Fresh Sperm Research Articles

Motility Subpopulations with Distinct Motility Characteristics Using Swim-Up-Selected Sperm Cells from Norwegian Red Bulls: Effects of Freezing-Thawing and Between-Bull Variation.

Discrete subpopulations of motile sperm cells have been found for several species and are implicated to be important for sperm functionality. The aim of this present study was to examine the motile subpopulations in swim-up-selected bull spermatozoa and the relationship between subpopulations in fresh and frozen-thawed sperm cells. In experiment 1, swim-up (SWUP)-selected and non-selected (control) sperm cells were analyzed using a Computer-Assisted Sperm Analyzer (CASA). In experiment 2, the semen from nine bulls was cryopreserved and analyzed using CASA both before and after freezing and after incubation at physiological temperatures. The SWUP population had a higher proportion of total motility, progressivity, and velocity compared to the control (p < 0.05). Likewise, both incubation over time and cryopreservation affected motility and motility parameters (p < 0.05). The population of rapid progressive (RapidP) sperm cells dominated the SWUP fraction and was higher than in the control samples (p < 0.05). Furthermore, RapidP was also the main part of fresh semen, but decreased significantly over time during incubation and due to cryopreservation. In conclusion, RapidP was the main population in SWUP-selected spermatozoa and seems to be an important subpopulation contributing to the differences between treatments and in response to the freezing of sperm cells.

Increase of the motility of buck semen with Andromed® extender and the addition of HTF

Objective: To test whether HTF (Human Tubal Fluid) is capable of protecting goat sperm cells increasing the semen m Objective: To determine if HTF (Human Tubal Fluid) can protect buck sperm cells, increasing semen motility when used by itself or combined with the Andromed® commercial extender. Design/Methodology/Approach: The semen of eight bucks of the Boer breed was used (10 ejaculates per male). Each ejaculate was diluted in three treatments (5 repetitions per treatment): 1) only Andromed®; 2) only HTF; and 3) Andromed® + HTF. The following variables were evaluated: mass motility (MM) and progressive motility (MP) in fresh (37 °C) and refrigerated (5 °C) semen. The differences between treatments were detected, based on the MM and MP in fresh and refrigerated semen variables, using the GLM procedure (SAS V9). In the case of the MM evaluated in three different periods, the measures repeated over time were analyzed using the Proc Mixed procedure (SAS V9). Results: Regarding the quality of fresh spermatozoa, MM and MP recorded similar values (p&gt;0.05) for the three treatments. On the one hand, MM registered 97.1% for Andromed®, 95.8% for HTF, and 97.5% for Andromed® + HTF; its highest value was achieved with the Andromed® + HTF combination, followed by Andromed® and HTF. On the other hand, the highest MP percentage was observed with the combination of Andromed® + HTF (91.0%), followed by Andromed® (90.4%), and HTF (89.0%). Regarding the quality of the sperm refrigerated at 5 °C, the combination of Andromed® + HTF had a higher MP value (p&lt;0.05) than other treatments. Study Limitations/Implications: In all the evaluated treatments, the motility of sperm decreased over time (0 to 24 hours after cooling); however, the treatments with Andromed®, and Andromed®+HTF maintained a high motility. Findings/Conclusions: The combination of Andromed® and HTF can be used to dilute both fresh and cooled semen.

Ability of Melon (Cucumis melo. L) Fruit Juices Based Tris Diluent on The Quality of Frozen Spermatozoa of Bali Cattle After Thawing

The high potassium content in melon plays a role in maintaining the normal function of the muscles, heart, nervous system, and potassium is also the main regulator of blood pressure. The aim of this study was to evaluate the effect of using melon juice (Cucumis melo L.) as a diluent on the quality of spermatozoa and semen of Bali cattle both before and after freezing. The Banyumulek Artificial Insemination Center produces sperm used in Bali cattle breeding. The technique used was a completely randomized design (CRD) with 4 treatments and 15 replications. The first treatment was 100% egg yolk tris buffer without the addition of melon juice (P0), the second treatment was 80% buffer without the addition of 20% melon juice (P1), the third treatment was 60% buffer with the addition of 40% melon juice (P2). The fourth treatment was 40% buffer with the addition of 60% melon juice (P3). The outcomes showed that the worth of flawless plasma film and mortality upsides of ox-like semen after weakening and subsequent to defrosting were not essentially unique. Be that as it may, not altogether disparate in that frame of mind of motility, level of reasonability, level of ordinariness and post-defrosting anomalies. According to the findings of this study, melon juice can be used as an alternative extender for fresh Bali cattle sperm and preserve the quality of spermatozoa.

Comparative iTRAQ proteomics identified proteins in fresh and frozen thawed yak spermatozoa

The changes in semen and cryodamage after the cryopreservation process negatively affect sperm function and motility. However, possible proteomic alterations of yak semen during cryopreservation have not yet been achieved. In this study, we compared proteomes of fresh and frozen thawed yak sperm using iTRAQ combined with LC-MS/MS proteome approach. Totally, 2064 proteins were quantitatively identified, including 161 in fresh sperm that showed significant differences compared to frozen thawed sperm. According to the Gene ontology (GO) enrichment analysis, differentially expressed proteins (DEPs) are predominantly associated with spermatogenesis, tricarboxylic acid cycle, ATP synthesis, and differentiation biological process. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEPs were mainly involved in metabolic pathways related to pyruvate metabolism, carbon metabolism, glycolysis/gluconeogenesis, together with the citrate (TCA) cycle. In the analysis of the protein-protein interaction (PPI) network, 15 potential proteins (PDHB, DLAT, PDHA2, PGK1, TP5C1, etc.) that could be related to the sperm quality of the yaks were obtained. Furthermore, 6 DEPs were validated by parallel reaction monitoring (PRM), confirming that the iTRAQ data were reliable. These results indicate that cryopreservation alters the proteome of yak sperm, which is possibly related to cryodamage and fertilization ability.

Clinical and neonatal outcomes of using a modified micro cryotube for cryopreservation of small numbers of spermatozoa for TESA-ICSI cycles

Cryopreservation of a small number of human spermatozoa is still a major challenge for embryologists. The aim of this study was to evaluate the clinical pregnancy and neonatal outcomes of intracytoplasmic sperm injection (ICSI) using a modified micro cryotube as freezing carrier for freezing small numbers of human spermatozoa collected by testicular sperm aspiration (TESA). We conducted a retrospective study to analyses the ICSI outcomes of using frozen-thawed few testicular spermatozoa in males with obstructive azoospermia (OA) from June 2017 to June 2021. Of 155 ICSI treatment cycles, 79 cycles were allocated to frozen sperm group and a modified micro cryotube was used for freezing testicular sperm, 76 cycles were allocated as fresh sperm group. No significant differences were observed in fertilization rate, good quality embryo rate, and blastocyst rate between the frozen sperm group and fresh sperm group (P > 0.05). Similarly, in the fresh embryo transfer cycles plus the first frozen-thawed embryo transfer cycles, the total clinical pregnancy rate (54.43% vs 57.89%), implantation rate (46.08% vs 49.47%), miscarriage rate (13.95% vs 13.64%) and live birth rate (45.57% vs 48.68%) were not statistically different between the frozen and fresh sperm groups (P > 0.05). In addition, there was no statistical differences in the mean gestational age (38.33weeks ± 1.74 vs 37.89weeks ± 1.87), preterm delivery rate (5.56% vs 10.81%), mean birth weight at delivery (3026.50 g ± 577.64 vs 2977.56 g ± 528.93), and low birth weight (12.50% vs 19.51%) between the two groups (P > 0.05 in all cases). Modified micro cryotube for cryopreservation of rare testicula rretrieved spermatozoa did not negatively affect the pregnancy and neonatal outcomes in TESA-ICSI cycles. The presented method may be a useful alternative for cryopreservation of small numbers of human spermatozoa in clinical setting.

P-057 Does sperm cryopreservation affect the clinical outcome? A retrospective study in an oocyte donation program

Abstract Study question Does sperm cryopreservation have a negative impact on pregnancy and live birth rate (LBR)? Summary answer The use of frozen sperm does not affect pregnancy rates and LBR significantly compared to fresh sperm. What is known already The use of frozen sperm, especially in oocyte donation cycles, is a common strategy in organizing a couple’s treatment. By now, contradictory data has been published concerning the impact of fresh sperm versus frozen on pregnancy and LBR. There is data indicating reduction of the LBR with the use of frozen sperm in a donation program, while in other studies no significant difference between fresh and frozen sperm was observed in the LBR. Study design, size, duration This is a retrospective cohort study conducted in Embryolab Fertility Clinic, Greece, from January 2019 to March 2020. Only oocyte donation cycles were included in the study. On the day of donor's oocyte retrieval ICSI was performed, either with fresh (group A) or frozen sperm (group B)All embryos were cultured to blastocyst stage, assessed for their development, vitrified and two were transferred post warming on a subsequent HRT cycle. Participants/materials, setting, methods For the present study, 135 cases were analyzed: in 28 cases fresh sperm was used, while frozen sperm was used in 107 cases[pd1] . The mean men’s age was 44.4 years, while all oocyte-donors were below 35 years old. The main outcome measure was LBR, while fertilization rate, blastulation rate as well as positive β-HCG were also analyzed. All patients with uterine factor, severe male factor or surgically obtained sperm were excluded from the study. Main results and the role of chance There was no significant difference between LBR of fresh vs frozen sperm (53.57% ±50.78 vs 50.47% ±50.23, p=0.086). Although positive β-CG reached a higher percentage in fresh sperm samples, there was no significant difference between two groups (85.71% ±35.63 in fresh vs 77.78% ±41.76 in frozen sperm, p=0.857). Similarly, both blastulation rate (68.93% ±25.22 in fresh vs 67.97% ± 28.72 in frozen sperm, p=0.873) and fertilization rate (85.36% ±16.92 in fresh vs 83.27% ±17.01 in frozen sperm, p=0.565) did not differ significantly between fresh and frozen sperm samples. [pd1] Regression analysis adjusting for relevant confounders (endometrial thickness the day of the embryo transfer, BMI, donor’s age) showed no association between LBR and sperm type (fresh vs cryopreserved). Limitations, reasons for caution This is a retrospective study, including sperm samples irrespectively to quality parameters (sperm count, motility, morphology). The number of previous treatments per couple was not included in the study. Wider implications of the findings The outcome of the study indicates that the use of frozen sperm is a viable choice in a donation program, as it does not induce any negative effects in LBR. However, specific subgroups might benefit from fresh sperm especially when the sperm parameters are low. Trial registration number N/A

P-711 Are there any differences among blastocyst euploid and mosaicism rate (high or low) using fresh or frozen ejaculate sperm in donated oocytes?

Abstract Study question To compare euploid and mosaicism rate (high or low) among blastocysts derived from fresh and frozen ejaculate sperm in donated oocytes. Summary answer No statistically significant differences were found on blastocyst euploid and mosaicism rate between blastocysts derived from fresh versus frozen. What is known already Even though 5%-10% autosomal aneuploidies and 5%-100% sex chromosomes aneuploidies are paternally derived, the clinical relevance of paternal contribution to chromosome aneuploidy is frequently overlooked. Besides, male infertility could contribute to the generation of chromosomal abnormalities in the resulting embryos. Sperm cryopreservation is a widely used technique in assisted reproduction for different reasons. However, little is known regarding the potential effect of sperm freezing on embryo aneuploidies. Study design, size, duration Retrospective, observational and multicenter study of patients undergoing Preimplantation Genetic Testing for Aneuploidies (PGT-A) from January 2017 to December 2022 in IVIRMA Madrid, Valencia and Barcelona. To elucidate the effect of advance maternal age, 1,780 biopsied blastocysts coming from the egg donation program were analyzed. Autologous sperm samples with less than 2 million spermatozoa per milliliter and male partners with altered karyotype, abnormal spermatozoa fish or any genetic disease were excluded from the study. Participants/materials, setting, methods Embryos were cultured in regular incubators, with an atmospheric concentration of 5% O2, 6.5% CO2 and a temperature of 37ºC. Blastocyst biopsy was performed either on day 5 or day 6 of embryo development and analyzed through Next Generation Sequencing (NGS). Sperm freezing was done following manufacturer’s instructions (Sperm freeze, Origio). Continuous variables were compared with a student´s t-test and categorical variables with a Chi-square using SPSS statistical program. Main results and the role of chance Mean paternal age (44.3±5.20 years old vs 43.2±5.20 years old), sperm concentration (46.67±33.05 mill/ml vs 35.1±32.92 mill/ml) were not significantly different among fresh versus frozen sperm group (p &amp;gt; 0.05). A total 1780 blastocysts tested for PGT-A were included: 1267 blastocysts coming from fresh ejaculate sperm (n = 194 patients) and 513 blastocysts from frozen sperm (n = 81 patients). No statistically significant differences were found in blastocyst euploid rate between fresh versus frozen ejaculate sperm (64.5% versus 67.2%, p = 0.278 ;) respectively. Blastocyst mosaicism rate: High mosaicism (&amp;gt;50% of aneuploidy trophectoderm cells) and low mosaicism (30-50% of aneuploidy trophectoderm cells) was comparable between fresh versus frozen sperm (2.7% versus 4.5%; p = 0.90) and (3.9% versus 3.7%), respectively. Limitations, reasons for caution This study is based on a retrospective data. Wider implications of the findings According to our findings, there are no statistically significant differences in the blastocyst euploid and mosaicism rate between fresh and frozen ejaculate sperm. 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: &amp;lt;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

P-223 Comparison of blastocysts’ iDAScore® (intelligent Data Analysis-Score) if in vitro fertilization is performed using ICSI instead of IMSI

Abstract Study question Does blastocysts’ iDAScore® differs when ICSI is performed instead of IMSI in case of teratozoospermia on the day of in vitro fertilization (D0)? Summary answer Blastocysts’ iDAScore® is unchanged when in ICSI or IMSI is performed in case of teratozoospermia at the day of fertilization (D0). What is known already In routine practice, IMSI for D0-teratozoospermia is sometimes not performed because of its higher cost and/or laboratory workload. Time-Lapse studies have shown that in vitro fertilization (IVF) using ICSI or IMSI in case of suboptimal sperm parameters lead to morphokinetically similar embryos. However, in patients with oligoasthenoteratozoospermia/teratozoospermia, evidence indicate that IMSI may improve embryo morphokinetics and further clinical outcomes. Since these studies rely on morphokinetical features assessed by an embryologist, the use of artificial intelligence may limit the human subjectivity for choosing the optimal blastocyst for transfer. Whether ICSI or IMSI for D0-teratozoospermia impact blastocysts’ iDAScore® has never been investigated. Study design, size, duration This retrospective monocentric study was performed between September 2021 and December 2022 in our reproductive medicine unit. A total of 605 oocytes (386 ICSI, 219 IMSI) from 58 patients (37 ICSI, 21 IMSI) were fertilized with fresh sperm using either ICSI or IMSI and then incubated for 5 days in Embryoscope8 (Vitrolife®) combined with iDAScore®. iDAScores® of 325 blastocysts (228 ICSI, 97 IMSI) were compared between IMSI and ICSI arms. Participants/materials, setting, methods For each couple 3 iDAScores® were available: i) mean iDAScore® of all blastocysts, ii) iDAScore® max, and iii) mean iDAScore® of useful blastocysts. These scores were compared according to the technique of IVF used (ICSI or IMSI). In addition, clinical pregnancy rates following blastocyst transfer were analyzed. In all, 53 blastocyst transfers (34 ICSI, 19 IMSI) have been realized during fresh or frozen cycles. Clinical pregnancy rates were defined by presence of fetal heartbeat on ultrasound. Main results and the role of chance Patients in the ICSI and IMSI groups were comparable in terms of women age (32.3 ± 5.6 and 32.2 ± 5.6 years), BMI (24.9 ± 4.3 and 25 ± 4.3 Kg/m2, respectively). In addition, mean IVF rank was 1.4 ± 1.5 and 2.8 ± 1.5 in ICSI and IMSI couples. The number of mature oocytes inseminated per patient was similar in ICSI and IMSI groups (10.9 ± 4.7 and 10.4 ± 4.6, respectively), as well as the mean number of blastocysts per couple (6.4 ± 3.5 and 4.6 ± 3.5) and the blastoformation rate (0.74 ± 0.2 and 0.66 ± 0.2, respectively). The mean iDAScore® of all blastocysts, iDAScore® max and mean iDAScore® of useful blastocysts in women having undergone ICSI and IMSI were comparable (7.60 ± 1.2 vs. 7.63 ± 1.1 (p = 0.92); 8.80 ± 1.1 vs. 8.78 ± 1.0 (p = 0.67) and 8.1 ± 1.0 vs.7.96 ± 1 (p = 0.77) respectively). Finally, cumulated pregnancy rates (CPR) were not different between ICSI and IMSI groups (22/34 and 10/18 respectively), (0.65 ± 0.49 vs. 0.55 ± 0.46, respectively (p = 0.53)). Limitations, reasons for caution These results should be interpreted with caution due to the retrospective design of the study and small population. Wider implications of the findings We observed no difference in iDAScores whether ICSI or IMSI is performed in case of D0-teratozoospermia. Since IMSI is more time-consuming and more expensive, if these results are confirmed by further studies, we may withdraw this indication of IMSI to avoid patients bear additional fees, especially in private clinics. Trial registration number not applicable

P-022 The ICSI Outcome of Frozen Thawed Testicular Sperm Utilization in Azoospermic Men with AZFc Microdeletion

Abstract Study question What is the efficacy of frozen thawed surgically retrieved spermatozoa in azoospermic men with AZFc microdeletion undergoing Introcytoplasmic sperm injection (ICSI)? Summary answer The comparison between frozen thaw to fresh testicular sperm did not show statistically significant difference in terms of fertilization, blastocyst development and pregnancy rates. What is known already In male infertility, Y chromosome microdeletion (YCM) may be the cause up to 10% of men with oligozoospermia or azoospermia. AZFc region deletion was mostly detected in YCM. In men with azoospermia and AZFc deletion spermatozoa can be retrieved via testicular sperm extraction (TESE). Today, couples suffering azoospermia due to AZFc deletion may have genetically own child by using testicular sperm with ICSI. Because of the rare incidence of AZFc deletion, the outcome of ICSI cycles in which frozen thawed spermatozoa utilized for assisted conception has not been widely known. Study design, size, duration The data of 611 men with non-obstructive azoospermia (NOA) who underwent microTESE surgery between 2014 and 2022 was retrospectively analyzed. Assisted reproduction treatment (ART) outcome of couples whose men with AZFc deletion was further analyzed according to the fresh or frozen thawed spermatozoa usage for ICSI. Participants/materials, setting, methods All of the patients were genetically screened with karyotype and Y chromosome microdeletion analysis and microTESE operation was performed for testicular sperm recovery. The patients who diagnosed AZFc deletion and had successful sperm recovery divided to two groups according to cryopreserved or fresh sperm utilization for ICSI. The spouse and men age, fertilization rate, blastocyst development, pregnancy and live birth rate was compared between the groups. Main results and the role of chance Twenty-seven (4.4%) men were diagnosed YMC deletion in total of 611 men with NOA who underwent microTESE. Spermatozoa was successfully retrieved in 14 men (51.8%) men who were diagnosed AZFc deletion. The mean age of women and men were 30.5 (3.5) and 32.8 (4.1) years, respectively. Oocyte retrieval and ICSI were accomplished in 18 cycles. In 7 (38.8%) cycles fresh testicular spermatozoa and in 11(61.2%) cycles frozen thawed testicular spermatozoa were used. In one cycle, embryo transfer was cancelled due to total fertilization failure. In total, fertilization rate as 2PN/M2 oocytes was 57% (119/206). In 5 cycles cleavage stage embryos were transferred whereas in remaining 13 cycles blastocysts were transferred. Blastocyst development rate as day 5 blastocyst/2PN was 45.2% (42/95). Comparison of fertilization and blastocyst development rates according to fresh TESE spermatozoa and frozen thawed TESE spermatozoa groups were 63.2% vs 54.4% and 50.0% vs 38.7% respectively (p &amp;gt; 0.5 for both). Clinical and live birth rates per embryo transfer cycle were 57.1% vs 40.0% and 42.8% vs 30.0% respectively (p &amp;gt; 0.5 for both). Limitations, reasons for caution Fresh and frozen thawed groups demonstrated no statistical difference in terms of ICSI outcome. However, relatively reduced fertilization and blastocyst development rates should be interpreted cautiously because beta type error. Further data is needed to clarify this difference. Wider implications of the findings The best of our knowledge our study is the largest in literature comparing frozen thawed and fresh sperm utilization for ICSI in men with AZFc deletion. This data may help for physicians and couples to give decision diagnostic microTESE surgery and cryopreservation of sperm further ICSI treatment. Trial registration number Not applicable

P-080 Enzymatic tissue processing after testicular biopsy in non-obstructive azoospermia enhances sperm retrieval and cumulative live birth rates

Abstract Study question What is the added value of enzymatic processing of testicular biopsies on testicular sperm retrieval rates in patients with non-obstructive azoospermia (NOA)? Summary answer In addition to mechanical mincing, enzymatic digestion increased sperm retrieval rates in testicular biopsies of NOA patients with 48.9%. What is known already Many studies focus on the surgical approach to optimize recovery of testicular sperm in NOA, and in spite of that, there is still controversy whether the type of surgery makes any difference as long as multiple biopsies are taken. Few studies, however, focus on the role of the IVF laboratory and the benefit of additional lab procedures, like enzymatic digestion, in order to optimize sperm retrieval rates and CLB rate per TESE. Study design, size, duration A retrospective single-center cohort study including all patients who underwent their first TESE by open multiple-biopsy method from January 2004 till July 2022. Only patients with a normal karyotype, absence of Y-q deletions and a strict diagnosis of NOA based on histology were included. Primary outcome was sperm retrieval after mincing or enzymes for intracytoplasmic sperm injection (ICSI). Secondary outcome was CLB after ICSI with fresh TESE, and subsequent ICSI cycles with frozen TESE. Participants/materials, setting, methods Multiple biopsies were obtained from the testis, unilateral or bilateral, on the day of oocyte retrieval. Upon mechanical mincing, dishes were searched for 30 min; when no or insufficient numbers of spermatozoa were observed, enzymatic treatment was performed using collagenase type IV. Multivariable regression analysis was performed to predict CLB by adjusting for the following confounding factors: male age, male FSH level, cryptorchidism, enzymatic digestion, number of oocytes and female age. Main results and the role of chance Hundred-eighteen patients were included of which 61.0% had successful sperm retrieval. Spermatozoa were retrieved after mechanical mincing (23.7%; 28/118) or after additional enzymatic digestion of the remaining 90 patients (48.9%; 44/90). Mean male characteristics were not different between patients with sperm retrieval after mincing or enzymes: age (34.5 vs 34.5 y), testis volume (10.2 vs 10.6 ml), FSH (17.8 vs 16.9 IU/l), cryptorchidism (21.4 vs 34.1%) and histological diagnosis (Sertoli Cell Only 53.6 vs 47.7%, maturation arrest 21.4 vs 38.6%, sclerosis/atrophy 25.0 vs 13.6%), respectively. Of the 72 patients with sperm available for ICSI, 23/72 (31.9%) obtained a LB after the injection with fresh testicular sperm (fresh and frozen embryo transfers). Forty-nine patients remained without LB, of which 34 had supernumerary testicular sperm frozen. Of these, 9/47 (47.4%) had a LB after ICSI with frozen testicular sperm, giving rise to a total CLB per TESE of 32/118 (27.1%) or 32/72 (44.4%) CLB per TESE with sperm. Of the female characteristics (couples with sperm available), only female age (30.3 vs 32.7 y – p = 0,042) was significantly lower in the group with a live birth. Multivariable logistic regression analysis showed that enzymatic digestion was associated with significant decrease of CLB per TESE. Limitations, reasons for caution Limitations of the study are related to the retrospective design. The selection of only NOA patients with specific characteristics (normal karyotype and absence Y-q deletion) and having their first TESE ever strengthens our findings. Whether enzymatic digestion after a failed TESE without digestion may improve retrieval rate remains undecided. Wider implications of the findings Enzymatic processing increases the sperm retrieval rate from testicular biopsies of NOA patients compared to mechanical mincing, demonstrating the importance of an appropriate laboratory protocol. NOA patients should be counselled that if sperm has been found after enzymatic digestion, their chances to father a genetically own child will be lower. Trial registration number not applicable

P-239 Comparison of reproductive outcomes for cleavage- and blastocyst-stage frozen embryo transfer: A retrospective study

Abstract Study question Does blastocyst-stage (day 5-6) embryo transfer improve pregnancy and live birth rates per frozen transfer compared to cleavage-stage (day 2-3) embryo transfer? Summary answer Pregnancy rate is significantly higher following frozen blastocyst-stage embryo transfer, while live birth rate does not differ. What is known already The extended culture of cleavage-stage (day 2-3) embryos to blastocyst-stage (day 5-6) embryos has been largely adopted in recent years. In fact, blastocyst culture may allow a better selection of the embryo to be transferred based on its developmental history and morphological criteria, and result in a lower number of exceeded embryos to be frozen for storage. However, the literature comparing pregnancy and live birth rates for cleavage- and blastocyst-stage transfer remains contradictory, and the benefits of frozen blastocyst transfer is still under discussion. Study design, size, duration This retrospective study investigated couples having frozen embryo transfer at the cleavage (days 2 or 3) or the blastocyst-stage (days 5 or 6) at our clinic between 2014–2021. Results from more cycles were analyzed individually. We excluded cycles based on fresh embryo transfer, oocyte or sperm donation. Cycles were further sub-classified based on the type of treatment, as natural or stimulated, and the pregnancy and live birth rates were compared. Participants/materials, setting, methods Data was obtained from patient records at our fertility clinic and sorted by two authors. Variables included female age, treatment plan, the stage of the embryo transferred, and the report of pregnancy and live birth. Statistical analysis was conducted by using MedCalc Software, and Chi-squared test applied to compare the outcomes’ frequency based on the day of frozen embryo transfer. P &amp;lt; 0.05 was considered significant. Main results and the role of chance A total of 2,922 cycles were included for analysis, with age of female patients being 37.2 ± 5.0 years at the time of transfer. Most cycles were stimulated (n = 2,217, 75.9%) compared to natural cycles (n = 705, 24.1%). In all cycles, IVF was used for fertilization. Approximately one third of the cycles (n = 954, 32.6%) underwent frozen embryo transfer at the cleavage-stage (days 2 or 3), while the remaining had transfer at the blastocyst-stage (n = 1,968, 67.4%). Pregnancy rate was significantly (P &amp;lt; 0.0001) higher for blastocyst-stage embryo transfer compared to cleavage-stage embryo transfer (36.2% vs 20.4%, respectively) when all cycles were analyzed. This significance was found when natural (23.3% vs 14.8%, P = 0.0052) or stimulated cycles (38.0% vs 25.8%, P &amp;lt; 0.0001) were sub-analyzed. While we observed 19.0% of successful live birth per embryo transferred in the entire cohort, this did not significantly vary depending on the stage of frozen embryo transferred (cleavage or blastocyst-stage, 13.6% vs 21.5%, respectively) and when patients were sub-classified based on the type of treatment chosen in natural (9.0% vs 14.2% respectively) or stimulated (18.0% vs 22.6%, respectively) cycles. With a large sample size and a very significant P-value for pregnancy rate, the role of chance is limited. Limitations, reasons for caution As a retrospective study, the non-random allocation to treatment groups might hide selection bias and limit the statistical power of the data analysis, especially for the live birth rate. Also, no adjustment for confounders was performed. Wider implications of the findings Frozen embryo transfer at blastocyst-stage may offer an improved chance of pregnancy in both natural and stimulated cycles, possibly resulting in less embryos stored and lower costs for patient treatment. Although no difference was observed, larger prospective studies are needed to clarify the impact on live birth rate. Trial registration number Not applicable

P-098 Cryopreservation process deregulates the expression of pathways important for fertility in human spermatozoa

Abstract Study question Does cryopreservation impact on transcriptome of conventional frozen human spermatozoa? Summary answer The cryopreservation of human sperm alters pathways important for fertility, although the upregulation of some genes can compensate for the harmful effects of freezing. What is known already Sperm cryopreservation is a widely used procedure for storing gametes for later use, for preserving fertility in patients before gonadotoxic treatments or surgery, and for sperm donation programs. The cryopreservation technique can cause damage to sperm through induction of DNA alterations, fragmentation and oxidation, and altered motility, mitochondrial activity and morphology. Transcriptomic alterations have been reported in cryopreserved sperm from various animal species, but no experimental data are available on the effect of cryopreservation on the transcriptome-wide profile of human sperm. Study design, size, duration Semen samples were obtained from 20 normospermic men from April to May 2022. Each sample was divided in two aliquots. From one aliquot total RNA was immediately extracted. The second aliquot was slowly cryopreserved and after a week of storage in liquid nitrogen total RNA was extracted. A total of 13 paired RNA samples passed quality control and were analyzed after randomization into 4 pools, each of 6 patients. Participants/materials, setting, methods The men who participated in this study had a median age of 35.0 years (range: 29.0-46.0). RNA was extracted by miRNeasy Micro Kit (Qiagen), analyzed by High Sensitivity RNA on 2200 Tape Station system (Agilent), amplified by Ovation Pico WTA SystemV2 (Nugen), labeled and hybridized on GE 4x44K v2 microarrays (Agilent). The paired Significance Analysis of Microarray (SAM) was performed using the limma hgug4112a.db and samr packages in R/BioConductor. Main results and the role of chance The expression profiles of cryopreserved sperm significantly differed from those of fresh sperm in 219 down-regulated and 28 up-regulated unique transcripts. The gene ontology analysis disclosed that cryopreservation downregulates genes involved in sperm motility by the mitochondria function (CABS1, SPATA19) and the correct organization of the sperm midpiece and flagellum cytoskeleton (ACTL7A, AKAP4, C9ORF24, CAPZB, CCIN, IQCG, ODF2, SPATA6, SPATA6L, TBC1D21), in fertilization (ACTL7A, AKAP4, PLCZ1, PRSS37, TUBGCP3), early embryo development (AGT, CLMN, DCC, DHRS3, EGFLAM, FAM20A, FREM1, GPI, HEMGN, KRTDAP, IFT172, MICAL2, MLF1, NF2, PGRMC2, PHACTR1, POPDC3, RO60, ROBO1, RORA, SGCA, SPRR2D, TCF4, TNC, TNNI3), oxidant detoxification (TXNDC29) and DNA repair (BRIP1, ERCC6, TRIP12), calcium ion binding and homeostasis (AMY1C, ANO1, ANO2, ASGR1, CABS1, CPNE9, FREM1, KCNIP2, ITPR3, PKD2L1, PLCZ1, SELENOK, SYN3, TNNI3). Upregulated genes were enriched in pathways related to the negative regulation of DNA damage response (ALOX15B, CD74, RPL10, SNAI1, THBS1), cellular response to calcium ion (ALOX15B, FOSB, HLA-DQB1, S100A4, THBS1), regulation of transcription (CD74, FOSB, HLA-DRB1, MAMLD1, RASD1, RPL10, SNAI1, TCF15, TLE1), protein stabilization (CD74), protein ubiquitination (BAG1, DCAF6, ERCC6, HERPUD2, KLHL7, MARCHF8, NF2, PSMA6, RNF133, SERGEF, TRIP12, UBE2DNL, UBL3, UBQLN3, ZNRF3) and ubiquitin protein ligase binding (GPI, HSPA1L, PRKAR2A, STX8). Limitations, reasons for caution Data validation in a larger sample cohort and by quantitative PCR would be useful, although we have applied stringent criteria for gene selection (FDR = 0). Further research should be undertaken to experimentally test whether the length of cryostorage can have any effect on the gene expression profile of sperm. Wider implications of the findings Specific pathways are deregulated by cryopreservation, in accordance with altered sperm morphology, motility and molecular integrity reported in literature. Functional enrichment analyses also revealed gene expression changes to compensate for the sperm cryoinjury. This finding is noteworthy for safety issue of sperm banking i.e., fertility preservation, gamete donation. Trial registration number not applicable

Association of A51703G and G51656T loci polymorphism in luteinizing hormone receptor (LHR) gene with sperm traits in Holstein bulls

Today, there is an increasing desire to use Candidate genes as molecular markers to improve reproductive traits. The luteinizing hormone receptor is expressed on the Leydig cells and plays a crucial role in gametogenesis initiation and maintenance. This study aimed to study the effects of luteinizing hormone receptor (LHR) gene polymorphisms (A51703G and G51656T) on bull sperm traits. Genomic DNA was extracted from the semen, blood, and samples of 150 AI Iranian Holstein bulls according to the established protocol [10] with minor modifications. Semen traits were routinely collected at bull semen collection centers from each animal between 1992 and 2015. The bull fertility traits include semen concentration (SC 108 per ml) sperm volume (SV ml), fresh sperm motility (FSM %), and post-tallow sperm motility (PTSM %). The mutation was detected with A PCR-RFLP method. The digestion of amplicons was also carried out with BfaI and HinfI restriction enzymes in A51703G and G51656T loci, respectively. The restriction profiles of PCR products indicated two alleles in each locus (A and G for A51703G and G and T for G51656T). The frequency of the A and T alleles were 0.6647 and 0.606, respectively. Analysis of variance showed that LHR loci had a significant effect on sperm characteristics in Iranian Holstein bulls (P < 0.05). Bulls with GG genotype surpassed those with GA and AA genotypes regarding SP, FSM, and PTSM in the A51703G locus. In the G51656T locus, the association result showed that bulls with AG genotype had higher than others in all traits (P < 0.05). In conclusion, these data suggest that LHR polymorphisms are an excellent genetic marker for breeding programs in male reproductive traits in the Iranian bull population.

Activation of caspase-3/7, an apoptotic-related marker, during incubation and cryopreservation of alpaca (Vicugna pacos) spermatozoa.

Caspases are crucial mediators of programmed cell death (apoptosis). Apoptosis can occur in spermatozoa during spermatogenesis or epididymal transit, as well as in ejaculated spermatozoa. A high proportion of apoptotic sperm would be a poor indicator of the freezability of a raw seminal sample. Alpaca spermatozoa are notoriously difficult to freeze successfully. Therefore, the objectives of this study were to study caspase activation during incubation (37°C) of fresh alpaca spermatozoa, as well as before and after cryopreservation, to gain some insight into the mechanisms behind the vulnerability of alpaca spermatozoa. Eleven sperm samples were incubated for 4 h at 37°C (Study 1), and 23 samples were frozen using an automated system (Study 2). Caspase-3/7 activation was assessed at 0,1,2,3, and 4 h in samples incubated at 37°C (Study 1); and before/after cryopreservation (Study 2) using CellEvent™ Caspase 3/7 Green Detection Reagent and flow cytometry. The proportions of alpaca spermatozoa with caspase-3/7 activated increased (p < 0.05) after 3-4 h of incubation at 37°C; however, caspase activation was similar before and after cryopreservation (36.2 ± 11.2% vs. 36.6 ± 33.7%, p > 0.05). The high standard deviation found after freezing could be explained by the existence of two subpopulations: one subpopulation where caspase-3/7 activation decreased during cryopreservation (from 36.6 ± 9.1% to 1.5 ± 2.2%), and the other subpopulation where caspase-3/7 activation increased after cryopreservation (from 37.7 ± 13.0% to 64.3 ± 16.7%). In conclusion, after 3-4 h of incubation, caspase-3/7 activation increased in fresh alpaca sperm, whereas cryopreservation affects alpaca sperm samples in different ways.

Evaluation of simple strategies to reduce the use of antibiotics in equine fresh sperm

According to the World Health Organization in 2021, the development of antimicrobial resistances is considered one of the top ten global public health threats. Microbial load negatively affects sperm quality and preservation (Ortega-Ferrusola and Peña, Reproduction in Domestic Animals. 2009; 44(3):518-522), which implies the use of antibiotics in semen extenders. The objective of this study was to study the effect of three semen processing techniques on quality and microbial load of fresh equine sperm to evaluate the possibility of reducing or eliminating the prophylactic use of antibiotics in the future. Ten adult stallions were included (one ejaculate per male). To avoid environmental contamination during sperm collection and processing, a protocol that maximizes hygiene measures was implemented. Aliquots of each ejaculate were processed following 4 protocols: Simple Centrifugation (450g x 7 min) in extender with antibiotics (SC+, as control group), Simple Centrifugation in extender without antibiotics (SC-), Single-Layer Colloidal Centrifugation (300g x 20 min) in extender without antibiotic (CC-) and Filtration (SpermFilter®) in extender without antibiotics (F-). Sperm motility (total and progressive motility, TM and PM, respectively, %), viability (Alives, %), and microbial load (on three different culture media (Columbia 5% Sheep Blood Agar, COS, general purpose medium for non-fastidious and fastidiousmicroorganisms, Sabouraud Dextrose Agar, SDA, for yeasts and molds, and Schaedler vitamin K1 5% Sheep Blood Agar, SCH, for fastidious anaerobic bacteria) were evaluated after processing. Statistical analysis was performed by Kruskal-Wallis, ANOVA and post hoc tests (p<0.05). Results are expressed as mean values ± SD. No significant differences were found between the control (SC+) and other groups (SC-, CC- and F-), neither in sperm characteristics (61,32a ± 20,84, 59,13a ± 21,84, 63,19a ± 18,87 and 52,54a ± 21,08 for PM and 84,60a ± 9,21, 85,05a ± 9,14, 84,90a ± 13,5 and 77,35a ± 13,27 for ALIVES respectively) nor in microbial load (216,50a ± 504,41, 348,30a ± 792,99, 731,30a ± 2180,11 and 51,70a ± 64,64 for COS; 1,00a ± 3,16, 11,00a ± 31,43, 4,00a ± 6,99 and 1,00a ± 3,16 for SDA and 25,00a ± 57,59, 41,00a ± 60,45, 10,00a ± 18,26 and 26,00a ± 42,48 for SCH respectively). The present processing methods, in combination with management measures that prevent environmental contamination, maintained sperm quality and microbial load of equine fresh semen to the same extent as traditional sperm processing protocols with antibiotics. Acknowledgements: Authors thank P. Cuesta for statistical assistance. This research was funded by Madrid Institute for Rural, Agricultural and Food Research and Development (IMIDRA).

Evaluation of long-term cryostorage on sperm function and fertility of brown-marbled grouper (Epinephelus fuscoguttatus)

Cryopreservation of fish semen is a valuable technique for reproductive management in aquaculture. The aim of this study was to evaluate the effect of long-term storage in liquid nitrogen on post-thaw sperm functional parameters of brown-marbled grouper (Epinephelus fuscoguttatus). Samples were cryopreserved in a simple freezing medium (10 % DMSO + 0.3 M Glucose), sperm quality parameters were assessed at different storage periods (one month, one year, 2 years and 3 years). Our results indicated that cryostorage periods had a significant effect on the quality of frozen-thawed spermatozoa. Compared with fresh semen, cryopreserved sample was characterized by an overall decline in motility, viability, mitochondrial membrane potential (MMP), ATP content and increased level of lipid peroxidation with the storage time going on. For the analyzed quality parameters, there were no significant differences in storage time cryopreserved over one month. After 3 years of storage, frozen sperm still showed relatively high total motility, viability and MMP (all over 80 %), yielding acceptable fertilizing capacity close to that of fresh sperm. Therefore, it can be concluded that brown-marbled grouper sperm could be effectively cryopreserved for at least 3 years. The results of this study would be helpful to optimize gamete management in routine hatchery practices.

The impact of cryopreserved sperm on intrauterine insemination outcomes: is frozen as good as fresh?

Frozen sperm utilization might negatively impact cycle outcomes in animals, implicating cryopreservation-induced sperm damage. However, in vitro fertilization and intrauterine insemination (IUI) in human studies are inconclusive. This study is a retrospective review of 5,335 IUI [± ovarian stimulation (OS)] cycles from a large academic fertility center. Cycles were stratified based on the utilization of frozen (FROZEN, n = 1,871) instead of fresh ejaculated sperm (FRESH, n = 3,464). Main outcomes included human chorionic gonadotropin (HCG) positivity, clinical pregnancy (CP), and spontaneous abortion (SAB) rates. Secondary outcome was live birth (LB) rate. Odds ratios (OR) for all outcomes were calculated utilizing logistic regression and adjusted (adjOR) for maternal age, day-3 FSH, and OS regimen. Stratified analysis was performed based on OS subtype [gonadotropins; oral medications (OM): clomiphene citrate and letrozole; and unstimulated/natural]. Time to pregnancy and cumulative pregnancy rates were also calculated. Further subanalyses were performed limited to either the first cycle only or to the partner's sperm only, after excluding female factor infertility, and after stratification by female age (<30, 30-35, and >35 years old). Overall, HCG positivity and CP were lower in the FROZEN compared to the FRESH group (12.2% vs. 15.6%, p < 0.001; 9.4% vs. 13.0%, p < 0.001, respectively), which persisted only among OM cycles after stratification (9.9% vs. 14.2% HCG positivity, p = 0.030; 8.1% vs. 11.8% CP, p = 0.041). Among all cycles, adjOR (95% CI) for HCG positivity and CP were 0.75 (0.56-1.02) and 0.77 (0.57-1.03), respectively, ref: FRESH. In OM cycles, adjOR (95% CI) for HCG positivity [0.55 (0.30-0.99)] and CP [0.49 (0.25-0.95), ref.: FRESH] favored the FRESH group but showed no differences among gonadotropin and natural cycles. SAB odds did not differ between groups among OM and natural cycles but were lower in the FROZEN group among gonadotropin cycles [adjOR (95% CI): 0.13 (0.02-0.98), ref.: FRESH]. There were no differences in CP and SAB in the performed subanalyses (limited to first cycles or partner's sperm only, after excluding female factors, or after stratification according to female age). Nevertheless, time to conception was slightly longer in the FROZEN compared to the FRESH group (3.84 vs. 2.58 cycles, p < 0.001). No significant differences were present in LB and cumulative pregnancy results, other than in the subgroup of natural cycles, where higher LB odds [adjOR (95% CI): 1.08 (1.05-1.12)] and higher cumulative pregnancy rate (34% vs. 15%, p = 0.002) were noted in the FROZEN compared to the FRESH group. Overall, clinical outcomes did not differ significantly between frozen and fresh sperm IUI cycles, although specific subgroups might benefit from fresh sperm utilization.

Effect of cryopreservation on post-thaw motility and viability of Grey mullet, Mugil cephalus sperm (Linnaeus, 1758)

In this study, we optimized a simple method of cryopreservation for Mugil cephalus sperm based on post-thaw motility and viability. A series of experiments were conducted by changing the extender, cryoprotectant and freezing height above the liquid nitrogen (LN) surface. First, we carried out the cryopreservation using the extender V2E and cryoprotective agents (CPAs) namely, propylene glycol (PG), methanol (MeOH), glycerol (GLY), ethylene glycol (EG), dimethylsulfoxide (Me2SO) and dimethylacetamide (DMA) at a final concentration of 5% and 10%. We found that 10% of GLY, EG and Me2SO were more suitable compared to other CPAs. Then, different freezing heights (6, 8, 10 and 12 cm) above the LN surface were experimented with extender V2E and optimized CPAs. Then, 0.3 M of glucose, sucrose and trehalose were tested as extender along with optimized CPAs and freezing height. Additionally, the effect of fast-rate freezing and storage days (7, 30 and 180) on post-thaw sperm quality was documented using the factors optimized in earlier experiments. For all experiments, the fresh sperm was diluted at a ratio of 1:1 with cryomedium (CPA + extender), loaded into cryovials (2.0 mL) and frozen. The cryopreserved sperm was thawed at 30 °C for 90–120 s and their quality was evaluated. Among the experimented factors, sperm diluted in cryomedium (0.3 M glucose + 10% EG) and frozen at 4 cm above the LN surface registered significantly (P < 0.05) highest post-thaw motility (73 ± 2%) and (71 ± 1%) viability. Fast-rate freezing has resulted in lower (about 30%) post-thaw motility and viability of sperm. The storage days (7, 30 and 180) did not have a significant effect on post-thaw sperm quality. Overall results show that using the factors optimized through this study, high-quality sperm can be obtained after cryopreservation.

Cryopreservation modifies the distribution of the prostate-derived lectin SL15 on the llama (Lama glama) sperm.

Lectin-like molecules play a key role in mammalian sperm functionality. These multifunctional proteins have been proven to be involved in sperm capacitation, sperm motility, and viability, formation of the oviductal sperm reservoir, and in sperm-oocyte interaction. In a previous study, we reported the presence of a novel seminal plasma lectin, sperm lectin 15kDa (SL15), adsorbed to the llama sperm. In order to gain knowledge in the understanding of SL15 and its functions, the aims of this study were to (a) elucidate the presence and localization of SL15 in the llama male reproductive tract and sperm, and (b) determine whether the sperm cryopreservation process of cooling and freeze-thawing affects the SL15 levels and distribution on llama sperm. We found that SL15 protein was expressed along the male reproductive system: testis, epididymis, prostate, and bulbourethral glands, being the prostate the main site of SL15 secretion. SL15 was localized on the sperm head, following different localization patterns. In order to understand if sperm cryopreservation induces modifications in the SL15 adsorption pattern, immunocytochemistry and flow cytometry analysis were carried out on fresh, 24h cooled, and frozen-thawed sperm. Both cooled and frozen sperm showed particular SL15 patterns, that were not observed in the freshly ejaculated, indicating loss of SL15. Flow cytometry analysis also exhibited a decrease of SL15 in the cooled sperm (P<0.05), whereas a tendency to decrease was found in frozen-thawed sperm (P<0.1) when compared with freshly ejaculated sperm. This study extends the knowledge about the SL15 in the llama male physiology and provides evidence that cryopreservation-related techniques disrupt SL15 adsorption to the sperm membrane, possibly affecting sperm functionality and fertility.