplasmic sperm injection (ICSI) is a valuable tool to optimize the remaining frozen sperm of subfertile or dead stallions. The possibility of thawing, diluting, and re-freezing, conventionally frozen spermatozoa has been previously studied, but not for vitrified sperm. We compared the effect of thawing/warming and re-freezing/re-vitrifying conventionally frozen/vitrified stallion sperm. For this purpose, 10 ejaculates from 2 stallions were divided into two aliquots and submitted to i)slow freezing (SF), extending sperm at 100 × 10 6 sperm/ml in permeable cryoprotectant medium (Botucrio, Betlabs, Lexington, KY), slow cooling for 2 hours and frozen 4cm over liquid nitrogen (LN 2 ) for 10 minutes; ii) or vitrification (VIT), extending sperm in permeable-cryoprotectant free medium (INRA96 + Trehalose 100mM + BSA 1%), cooled for 1 h and plunged directly into LN 2 . Two SF straws were thawed at 37 °C for 30 s, whereas two VIT straws were warmed at 60 °C for 5s. One straw was used for sperm parameters assessment while the second straw was re-frozen (RE-SF) or re-vitrified (RE-VIT) andthawed for sperm analysis. Sperm were analyzed for total (TMOT) and progressive (PMOT) motility (CASA, SCA, Microptic, Barcelona, Spain) and percentage of viable, non-reacted sperm (VNR, PI/PNA staining using flow cytometry). TMOT (80.6 ± 2.2 % vs. 70.2 ± 2.9% and 79.4 ± 2.5% vs. 70.1 ± 3.0%), PMOT (46.9 ± 6.1% vs. 31.3 ± 3.4% and 47.8 ± 6.4% vs. 38.1 ± 3.9%) and VNR (81.4 ± 2.4% vs. 71.2 ± 3.1% and 79.1 ± 3.1 vs. 72.2 ± 3.9%) showed significantly (p<0.05) lower values after SF vs. RE-SF and VIT vs RE-VIT, respectively. RE-VIT showed significantly (p<0.05) higher PMOT values than RE-SF (38.1 ± 3.9% vs. 31.3 ± 3.4%). In conclusion, both re-freezing and re-vitrification decrease sperm parameters after warming. Interestingly, re-vitrified sperm obtained higher progressive motility values than conventionally re-frozen sperm. Although further studies are needed to evaluate the effect of using vitrified sperm for ICSI, re-vitrification seems an interesting alternative for the optimization of valuable stallion ejaculates.

Hyaluronic acid-sperm selection significantly improves the clinical outcome of couples with previous ICSI cycles failure.

Beneficial effects of hyaluronic acid (HA)-based selection of spermatozoa for intracytoplasmic sperm injection (ICSI) are still controversial, and further studies are needed to categorize patients that might benefit from such a method. We investigated whether HA sperm selection improved ICSI outcome of couples with previous ICSI cycle failure. In this retrospective study, we prospectively collected data of (i) Group 1: 96 couples who performed one failed ICSI cycle ("1st procedure," n=96) followed by another ICSI cycle ("2nd procedure," n=101); ii) Group 2: 87 couples who performed one failed ICSI cycle (n=87) followed by an HA-ICSI cycle (n=104). Differences between procedures and groups were measured by paired and independent statistical tests, respectively. A generalized linear mixed model analyzed the effect of procedure on the outcomes and the interaction between procedures and groups. Injection of HA-bound sperm significantly improved cleavage rate with respect to standard ICSI (p=0.026). No evolutive pregnancies were obtained in the 1st ICSI attempts. The 2nd ICSI cycles resulted in successfully seven pregnancies. In HA-ICSI cycles, the better quality of embryos with respect to ICSI (p=0.034) increased the choice of day 5 embryo transfer (p=0.030), which resulted in successfully 28 pregnancies. No differences were observed in clinical outcomes of the two ICSI procedures in Group 1, while pregnancy and implantation rates were significantly higher in HA-ICSI with respect to ICSI cycles (p=0.001, p<0.0001, respectively). No negative perinatal outcomes were recorded. In couples where previous 1st ICSI failed, selection of HA-bound spermatozoa significantly improved clinical outcomes with respect to further standard ICSI. This study identified couples with previous ICSI cycles failure as a category of infertile patients that really may benefit from HA sperm selection before ICSI.

Does Choosing Microfluidics for Sperm Sorting Offer an Advantage to Improve Clinical Pregnancies in Donor Egg Recipients?

ABSTRACTBackground:Microfluidics (MF), an advanced sperm sorting technology results in the extraction of spermatozoa with higher DNA integrity and lower DNA damage compared to existing conventional sperm sorting methods.Aims:The aim of the present study is to assess the efficiency of MF and to isolate the best spermatozoa for intracytoplasmic sperm injection (ICSI) over the density gradient (DG) technique.Study Setting and Design:We recruited couples who choose the oocyte donation programme for this study to eliminate confounding factors associated with oocyte quality.Materials and Methods:Sperm was processed by MF (n = 180) and DG (n = 151). ICSI was performed and positive pregnancy, miscarriage and clinical pregnancy rates were compared.Statistical Analysis:All variables were analysed using Graph Pad Prism 5. The unpaired two-tailed t-test was used to assess the significance. A value of P < 0.05 was considered statistically significant.Results:There was no significant difference in pregnancy rates between the groups. However, a clear demarcation is seen in terms of clinical pregnancy rates, where the DG group achieved higher clinical pregnancies (91.7%) compared to the MF group (80.7%). Further, we compared miscarriage rates and biochemical pregnancies, and found a significantly higher miscarriage and biochemical pregnancy rate in the MF group (14.5% and 4%, respectively) compared to the DG group (6% and 1%, respectively).Conclusions:Based on the available literature, we anticipated a higher clinical pregnancy rate with MF compared with conventional processing. Our results show MF does not have any add-on positive effect on clinical pregnancy rate.

Clinical benefits of a modified Cryopiece system for cryopreservation of rare ejaculated and testicular spermatozoa for ICSI.

Cryopreservation of rare testicular-retrieved spermatozoa for intracytoplasmic sperm injection (ICSI) in patients with severe oligozoospermia and azoospermia remains a major challenge in clinical practice. This study evaluated the Cryopiece system as a potential technique to cryopreserve rare human spermatozoa for ICSI. Small numbers of ejaculated (24 patients) and testicular (13 patients) spermatozoa were cryopreserved using the Cryopiece system. The total number of recovered spermatozoa and motility were assessed after thawing. Thirty-seven couples underwent ICSI using spermatozoa cryopreserved by the Cryopiece system, and ICSI outcomes (rates of fertilization, embryo cleavage, and clinical pregnancy) were evaluated. The average sperm post-thaw retrieval rate was 79.1%, and motility was 29.7%. Ejaculated spermatozoa had a higher post-thaw motility (32.5%) than testicular spermatozoa (21.8%; P = 0.005). ICSI achieved a fertilization rate of 61.9%, embryo cleavage rate of 84.6%, and clinical pregnancy rate of 43.3%. The ICSI outcomes in the ejaculated and testicular frozen-thawed spermatozoa were similar. Assisted oocyte activation (AOA) after ICSI with motile (72.1%) or immotile (71.9%) spermatozoa resulted in a significantly higher fertilization rate than that when using motile spermatozoa without AOA (52.0%; P = 0.005). However, AOA did not enhance the clinical pregnancy rate (55.6% or 40.0% vs 35.3%; P = 0.703). The Cryopiece system is simple and useful for the cryopreservation of small numbers of ejaculated or testicular spermatozoa for ICSI in patients with severe oligozoospermia or nonobstructive azoospermia.

Sperm Selection Technology in ART.

Intracytoplasmic sperm injection (ICSI) is a commonly used in vitro fertilization technique. Selection of sperm for injection is currently done using subjective assessment of morphology, which may not accurately identify the best-quality sperm. Hyaluronic acid receptors on sperm plasma membranes are a marker of maturity, and sperms which are capable of binding immobilized hyaluronic acid in vitro are of higher quality. This can be used as an advanced sperm selection technique to choose sperm for ICSI, termed physiological ICSI (PICSI). Several studies reported improved fertility treatment outcomes when using PICSI compared with conventional ICSI; however, the majority of studies are underpowered. Recently, a large, multicenter, randomized controlled trial, known as the Hyaluronic Acid Binding Sperm Selection (HABSelect) trial, found a significant reduction in miscarriage rates with PICSI, but no significant effect on live birth rate. There are still many avenues through which PICSI may provide an advantage, subject to confirmation by future research, such as improved long-term health of offspring. Other advanced sperm selection techniques include intracytoplasmic morphologically selected sperm injection, magnetic-activated cell sorting, and Zeta potential sperm selection; however, the most recent Cochrane review concluded that there is currently insufficient evidence to ascertain whether these techniques improve clinical outcomes, such as live birth rates.