Fresh Sperm Research Articles

Investigation of the efficacy of different cryoprotectants in the freezing of testicular tissue and epididymal sperm: Spermatological parameters, tissue viability and PARP-1 gene expression

The presented study covers testicular tissue and epididymal spermatozoa cryopreservation processes in bulls and aims to investigate the effects of these applications on spermatological parameters, cell viability in testicular tissue, and the expression of the PARP-1 gene, a DNA repair enzyme. Testes of 20 bulls over 2 years old, slaughtered in a slaughterhouse, were used in the study. After spermatological evaluations, the semen obtained from the cauda epididymis was frozen in liquid nitrogen vapor according to the straw method and stored in liquid nitrogen (−196 °C). Testicular tissue pieces obtained from the testicles were frozen by the slow freezing method in cryotubes in diluents containing Dimethylsulfoxide (DMSO) and Ethylene Glycol (EG) cryoprotectants and stored in liquid nitrogen (−196 °C). The total motility (TM) (85.89 ± 12.83 %), progressive motility (PM) (54.02 ± 15.77 %), and kinematic parameter values of fresh sperm were significantly higher compared to the TM (57.62 ± 13.13 %), PM (29.60 ± 10.76 %), and kinematic parameter values after thawing (P < 0.05). Significant decreases in plasma membrane integrity (PMI) and viability and an increase in chromatin condensation and morphological disorders in the head, middle part, and tail regions were observed in post-thaw semen samples (P < 0.05). When the effects of DMSO and EG on cell viability after thaw in frozen testicular tissue were evaluated, it was observed that the cell viability values of testicular tissues frozen with EG (45.70 ± 10.00) were statistically significantly lower than those frozen with DMSO (51.20 ± 7.70) (P < 0.05). When the effects of both cryoprotectants on gene expression in tissue and semen samples were examined, it was determined that gene expression increased on average 0.19 ± 0.27 times in the tissue samples in the DMSO group compared to fresh tissue samples and 0.17 ± 0.19 times in the tissue samples in the EG group. It was determined that gene expression levels increased by an average of 1.20 ± 1.08 times in post-thaw epididymal spermatozoa samples compared to fresh semen samples. The results show that cryopreservation can activate cellular repair mechanisms by stimulating PARP-1 gene expression and affect gene expression by activating specific pathways in tissues and cells.

Pregnancy outcomes in patients with non-obstructive azoospermia undergoing micro-TESE: comparison of fresh vs. frozen-thawed testicular sperm.

The relative merits of fresh or frozen testicular sperm in ICSI remain a matter of contention. This study aims to compare the reproductive outcomes of non-obstructive azoospermia patients undergoing ICSI using fresh and frozen-thawed microdissection testicular sperm extraction (micro-TESE) sperm. A total of 223 men with non-obstructive azoospermia underwent micro-TESE to collect testicular spermatozoa. ICSI cycles were performed using fresh and frozen-thawed spermatozoa. The cleavage states and grading of embryos, fertilization, and pregnancy outcomes were compared between the groups to assess the impact of cryopreservation of testicular spermatozoa on embryo quality and ICSI outcomes. A total of 223 cases were evaluated, with fertilization observed in 208 cases and no fertilization observed in 15 cases. The number of day 3 total embryos and the number of cleavage embryos differed between the fresh and frozen micro-TESE groups, whereas the number of two-pronuclei oocytes, grading of embryos, fertilization, pregnancy, and live birth rates were found to be similar between the two groups. The cryopreservation of spermatozoa obtained by micro-TESE does not affect the fertilization rate or pregnancy outcome in cases of non-obstructive azoospermia. The present findings, when considered in conjunction with the extant evidence, may serve to alleviate concerns regarding the utilization of frozen-thawed micro-TESE sperm in patients with non-obstructive azoospermia.

Enhancing Cryopreserved Sperm Quality in Chinese Rare Minnow Gobiocypris rarus: The Impact of Antifreeze Proteins.

The Chinese rare minnow (Gobiocypris rarus), an important model fish in China, faces endangerment in the wild. Sperm cryopreservation facilitates the development of new strains and germplasm conservation, but the quality of its cryopreserved sperm remains low. This study evaluates the protective effects of different concentrations of antifreeze proteins (AFP I and AFP III) on the cryopreservation of Chinese rare minnow sperm. Cryopreserved sperm showed significant declines in progressive motility, curvilinear velocity (VCL), average path velocity (VAP), and lifespan compared to fresh sperm, except for straight-line velocity (VSL). The cryomedium containing 10 μg/mL AFP I improved these parameters to their highest levels. However, no significant difference was found in progressive motility and kinetic parameters between cryopreserved sperm with and without AFPs. Cryopreserved sperm with 10 μg/mL AFP I showed the highest plasma membrane integrity, mitochondrial activity, and DNA integrity, significantly better than without AFPs; importantly, the fertilization rate of cryopreserved sperm with 10 μg/mL AFP I was not significantly different from that of fresh sperm. These results indicate that the addition of 10 μg/mL AFP I to the cryomedium for Chinese rare minnow sperm does not improve kinetic parameters but significantly enhances sperm quality, aiding in its new strain development and germplasm conservation.

Study on sperm cryopreservation of hybrid fish derived from Carassius cuvieri (♀) × Carassius auratus red var (♂)

Cryopreservation of sperm is an effective method for conserving germplasm resources in fish genetic breeding. The high-quality hybrid fish (WR) derived from white crucian carp (Carassius cuvieri, WCC, ♀) and red crucian carp (C. auratus red var., RCC, ♂), possesses valuable traits such as high survival rates, strong resistance, and rapid growth, representing an important germplasm resources of crucian carp. This study compared the effects of different antifreeze solutions on sperm viability among three varieties (WR, WCC, and RCC) and examined changes in enzyme activity, fertilization rates, and hatching rates after cryopreservation, aiming to enhance the cryogenic sperm cryopreservation technique in hybrid fish and investigate the mechanisms underlying spermatozoa damage caused by cryopreservation. The results showed that the antifreeze combination of D14 with 15 % dimethyl sulfoxide (DMSO) had the best effect in preserving the sperm of WR and WCC, while D20 with 10 % DMSO was the optimal combination for RCC sperm. After ultra-low temperature preservation, the longevity, fertilization, and hatching rates of frozen sperm were significantly lower (P < 0.05) compared to fresh sperm. The enzyme activities of superoxide dismutase (SOD), lactate dehydrogenase (LDH), and creatine kinase (CK) were significantly decreased (P < 0.05) in spermatozoa, whereas they showed a significant increase (P < 0.05) in sperm plasma. Succinate dehydrogenase (SDH) activity was significantly reduced (P < 0.05) in frozen spermatozoa of RCC compared to fresh spermatozoa, and exhibited lower activity in frozen spermatozoa of WCC and WR. Additionally, SDH activity was significantly elevated (P < 0.05) in frozen sperm plasma, and glutathione reductase (GR) activity was significantly lower (P < 0.05) in both frozen sperm plasma and spermatozoa across all three species. The study demonstrated that cryopreservation had a significant effect on the enzyme activities of spermatozoa and sperm plasma in all three species. These findings provide important technical support for the conservation of high-quality fish germplasm resources, particularly for novel varieties resulting from distant hybridization in fish.

Hormone-Driven Temperature Optimization for Elevated Reproduction in Goldfish (Carassius auratus) under Laboratory Conditions.

This study investigates the efficacy of hormone-induced artificial reproduction in goldfish (Carassius auratus) under controlled temperatures. Ovaprim injections significantly enhanced ovulation and sperm production compared to controls. Medium temperature (22 °C) produced the highest ovulation rates, fastest ovulation timing, and optimal sperm quality (motility and morphology) compared to high (28 °C) and low (16 °C) temperature groups. The low-temperature group exhibited reduced sperm motility duration and higher rates of sperm and larvae damage. The sperm volume of the high-temperature group was higher, but their post-injection survival rates were lower. Furthermore, the lowest spawning rate and low egg quality were noted in the high temperature. Cryopreservation using extender E4 (15% DMSO) exhibited superior post-thaw sperm motility and achieved higher fertilization rates. Fertilization rates, embryo development, and larval survival were all highest at the medium temperature. Larvae hatched from fresh sperm at medium temperature exhibited faster growth and fewer deformities. These findings suggest that hormone stimulation coupled with a medium temperature regimen is critical for successful artificial reproduction in goldfish. Cryopreservation with extender E4 holds promise for sperm banking; however, further optimization is necessary to improve fertilization success with thawed sperm. Future research could explore the influence of temperature on sperm physiology and refine cryopreservation protocols to enhance fertilization rates.

Comparative motility assessment of sperm retrieved from micro-testicular sperm extraction: A single-centre study comparing fresh and frozen-thawed sperm.

Microsurgical testicular sperm extraction (microTESE) is crucial for treating non-obstructive azoospermia (NOA), offering both 'fresh' and 'frozen' options. This study evaluates the impact of fresh versus frozen microTESE on the progression to intra-cytoplasmic sperm injection (ICSI) cycles, focusing on sperm motility. We conducted a retrospective analysis of microTESE procedures at a major medical centre from 2007 to 2021, excluding cases of obstructive azoospermia and cryptozoospermia. Patients were divided into two groups: fresh microTESE (Group FR) and frozen microTESE (Group FZ). Sperm motility was assessed, and ICSI outcomes were compared between groups. Out of 128 microTESE procedures on 113 NOA patients, 31 were fresh and 97 were frozen. Sperm was found in 67.7% of fresh cases and 45.3% of frozen cases. In fresh cases, 85.7% had motile sperm for ICSI, whereas in frozen cases, 81.8% had motile sperm initially, but only 52.7% retained motility post-thaw. Our findings indicate a significant drop in motile sperm availability for ICSI in frozen microTESE cases compared to fresh ones. This suggests a potential advantage of fresh microTESE for certain couples, despite the logistical challenges, highlighting the need for careful patient selection and counselling.

High temperature induces motility of zebrafish sperm after short-term storage

Short-term storage on ice of zebrafish (Danio rerio) sperm has been successfully applied in diverse basic experimental research. The optimal and economically efficient short-term storage method to date, however, can only maintain the fertilization ability of the sperm for a few hours. In this study, we aimed to improve zebrafish sperm quality after short-term storage and to test whether this affects the nucleotide cargo of spermatozoa. Zebrafish sperm was diluted with E400 extender (130 mM KCl, 50 mM NaCl, 2 mM CaCl2, 1 mM MgSO4, 10 mM D-(+)-Glucose and 30 mM HEPES-KOH, pH 7.9, 400 mOsmol/kg) at a dilution ratio 1:20 (v/v) in tubes (500 μL) and stored for 24 h at 0–2 °C under aerobic conditions. Elevating the temperature of sperm suspension at 28 °C for 20 min significantly enhanced sperm motility during activation in both distilled water and Perchec solution with 0.25 % Pluronic F-127. Despite the observed decline in sperm motility during short-term storage, Perchec solution (45 mM NaCl, 5 mM KCl, 30 mM Tris, pH 8.0, 160 mOsmol/kg) enhanced activation of the aged sperm compared to activation of distilled water. Following 24 h of sperm storage, higher fertilization, and hatching rates were observed when sperm suspension was incubated at 28 °C for 20 min and activated in Perchec solution at a ratio of approximately 6120 spermatozoa per egg, compared to activation in hatchery water. A tentative RNA-seq study identified a set of 2180 differentially expressed genes after 24 h of sperm storage, significantly enriched in the glycosaminoglycan degradation and galactose metabolism pathways. The validity was discussed due to the limited mRNA content in spermatozoa. No alterations were identified in the DNA methylation level of 24 h stored sperm and neither differentially methylated CpGs (DMCs) nor differentially methylated regions (DMRs) were detected between fresh and aged sperm. Based on these phenotypic and molecular investigations and the successful implementation of high-temperature incubation prior to sperm activation in artificial reproduction, we propose that high-temperature incubation following short-term sperm storage at low temperatures is a safe and significant approach in zebrafish and holds potential benefits for other economically important warm-water species in aquaculture.

Addition of Cryoprotectant DMSO Reduces Damage to Spermatozoa of Yellow Catfish (Pelteobagrus fulvidraco) during Cryopreservation: Ultrastructural Damage, Oxidative Damage and DNA Damage.

Spermatozoa cryopreservation protocols have been established for yellow catfish (Pelteobagrus fulvidraco), but cryopreservation can still cause cellular damage and affect spermatozoa viability and fertility. Therefore, the aim of this paper was to evaluate the effects of adding or not adding cryoprotectants during low-temperature storage on the ultrastructural damage, oxidative damage, and DNA damage of thawed yellow catfish spermatozoa. The mixed semen of three male yellow catfish was divided into a fresh spermatozoa group, a frozen spermatozoa group (DMSO+) with a cryoprotectant (10% DMSO), and a frozen spermatozoa group without a cryoprotectant (DMSO-). Ultrastructural of the spermatozoa after thawing were observed under an electron microscope and the spermatozoa were assayed for SOD, MDA, and T-AOC enzyme activities, as well as for DNA integrity. In terms of movement parameters, compared with DMSO-, the addition of DMSO has significantly improved sperm motility, curve line velocity (VCL), and straight line velocity (VSL). The ultrastructural results showed that although thawed spermatozoa exhibited increased damage than fresh spermatozoa, 10% DMSO effectively reduced the damage to the plasma membrane, mitochondria, and flagellum of spermatozoa by cryopreservation, and most of the spermatozoa were preserved with intact structure. The results of oxidative damage showed that compared with frozen spermatozoa, 10% DMSO significantly increased the activities of SOD and T-AOC enzymes and clearly reduced the activity of the MDA enzyme. The antioxidant capacity of spermatozoa was improved, lipid peroxidation was reduced, and the oxidative damage caused by cryopreservation was mitigated. The DNA integrity of spermatozoa showed that 10% DMSO clearly reduced the DNA fragmentation rate. In conclusion, 10% DMSO can effectively reduce the ultrastructural damage, oxidative damage, and DNA damage of yellow catfish spermatozoa during cryopreservation; it can also further optimize the cryopreservation protocol for yellow catfish spermatozoa. Meanwhile, it also provides a theoretical basis for the future optimization of the cryopreservation protocols.

Sperm cryopreservation protocol for micro-TESE-retrieved sperm.

Azoospermia is characterized by the absence of sperm in the ejaculate and is categorized into obstructive azoospermia (OA) and nonobstructive azoospermia (NOA). For men with NOA, testicular sperm extraction (TESE) is the only method to obtain sperm for assisted reproductive technology (ART). Given the rarity of these sperm and the unpredictable success of subsequent retrieval attempts, cryopreservation of microdissection-TESE-obtained sperm is essential. Effective cryopreservation prevents the need for repeated surgical procedures and supports future ART attempts. After first delving into the physiological and molecular aspects of sperm cryopreservation, this review aims to examine the current methods and devices for preserving small numbers of sperm. It presents conventional freezing and vitrification techniques, evaluating their respective strengths and limitations in effectively preserving rare sperm, and compares the efficacy of using fresh versus cryopreserved testicular sperm.

Use of a sperm-Hyaluronan binding assay for evaluation of sperm quality in dromedary camels

The objective of this study was to assess the ability of camel spermatozoa to bind in the Hyaluronan Binding Assay (HBA), to determine if conventional sperm quality parameters, in vitro fertilization capacity, and precursor of A-Kinase Anchoring Protein 4 (proAKAP4) values correlate with HBA results. The potential to predict post-thaw fertilization performance from HBA for fresh dromedary camel sperm was also evaluated. Semen samples were collected and assessed both fresh and post thawing, at 0 h and 1.5 h. Conventional semen analysis, HBA, and a proAKAP4 biomarker-test were used to validate sperm quality. A heterologous sperm penetration assay using zona pellucida-free goat oocytes was used to assess in vitro sperm fertilizing capacity. The results showed that dromedary camel spermatozoa bound to hyaluronan with no correlation between results from fresh samples and after thawing. Furthermore, the proAKAP4 test results showed a negative correlation with HBA at 0 h after thawing (r = - 0.62; P = 0.03). In the conventional analysis, only progressive motility (r = 0.65; P = 0.02) and straightness correlated with HBA for fresh semen (r = 0.69; P = 0.01). In the sperm penetration assay, a moderate but non-significant correlation was identified between fresh sperm HBA and penetration (r = 0.52; P = 0.07). In conclusion, results suggested that HBA can be used to assess camel sperm properties, but further investigation is needed to understand its correlation with other sperm quality parameters. The HBA score from fresh dromedary camel sperm was unable to predict post-thaw fertilization performance.

Addition of Mitoquinone (MitoQ) to Fresh Human Sperm Enhances Sperm Motility without Attenuating Viability.

The preparation of human sperm in an andrology laboratory subjects it to oxidative stress. Reactive oxygen species are produced by mitochondria, making it susceptible to oxidative damage; hence, mitochondria-targeted antioxidants like Mitoquinone (MitoQ) might have therapeutic potential for oxidative-damage-associated disorders. The current research aims to establish whether MitoQ has any positive effects during in vitro preparation of fresh human sperm. Viability and motility are evaluated to determine the effective MitoQ concentration and to assess whether MitoQ supplementation is affected by sperm concentration by incubating normospermia semen samples at 37 °C for 2 h and 4 h, respectively. The effect of semen centrifugation following supplementation of 20 × 106 sperm/mL with 200 nM MitoQ is also assessed by measuring viability, motility and sperm DNA fragmentation. The best sperm motility is achieved after 2 h of incubation with 200 nM MitoQ at 37 °C. Sperm concentration of 20 × 106 sperm/mL is the best concentration where 200 nM MitoQ works efficiently. For semen centrifugation at 300× g for 20 min, supplementation with 200 nM MitoQ shows higher sperm motility. The current results demonstrate that MitoQ supplementation during in vitro human semen preparation procedures positively affects fresh sperm motility without affecting viability or increasing DNA fragmentation.

Revisiting the Injury Mechanism of Goat Sperm Caused by the Cryopreservation Process from a Perspective of Sperm Metabolite Profiles.

Semen cryopreservation results in the differential remodeling of the molecules presented in sperm, and these alterations related to reductions in sperm quality and its physiological function have not been fully understood. Given this, this study aimed to investigate the cryoinjury mechanism of goat sperm by analyzing changes of the metabolic characteristics in sperm during the cryopreservation process. The ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) technique was performed to explore metabolite profiles of fresh sperm (C group), equilibrated sperm (E group), and frozen-thawed sperm (F group). In total, 2570 metabolites in positive mode and 2306 metabolites in negative mode were identified, respectively. After comparative analyses among these three groups, 374 differentially abundant metabolites (DAMs) in C vs. E, 291 DAMs in C vs. F, and 189 DAMs in E vs. F were obtained in the positive mode; concurrently, 530 DAMs in C vs. E, 405 DAMs in C vs. F, and 193 DAMs in E vs. F were obtained in the negative mode, respectively. The DAMs were significantly enriched in various metabolic pathways, including 31 pathways in C vs. E, 25 pathways in C vs. F, and 28 pathways in E vs. F, respectively. Among them, 65 DAMs and 25 significantly enriched pathways across the three comparisons were discovered, which may be tightly associated with sperm characteristics and function. Particularly, the functional terms such as TCA cycle, biosynthesis of unsaturated fatty acids, sphingolipid metabolism, glycine, serine and threonine metabolism, alpha-linolenic acid metabolism, and pyruvate metabolism, as well as associated pivotal metabolites like ceramide, betaine, choline, fumaric acid, L-malic acid and L-lactic acid, were focused on. In conclusion, our research characterizes the composition of metabolites in goat sperm and their alterations induced by the cryopreservation process, offering a critical foundation for further exploring the molecular mechanisms of metabolism influencing the quality and freezing tolerance of goat sperm. Additionally, the impacts of equilibration at low temperature on sperm quality may need more attentions as compared to the freezing and thawing process.

Seasonal seminal quality variations and vitrification prospects in black flounder Paralichthys orbignyanus

Long-term preservation of gametes has been identified as a tool to improve broodstock management and increase the number of juveniles produced by artificial fertilization. Paralichthys orbignyanus is an important commercial and recreational species distributed in marine and estuarine waters from Rio de Janeiro (Brazil) to the San Matías Gulf (Argentina). This work focused on studying the seminal quality of tank-reared P. orbignyanus, demonstrating that males are fluent year-round, with the highest yields at the early reproductive season. Fresh sperm exhibited good forward swimming, and samples could be refrigerated up to 48 h while retaining their motility after activation. The optimal conditions for P. orbignyanus sperm motility activation were established as 950 mOsmol/Kg and pH values between 7 and 7.9. Additionally, a well-defined protocol for semen vitrification was developed to assess the cryotolerance of this species’ sperm. We successfully produced high-quality sperm samples, using two vitrification formulations containing trehalose and both z-1000 and x-1000 polymers, that can be used in a near-future in vitro embryo production program.

Decreased human sperm motility and vitality after fast gravity load changes in a parabolic flight

Little is known about the effects of low gravity on human gametes. The aim of this study was to analyze if fresh human sperm samples after fast gravity load changes suffered any detrimental effect in comparison to the splits maintained in Earth's gravity. Fifteen fresh samples from normozoospermic donors were analyzed. Statistically significant differences in vitality (69.7 ± 9.9 % vs 72.4 ± 9.7 %, [95 % CI: 0.002,0.07]); motile sperm concentration (23.7 ± 15.3 M/ml vs 31.5 ± 25.1 M/ml, [95 % CI: 1.03,14.65]); grade “a” sperm concentration (8.7 ± 6.5 M/ml vs 11.7 ± 9.9 M/ml, [95 % CI: 0.71,5.28]); percentage of progressive motility sperm (30 ± 12.9 % vs 36 ± 14.3 %, [95 % CI: 0.10,0.37]) and curvilinear velocity VCL: 45.7 ± 12.8 μm/s vs 47.7 ± 13.3 μm/s, [95 % CI: 0.79,3.22]) were observed. No statistical differences were observed in other sperm kinematic parameters, morphology, DNA fragmentation, apoptosis, and oxidative stress. In conclusion, even though it did not result in a total loss, heavy gravity load changes including microgravity causes a significant decrease in sperm vitality and motility suggesting that negative consequences would be even higher if the exposure were longer. The results obtained indicate that further research is really needed before Assisted Reproduction will be considered for the future human reproduction outside the Earth.

P-072 Fresh testicular sperm seems to yield more fertilization abnormalities and early pregnancy loss than frozen testicular sperm

Abstract Study question How do ICSI outcomes using fresh testicular sperm, compare to those using frozen samples cryopreserved from the previous fresh testicular sperm extraction (TESE)? Summary answer Although fresh testicular sperm led to a better fertilization and pregnancy rate,it yielded more fertilization abnormalities and miscarriages than frozen sperm for the same couples. What is known already Testicular biopsies can be performed in cases of severe male infertility to search for spermatozoa. In the current era of advanced reproductive techniques, ICSI in association with TESE may be an effective treatment for patients with azoospermia. TESE could be performed the day of oocyte pick-up, or instead, it may be accomplished previously, and in this case TESE spermatozoa are cryopreserved for the future ICSI cycles. However, when reviewing the literature, not a lot of papers tried to compare the ICSI outcomes between fresh and frozen testicular sperm in the same couples. Study design, size, duration This retrospective study included the clinical and embryological data of pairs of fresh and frozen sperm injection cycles from couples after TESE between 2010 and 2023. The first ICSI was performed with fresh spermatozoa retrieved from TESE. The subsequent ICSI cycles used frozen spermatozoa from the first TESE. Participants/materials, setting, methods All infertile couples that underwent ICSI+TESE with testicular sperm cryopreservation in the study period were identified (110 couples). Of whom, 34 couples who underwent 40 subsequent ICSI cycle using frozen spermatozoa from the previous TESE were included. ICSI cycles were divided in: Group 1:ICSI with TESE and fresh testicular sperm injection Group 2:ICSI with frozen testicular sperm injection ICSI outcomes were compared for: fertilization/fertilization abnormalities rate, embryo quality, pregnancy and miscarriages rate, and live birth rate. Main results and the role of chance The average age of men who underwent ICSI was 42 years, while that of women was 33 years. On average, the couples had their ICSI with frozen sperm within 1.7 years after the TESE. There were no significant differences in women’s clinical parameters, the type of ovarian stimulation used nor its primary outcome between the two ICSI attempts. Although the primary outcome of ICSI showed a significantly higher fertilization rate for fresh testicular sperm compared to frozen sperm (64.4% vs 44.8%; p = 0.003), there was a tendency for more fertilization abnormalities when fresh testicular sperm was used (8.3% vs 6.2%, p = 0.4). Despite a better quality of cleavage embryos in ICSI with fresh testicular sperm (43.9% vs 39.2%, p = 0.6) and a higher pregnancy rate for the same group (21.9% vs 15.9%, p = 0.52), the miscarriage rate was twice as high when compared to the miscarriage rate of ICSI with frozen testicular sperm (6.7% vs 3.4%, p = 0.8), indicating that cryopreservation of testicular sperm could play a role in selecting better sperm for micro-injection. As for the live birth rate, ICSI with frozen sperm led to not statistically inferior rates when compared with ICSI with fresh sperm in the same couples (6.7% vs 10%, p = 0.6). Limitations, reasons for caution Due to our inclusion criteria, the study population may seem to be too reduced to reach significant results. To gain more informative insights, we recommend combining results from different centers and referring to the etiology of azoospermia that led to the TESE procedure. Wider implications of the findings Genetic testing and chromosomal aberration detection on testicular spermatozoa before and after cryopreservation could be proposed to compare the proportion of defected spermatozoa in both types of samples. Trial registration number not applicable

Sperm cryotolerance in mature male parr of Atlantic salmon (Salmo salar L. 1758): Effects of dietary lipid content and n-3 essential fatty acids composition

The cryoresistance of sperm is a significant concern issue in conservation and aquaculture. The nutrition of broodstock fish affects the quality of gametes. This study aimed to assess the impact of four diets on the quality of fresh and cryopreserved sperm of sexually mature salmon parr during spermatogenesis. Two dietary factors were modulated: lipid intake (12% and 20%) and n-3 essential fatty acid composition (DHA and ALA). No significant difference in parr growth rate or sperm concentration following the feeding period was observed. Viability and mitochondrial membrane potential of fresh sperm were also unaffected. However, fresh sperm motility was significantly higher in fish fed the 20% ALA diet compared to those fed the 12% DHA diet (p = 0.001). Cryopreservation reduced viability, motility, curvilinear velocity (VCL), and average path velocity (VAP), and increased linearity, regardless of the diet (p < 0.01). However, cryopreserved spermatozoa from parr fed diets enriched with DHA and containing 20% lipids showed a higher percentage of viability compared to spermatozoa from fish fed diets containing 12% lipids (p = 0.044). The study found that mitochondrial membrane potential, motility, VCL, and VAP was higher and that linearity was lower in salmon fed diets with higher levels of lipid and plant-based n-3 fatty acids compared to those fed 12% lipid diets, regardless of the n-3 fatty acid composition. This suggests that the higher levels of lipid and plant-based n-3 fatty acids provided greater resistance of sperm to damage by cryopreservation.

P-020 Clinical outcomes of 583 treatment cycles with fresh and cryopreserved testicular sperm in cases without karyotype abnormalities and Y chromosome microdeletions needing testicular sperm extraction

Abstract Study question What are the embryological, clinical and newborn outcomes using fresh and cryopreserved testicular sperm in non-obstructive azoospermia patients? Are the differences between both techniques significant? Summary answer In this population there was a high live birth delivery rate with no significant differences observed in the outcomes using fresh and cryopreserved testicular sperm. What is known already The use of cryopreserved testicular sperm has been considered of lower quality due to freezing/thawing consequences (1). Nevertheless, cryopreserved testicular sperm presents the advantages of avoiding female stimulation in cases of failed testicular sperm retrieval, or multiple testicular biopsies (2). However, the majority of the studies evidenced no significant differences in the clinical outcomes between the use of cryopreserved and fresh sperm (3). As only a few studies are dedicated to compare both techniques (4) we expanded the number of cycles and provided full demographic, stimulation, embryological, clinical and newborn outcomes.

P-087 Obstetrical and perinatal outcomes from intracytoplasmic sperm injection couples with oocyte donation are not affected by advanced paternal age measured as a continuous variable

Abstract Study question Does paternal age (PA), considered a continuous variable, have any effect on obstetrical and perinatal outcomes in intracytoplasmic sperm injection (ICSI) with donated oocytes? Summary answer PA does not affect clinically relevant obstetrical and perinatal outcomes, but it significantly increases the pregnancy’s duration in ICSI with autologous sperm and donated oocytes. What is known already Currently there has been an evident delay in childbearing, therefore studies relating advanced PA with reproductive risks are increasing and results are controversial. These studies frequently study PA by dividing it in groups arbitrarily constructed, but probably a more precise evaluation is obtained using PA as a continuous variable. Furthermore, oocyte donation standardizes female factor, to some extent, allowing a more realistic male factor evaluation. In this regard, we aimed to evaluate the effect of PA at conception (continuous variable) on obstetrical and perinatal outcomes in ICSI couples with autologous sperm and donated oocytes in a large population of patients. Study design, size, duration This retrospective observational multicentric cohort study evaluated data from couples that performed an ICSI between January 2008 and March 2020 using autologous sperm and donated oocytes. We included only singleton deliveries and the first delivery of each couple. PA was analysed as a continuous variable, considered more informative in comparison to its analysis as categorical. Data available and included consisted of 15,696 patients who had a delivery with or without a live birth. Participants/materials, setting, methods PA ranged from 21-54 years. Clinical database was constructed with reproductive outcomes from electronic medical records. Erroneous or incomplete data were removed. Statistical analysis included a descriptive analysis, followed by univariate and multivariate models using binary logistic regression or linear regression. We adjusted for maternal age and body mass index, PA, fresh sperm concentration and progressive motility, cycle type, gestational age, transfer on day 5 and delivery type (when appropriate). P &amp;lt; 0.05 was considered statistically significant. Main results and the role of chance Mean PA was 41.45 (41.38-41.51). We included a total of 15,696 deliveries and 15,673 live births. Results from multivariate models are presented as adjusted odd ratio or regression coefficient with 95% confidence intervals and adjusted p. In terms of obstetrical outcomes, PA results were 1.086 (0.959-1.229) p = 0.194 for gestational diabetes, 0.963 (0.849-1.093) p = 0.561 for anemia, 0.947 (0.837-1.072) p = 0.388 for hypertension, 0.959 (0.878-1.048) p = 0.356 for preterm birth and 0.986 (0.929-1.046) p = 0.641 for caesarean delivery. Regarding perinatal outcomes, PA results were 0.442 (0.005-0.879) p = 0.047 for gestational age, -0.216 (-15.559-15.126) p = 0.978 for infant weight, 1.101 (0.941-1.288) p = 0.231 for low birth weight (&amp;lt;2,500 g), 0.710 (0.463-1.088) p = 0.116 for very low birth weight (&amp;lt;1,500g), 0.988 (0.933-1.047) p = 0.684 for female sex, 0.002 (-0.076-0.08) p = 0.96 for length, -0.012 (-0.088-0.064) p = 0.763 for cranial perimeter, 0.991 (0.876-1.12) p = 0.88 for NICU admission, and 0.027 (-0.028-0.083) p = 0.337, 0.025 (-0.012-0.061) p = 0.18 and -0.045 (-0.096-0.007) p = 0.089 for Apgar 1, 5 and 10 minutes, respectively. According to our results, PA does not affect clinically relevant outcomes for the health during pregnancy and of the infant. However, PA significantly increases the duration of the pregnancy in ICSI with autologous sperm and donated oocytes. Limitations, reasons for caution The study’s major strengths are the consideration of PA as a continuous variable, the use of oocyte donation (controlling female factors) and the inclusion of a huge sample size. However, due to its retrospective nature there are some biases derived from the clinical practice and the presence of missing data. Wider implications of the findings Current evident delay in childbearing merits further research on the effect of PA on reproductive outcomes, addressed in this study. Our study suggests that PA is not associated with obstetrical and perinatal outcomes, except for gestational age, which sends a hopeful message to fathers of advanced paternal age. Trial registration number not applicable

Cryopreservation alters buffalo sperm kinematics and mitochondrial parameters, acrosome and intra-cellular calcium status

BACKGROUND: Little is known about the effects of different seasons on the cryopreservation success of buffalo sperm in terms of kinematics and sperm functional parameters. OBJECTIVE: To study the effect of three seasons (winter, comfort and summer) and cryopreservation on sperm kinematics and functional properties in buffalo bulls. MATERIALS AND METHODS: Semen ejaculates (n = 90) collected during three seasons i.e. winter (n = 30), comfort (n = 30), summer (n = 30) were evaluated for sperm kinematics and functional properties. RESULTS: Sperm kinematics with respect to total (TM), progressive (PM) and rapid motility (RM) was higher (P&lt;0.05) in fresh sperm compared to sperm that had been frozen-thawed. Similarly, all kinematic parameters [viz. average path velocity (VAP), straight linear velocity (VSL), curvilinear velocity (VCL), beats cross frequency (BCF), lateral head displacement (ALH), linearity (LIN) and straightness (STR)] were higher (P&lt;0.01) at the fresh stage. With respect to season, frozen-thawed semen TM (57.67 ± 115%), PM (50.2 ± 1.15%) and RM (51.6 ± 1.19%) were higher (P&lt;0.01) when using sperm collected during winter. The stage of cryopreservation (i.e., equilibration and freeze-thawing) also showed significant effects (P&lt;0.01) on mitochondrial superoxide positive status (MSPS), mitochondrial membrane potential (MMP), acrosome status and intra-cellular calcium status. CONCLUSION: The season of sperm collection and cryopreservation have significant effects on buffalo bull sperm kinematics and functional properties.

Totally extraperitoneal laparoscopy-assisted microsurgical vasovasostomy for the treatment of obstructive azoospermia caused by pediatric bilateral inguinal herniorrhaphy.

Pediatric inguinal hernia repair (IHR) is a common cause of obstructive azoospermia (OA). Yet, the surgical treatment for this kind of OA remains difficult with poor fertility outcome. To evaluate the safety and effectiveness of totally extraperitoneal laparoscopy-assisted microsurgical vasovasostomy (VV) in the treatment of OA caused by pediatric bilateral IHR. Totally, 37 patients with OA caused by pediatric bilateral IHR were enrolled in this study from March 2015 to December 2020 in Shanghai General Hospital. The clinical data and fertility outcomes were collected and analyzed. All patients enrolled had a history of bilateral IHR at the age of 1-10 years old. The mean age of patients was 27±4.31 (range: 18-35) years. Totally extraperitoneal laparoscopy (TEP) was applied in 31 patients for the exploration and retrieval of pelvic vas deferens end, and 30 of them underwent microsurgical VV successfully. Among the six cases where TEP was not applied, five cases underwent microsurgical anastomosis. Intraoperative exploration revealed that the location of vas deferens injuries included scrotum (2.70%, 1/37), inguinal canal (5.41%, 2/37), pelvic cavity (78.37%, 29/37), and multiple sites (13.51%, 5/37). The mean operation time was 339±96.73 min (range: 130-510 min). There were no surgical complications. Thirty-three cases were followed up for 5-48 months with four cases lost to follow-up. The overall patency rate, pregnancy rate, and natural pregnancy rate were 75.86% (22/29), 46.67% (14/30), and 36.84% (7/19, 3 patients without family planning), respectively. And seven couples conceived through the assisted reproductive technique, two of which using fresh sperm in the ejaculate. TEP laparoscopy-assisted microscopic VV is an effective treatment for patients with OA caused by pediatric bilateral IHR.