Sperm Recovery Rate Research Articles

Post-Thaw Sperm Recovery Rates; A Retrospective Study

Background: Cryopreservation is currently the only fertility preservation option available to patients undergoing gonadotoxic treatment. Much research in the field focusses on techniques, media and equipment employed to cryopreserve cells, and demonstrating cell viability after using such. Comparatively, there is a death of information available on the procedures and equipment used to thaw cryopreserved cells, or estimates for post-thaw recovery. Aim: This is a retrospective study assessing semen parameters in post-thawed specimen from patients who attended the Royal Women’s Hospital Andrology Unit for sperm cryopreservation prior to gonadotoxic or other treatments. The study aims to examine the relationship between sperm cryopreservation technique, media and period of storage with post-thaw sperm parameters. Method: Patients with written consent to discard their stored semen were selected for inclusion in the study (n=59); samples with sperm concentration [Formula: see text]1 million/ml prior to freezing (n=11) were excluded. Semen samples were thawed accordingly: 5 minutes at room temperature followed by incubation at 37[Formula: see text]C for sperm straws; vials were transferred directly to a mixer incubator at 37[Formula: see text]C. All samples were immediately assessed for sperm concentration and motility. Recovery rates were calculated and data were analysed using SPSS (IBM, USA) version 25. Results: Progressive motility recovery was 32.4% ± 20.6. Patients were divided into groups according to period of storage, and showed no significant differences for recovery of motility and concentration (p=0.44, 0.96, respectively). Furthermore, progressive motility recovery rates were not significantly different between freezing techniques (p=0.116) and media (p=0.099). Conclusion: Data from this study are preliminary and further study is warranted to fully extrapolate the relationship between sperm cryopreservation protocols and period of storage on recovery rates. The lack of relationship between these factors exhibited in the data could suggest that research and development in thaw technologies may be more pertinent to improving sperm recovery rates.

Is testicular microdissection the only way to retrieve sperm for non-obstructive azoospermic men?

Men presenting with non-obstructive azoospermia are the most challenging clinical scenario for an infertile couple. Intracytoplasmic Sperm Injection (ICSI) with testicular sperm retrieval gave a chance for biological fatherhood once sperm can be found, but unfortunately sperm recovery rate (SSR) is something near 50%, leading to a discussion about what surgical retrieval technique is the best. Historically sperm have been retrieved using conventional Testicular Sperm Extraction (c-TESE), Testicular Sperm Aspiration (TESA), a combination of Testicular Fine Needle Aspiration (TfNA)/c-TESE, Testicular Microdissection (TM) and Open Testicular Mapping (OTEM). c-TESE published in 1995 by Devroey and cols. consists of testis delivery, a large unique albuginea incision and extraction of a portion from the majority of testicular tubules. TESA published in 1996 by Lewin and cols. is done percutaneously using a 21–23 gauge needle and a syringe to aspire testicular tubules. TfNA was published in 1965 by Obrant and Persson as an aspiration biopsy and cytological exam to verify sperm production. In 1999 Turek and cols. published the use of TfNA combined with c-TESE for sperm retrieval. In 1999, Peter Schlegel published a technique using a microsurgical approach to identify more probable sperm production areas inside the testicle that could be excised with better precision and less tissue. OTEM is a multiple biopsy approach, published in 2020 by Vieira and cols., based on TfNA principles but done at the same time without albuginea opening or surgical microscope need. Since Testicular Microdissection publication, the method became the gold standard for sperm retrieval, allowing superior SSR with minimal tissue removal, but the amount of testicular dissection to find more probable spermatogenesis areas, difficulties in comparative design studies, diversity TM results among doctors and other methods that can achieve very similar results we question TM superiority. The objective is review existing literature and discuss advantages and disadvantages of all the methods for sperm retrieval in non-obstructive azoospermia.

An optimized centrifugation protocol for ram sperm ensuring high sample yield, quality and fertility

The optimization and implementation of artificial insemination (AI) in sheep is necessary to increase the livestock productivity through enhanced control of reproductive function. Sperm centrifugation is a common procedure in the ejaculate handling in AI and other assisted reproductive technologies (ART), as part of new methods of sperm analysis, selection or preservation. However, our research group previously established that this simple procedure might cause a large sperm loss and induce deleterious effects on the sperm function of the ovine species when high centrifugation forces are employed. To our knowledge, there are no studies on combined effect of extender and different centrifugal forces on ram sperm yield and quality. Furthermore, evidence of in vivo fertility rate using sperm obtained with various centrifugation forces is also lacking in this species. Thus, the objective of this work was to define the ideal conditions for ram semen centrifugation that will achieve the best quantity and quality sample to ensure unaffected fertilization ability of centrifuged ram sperm. The Experiment 1 evaluated the effect of the centrifugation procedure of two extenders (INRA 96 and Tyrode's) and two cooling protocols (Rapid and Slow Refrigeration −35 °C to 15 °C–) on sperm recovery rate and quality (motility and kinetic parameters, viability, apoptosis and mitochondrial activity). INRA 96 combined with Slow Refrigeration and Tyrode's at room temperature registered the highest sperm recovery and quality values (P ≤ 0.05). In Experiment 2, the influence of three centrifugal forces (600, 1200 and 6000×g for 10 min) was assessed immediately after centrifugation on the technical performance and sperm functionality in diluted samples with INRA 96 and Tyrode's at the conditions set out in Experiment 1. The lowest pellet weight (P ≤ 0.05) without harmful effect on sperm physiological status (P > 0.05) was achieved at 1200×g, since 6000×g induced sperm motility damage (P ≤ 0.05) with both extenders. Finally, to ensure the total safety of the centrifugation protocol, Experiment 3 tested in a combined in vitro and in vivo test the effect of these three centrifugal forces on ram sperm quality after dilution (INRA 96) and liquid storage (6–8 h at 15 °C). The damage produced by 6000×g on sperm motility (P ≤ 0.05) was maintained over time, coinciding with a lower fertility (P ≤ 0.05). In conclusion, ram sperm can be centrifuged in INRA 96 extender up to 1200×g for 10 min at 15 °C as secure values with high recovery rates and without detrimental effects on sperm quality and fertility.

P-027 which is the best method of testicular sperm retrieval for NOA? an answer from 1071 TESE cases in my clinics over 25 years

Abstract Study question Which improves sperm recovery rate (SRR) for men with non-obstructive azoospermia (NOA), microdissection TESE (m-TESE) or conventional TESE (c-TESE)? Summary answer SRR of c-TESE vs. m-TESE for NOA was not significant. We need to develop a safer, less-invasive technique with fewer complications and higher SRR. What is known already m-TESE has been performed for men with NOA since Schlegel P’s report (Hum Reprod, 1999). Seminiferous tubules containing many developing germ cells were likely to be larger and more opaque and the ability to find spermatozoa increased from 45% (10/22) to 63% (17/27) after introduction of the microdissection technique. However, Jarvis S et al. (Turek PG group, 2019) reported that at least one site revealed mature sperm in 24 (29.3%) of 82 men with prior failed micro TESE procedures, and sperm were more likely to be found in the testis periphery rather than centrally with FNA mapping. Study design, size, duration TESE was performed in 1071 azoospermic patients in my clinics; c-TESE was performed for all azoospermic patients between 1996 and 2005 in Sendai; c-TESE for OA and m-TESE for NOA was performed from 2006 in Sendai, and from 2012 in Takanawa. The patients were divided into four groups; 1) FSH≦9.2mIU/ml, Testicular volume(TV)≧15/ml (OA), 2) FSH≦9.2mIU/ml, TV < 15ml, 3) FSH>9.2mIU/ml, TV≧15ml, 4) FSH>9.2mIU/ml, TV < 15ml (NOA). Participants/materials, setting, methods c-TESE was performed in 178 azoospermic men (50, 12, 7, 109, respectively) in 1996-2005. c-TESE was performed in 181 patients (131, 26, 15, 9, respectively) and m-TESE was performed in 541 patients (56, 33, 35, 416, respectively) in 2006 -2020. SRR, pregnancy rate (PR), and birth rate (BR) in 1996-2005 were compared with those in 2006-2020. Main results and the role of chance Statistical analysis was performed by chi-square analysis. SRR in c-TESE was 96.0% (48/50) in group 1 (OA) vs. 36.7% (40/109) in group 4 (NOA) in Sendai between 1996 and 2005 (P < 0.001). SRR of c-TESE was 36.7% (40/109) in group 4 (NOA, in Sendai, 1996-2006), whereas SRR of m-TESE was 31.3% (130/416) in group 4 (NOA, in Sendai and Takanawa, 2006-2020). There was no significant difference (np). Total SRR of conventional TESE for all azoospermic patients between 1996 and 2005 was 54.9% (96/175), whereas total SRR of c-TESE for OA and m-TESE for NOA between 2006 and 2020 was 51.7% (373/721). NOA rate 4) of azoospermic patients of 1996-2005 and that of 2006-2020 was 61.2% (109/178) and 58.9% (425/721), respectively. There was no significant difference (np). PR was 37.4% (61/163) and BR was 31.9% (52/263) in c-TESE (1996-2005), PR was 39.6% (388/980), and BR was 27.6% (270//980) in both c-TESE and m-TESE (2006–2020). There was a significant difference in birth rate (P < 0.05). In conclusion, SRR, PR, and BR were not improved even after m-TESE was performed. Considering the fact that m-TESE cannot find sperm thoroughly, adopting other techniques such as sperm fine-needle aspiration may be required. Limitations, reasons for caution Not applicable. Wider implications of the findings We could not show the effectiveness of m-TESE compared with c-TESE. However, the total volume of seminiferous tubules collected by c-TESE was the higher. The new one-step fine-needle aspiration has the potential to achieve higher sperm yield with fewer complications. Further advanced technology will hopefully improve the SRR in NOA. Trial registration number none

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

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

P-117 A novel method to select spermatozoa with the best morphokinetic characteristics and superior genomic integrity

Abstract Study question Can an automated device based on membrane electrophoresis be used to select spermatozoa with superior morphokinetic characteristics and lower sperm chromatin fragmentation (SCF)? Summary answer Albeit at an expense of concentration, a membrane-electrophoretic device was able to isolate a proportion of spermatozoa with the highest motility, morphology, and genomic integrity. What is known already Conventional sperm selection based on density gradient centrifugation (DGC) can enhance the proportion of progressively motile spermatozoa; however, DGC methods are limited in their ability to select spermatozoa with higher genomic integrity. Membrane-based microfluidic technologies (MFSS) have already been used clinically to select for spermatozoa with a superior chromatin status; however, these methods are based on the intrinsic characteristics of motile spermatozoa capable of selecting themselves. By incorporating an extrinsic electrophoretic drive, membrane electrophoresis would overcome a limitation of progressive motility in the semen sample and be able to isolate viable gametes with better morphology and reduced SCF. Study design, size, duration From August 2020 to December 2021, semen samples from 68 men were evaluated by standard semen analysis and simultaneously processed by DGC or a novel membrane-electrophoretic sperm sorter (EPSS) to select for progressively motile spermatozoa. Concentration, motility, progressive motility, morphology, and SCF were measured and compared between raw, DGC-, and EPSS-processed specimens. Participants/materials, setting, methods Fresh ejaculates were evaluated by standard semen analysis according to WHO 2021 criteria. Following complete liquefaction, specimens were divided into two equal aliquots for DGC and EPSS sorting. SCF was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay under fluorescent microscopy; at least 500 spermatozoa were evaluated for each specimen with a normal threshold of ≤ 15%. Main results and the role of chance A total of 68 men with an average age of 38.1±6 years had the following average semen parameters: volume of 3.1±1 mL, concentration of 58.2±33 x106/mL, 43.3±9% motility, 40.5±9% forward progression motility, and a normal morphology of 2.9±0.8%. When comparing the final parameters between the EPSS and DGC methods, despite a reduced sperm concentration (7.3±9 x106/mL vs 45.0±36 x106/mL, P<0.0001, respectively), EPSS was highly selective for motile spermatozoa, yielding significantly higher motility (93.1±16% vs. 86.8±15% P<0.0001) and forward progressive motility (92.3±17% vs 85.2±15%, P<0.0001). Despite an overall lower sperm recovery rate (21.4±25% vs 75.4±17%, P<0.0001), normal morphology improved to 3.4±0.8% (P<0.0001) after EPSS but remained unchanged in the DGC-processed sample. Although both EPSS and DGC improved SCF from 12.2±9% in the raw specimen to 4.8±6% and 6.6±7% (P < 0.0001), respectively, EPSS outperformed DGC (P < 0.05). Moreover, the DGC method took up to 60 min to process, whereas the EPSS technique took a total of 6 min. Limitations, reasons for caution The selection of spermatozoa by EPSS is a promising technique to isolate progressively motile spermatozoa with enhanced morphology and superior chromatin integrity, albeit at a lower concentration. This is a preliminary study; the benefit and safety of this method must be further demonstrated by insemination or IVF treatment. Wider implications of the findings A membrane electrophoresis device may be a viable alternative method to MFSS to identify spermatozoa with superior morphokinetic characteristics and intact chromatin. Moreover, incorporating an automated device can reduce gamete processing time while minimizing labor costs and inter-operator errors. Trial registration number N/A

Varicoceles in Men With Non-obstructive Azoospermia: The Dilemma to Operate or Not.

The knowledge on male reproduction is constantly expanding, especially in treating infertility due to non-obstructive azoospermia (NOA). Varicocele is occasionally diagnosed in a subpopulation of males with NOA. Varicocele repair in NOA-men may contribute to the reappearance of spermatozoa in semen. However, spontaneous pregnancies are observed in only a small percentage of NOA-men post-varicocelectomy. Additionally, it has been reported that the repair of varicocele in NOA-men (before the performance of sperm retrieval techniques) may increase the testicular sperm recovery rate. In addition, it increases the pregnancy rate in intracytoplasmic sperm injection (ICSI) programs in NOA-men without spermatozoa in the semen post-varicocelectomy. In addition, to the improvement in Sertoli cellular secretory function, varicocelectomy may increase the secretory function of Leydig cells, which subsequently results in improved androgen production, raising the probability to negate the need for testosterone replacement therapy in cases of late-onset hypogonadism. On the other hand, the benefit of varicocelectomy in patients with NOA is still debatable. The current review study aims to provide a critical and extensive review of varicocele repair in males with NOA. This study additionally focuses on the impact of varicocele repair on sperm retrieval rates and its influence on the ICSI outcomes for those couples who remain negative for spermatozoa in their semen samples post-varicocelectomy.

Pengaruh Waktu Thawing Terhadap Kualitas Spermatozoa Ayam Buras

The purpose of this study was to determine the effect of thawing time on the quality of native chicken spermatozoa. The research design used in this study was a completely randomized design (CRD) with 5 treatment levels and 5 replications. The treatments used were P1 = thawing time of 20 seconds, P2 = thawing time of 30 seconds, P3 = thawing time of 40 seconds, P4 = thawing time of 50 seconds, P5 = thawing time of 60 seconds. Semen that has been diluted to be packed in 5 ministraw 0.25 ml, then equilibrated for 2 hours. After equilibration, proceed with the freezing process (prefreezing). Then the straws were stored in a liquid N2 container (-196°C) for 24 hours. frozen semen was thawed again with warm water at 37°C according to the treatment. Parameters observed were sperm motility and recovery rate. The results of this study showed that thawing time had no significant effect (P>0.05) on sperm motility and recovery rate. It was concluded that the thawing time of 20 to 60 seconds could be used in freezing native chicken semen.

Repeated microdissection testicular sperm extraction in patients with non-obstructive azoospermia: Outcome and predictive factors

ABSTRACT Objective To assess the feasibility of repeated sperm recovery in patients with non-obstructive azoospermia (NOA), as little is known about the extraction rate in repeated microdissection testicular sperm extraction (microTESE) in these patients. Patients and Methods A total of 134 men with NOA had their first sperm recovery between January 2013 and February 2020. Repeated microTESE had been done mostly for patients with a successful initial retrieval. Results In the 323 procedures performed on the 134 men with NOA, sperm could be retrieved in 236 procedures (73.1%). A total of 88, 61 and 40 men underwent two, three and four sperm retrievals, respectively. In these cycles, sperm could be extracted in 65 (73.9%), 53 (86.9%) and 37 (92.5%) men, respectively. During the first microTESE procedure, sperm could be extracted in 81 (60.4%) men with NOA. In all, the success rate was significantly different between subgroups, showing highest rate in hypospermatogenesis cases (95.6%), followed by maturation arrest (58.5%), and Sertoli cell-only syndrome (56.0%). However, this difference was not significant at the third and fourth repeated microTESE. The FSH levels and testicular volume were among the noticeable factors affecting success of sperm retrieval. The duration between the first and second biopsies significantly increased the success rate by a factor of 1.3-fold/month; however, afterwards, the duration did not play any role in the success of microTESE. The success of previous trial significantly increased the probability of success by 10.1-fold in the second trial, 5.6-fold in the third trial, and 16.5 folds in the fourth. Conclusion Repeated MD -TESE ensures a high sperm recovery rate in patients with NOA. These data also show that when no spermatozoa can be obtained after thawing cryopreserved testicular sperm for ICSI in NOA patients, a repeat microTESE procedure can be planned. Abbreviations ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilisation; MA: maturation arrest; (N)OA: (non-)obstructive azoospermia; OR: odds ratio; SCOS, Sertoli cell-only syndrome; SRR: spermatozoa retrieval rate; (micro)TESE: (microdissection) testicular sperm extraction

The Effectiveness of Sperm Preparation Using Density Mini-Gradient and Single-Layer Centrifugation for Oligospermia Samples

Background:Sperm preparation is an important step during assisted reproduction, and different assisted reproductive techniques have different sperm quality requirements. For intrauterine insemination (IUI), the total motile sperm count is a predictor of a patient’s fertility.Objective:The aim of this study was to compare the sperm recovery rate and DNA fragmentation index (DFI) outcomes following density mini-gradient and single-layer centrifugation in preparation for intrauterine insemination (IUI).Methods:A total of 30 semen samples with concentrations under 15 million cells/ml were obtained, and each sample was divided into 3 aliquots, with each aliquot subjected to 1 of 3 separation methods: mini-gradient, single-layer using a 90% density layer (single 90-layer), and single-layer using a 45% density layer (single 45-layer). Total sperm motility and sperm recovery rates were compared before and after preparation using each method.Results:The sperm concentration obtained using single 45-layer was higher than the other groups (p<0.05), but sperm motility was higher using the mini-gradient and single 90-layer methods higher than the single 45-layer method (p<0.05). The recovered sperm motility rates for the mini-gradient, single 90-layer, and single 45-layer methods were 57.6% ± 20.6%, 62.8% ± 18.5%, and 78.7% ± 12.4%, respectively, indicating a better outcome for the single 45-layer method than for the other methods.Conclusion:All of these methods can be applied to sperm preparation for IUI, and the optimal method can be selected based on initial sperm quality to collect sperm with good motility and DNA integrity to achieve a satisfactory pregnancy rate.

Comparative sperm recovery rate after density gradient centrifugation with two media for in vitro fertilization.

To compare the efficacy of two density gradient centrifugation media for retrieving spermatozoa from semen samples by evaluating the total motile sperm count (TMSC) and the percentage recovery. Twenty-two men with different sperm counts participated in the study. The samples were divided into two equal aliquots and processed using the commercial ISolate Sperm Separation Medium (Irvine Scientific, United States) and the GV Gradiente (IngáMed, Brazil). After separation, samples were counted and evaluated for motile sperm recovery. The mean TMSC in the fresh sample was 19.65±21.08 million/mL. After the ISolate separation the TMSC was 6.71±7.29 million/mL, and for the GV Gradiente it was 6.27±6.82 million/mL. The percentage of motile spermatozoa recovered was 36.47%±21.61 for ISolate and 35.22%±21.24 for the GV Gradiente (p>0.05). The samples from 6 oligospermic patients (27%) were evaluated separately and the TMSC for ISolate was 4.83±2.92 million/mL, and for the GV Gradiente, it was 4.16±3.12 million/mL (p=0.54). When evaluating only normospermic patient samples, the TMSC for ISolate was 9.05±7.29 million/mL, and for the GV Gradiente, it was 8.47±6.79 million/mL (p=0.83). There was no statistical difference in retrieving motile sperm using the GV Gradiente and the ISolate Separation Medium.

Action of swim-up and caffeine on equine frozen sperm.

Cryopreservation of equine semen is crucial to semen commercialization. However, it reduces sperm motility and longevity. Thus, sperm selection methods and addition of motility-activating substances to sperm, such as caffeine, may improve sperm quality of equine frozen semen. The objective of the current work was to evaluate the effects of caffeine on recovery and quality parameters of frozen-thawed sperm subjected to swim-up selection to be used in intracytoplasmic sperm injection (ICSI) in assisted reproductive techniques. Stallion semen were frozen and after thawing different caffeine concentrations were added to the samples performing four treatments control (no caffeine), 3, 5, and 7.5 mM caffeine. Sperm kinematic and motility were assessed by computer-assisted sperm analysis (CASA). Then, the four treated samples were submitted to the swim-up sperm selection, and the number of recovered sperm and morphology were evaluated at four times 20, 40, 60, and 80 min. The swim-up increased the recovery proportion of normal morphology sperm without (80.1±1%) or with caffeine addition (3mM: 81.2±1%, 5mM: 79.9±1% and 7.5 mM 78.9±1%) compared to the thawed semen (70±2%). However, the addition of 5 mM caffeine induced an increase in sperm motility (38.9±2.8 vs. 32.6±3.4%, P<0.05), and sperm recovery after swim-up (7.9x106 vs. 3.4x106 sperm/ml, P<0.05) compared to the control. The addition of 5 mM caffeine to frozen-thawed equine semen before swim-up selection improved sperm motility and increased the sperm recovery rate while not decreasing the percentage of morphologically normal sperm. Thus, caffeine addition to frozen-thawed equine semen before swim-up selection has potential clinical application in improving sperm quality for use in ICSI.

Does Microfluidic Sperm Sorting Affect Embryo Euploidy Rates in Couples with High Sperm DNA Fragmentation?

Male infertility contributes as the main factor in 30-50% of infertility cases. Conventional methods for sperm preparation have induced questioning of sperm recovery rates. The microfluidic sperm sorting (MSS) technique selects highly motile sperm with lower levels of SDF (sperm DNA fragmentation) compared to conventional sperm sorting techniques. This study aimed to determine whether utilizing this technique will reveal better embryo quality and euploidy rates in couples with repeated implantation failure (RIF) and high SDF in a new PGT-A (preimplantation genetic testing for aneuploidies) cycle. This retrospective study included couples referred to PGT-A for previous repeated ART (assisted reproductive techniques) cycle failures and with high SDF. In their new cycles, couples who accepted the technique were assigned to the MSS group, and the rest were managed with DGC (density-gradient centrifugation). Two groups were compared in terms of fertilization and euploidy rates, clinical miscarriage and live birth rates, the total number of blastocysts, and top quality blastocysts. There was no difference between the groups regarding fertilization rates, euploidy rates, clinical miscarriage, and live birth rates. The total number of blastocysts and top quality blastocysts were significantly higher in the MSS group. The MSS technique provides a higher number of top-quality blastocysts than DGC; however, neither euploidy nor live birth rates improved. Studies focusing on confounding factors to embryonic genomic status in the presence of high SDF are needed.

Las subpoblaciones de espermatozoides y su calidad en fracciones producidas por la centrifugación de una sola capa en muestras frescas y normospérmicas de esperma de cordero

Single layer centrifugation (SLC) technique has been developed to select the best sperm population in the ejaculate in order to increase the fertilization rates by artificial insemination or in vitro fertilization. Normospermic ram semen samples containing 800 and 3,000 × 106 sperms/ml (C800 and C3000, respectively) were processed by SLC. Three sperm fractions were separated in each sample following silica-coloidal sperm centrifugation and sperm yield, quality and subpopulations were analyzed in each one. In C800 group, the sperm recovery rate did not vary in any studied fraction, but when samples were highly concentrated (C3000) the top fraction (F1) contained significantly higher spermatozoa than bottom fraction (F3). Also, it was observed that F1 in C3000 had got a significantly higher percentage of spermatozoa (53.2 %) than in C800, while the quantity of spermatozoa recovered in fraction 2 was lower (25.2 % vs 45.4 %). Based on the sperm motility parameters, three sperm subpopulations were identified: SP1, low velocity spermatozoa showing no progressive movement (19.1 %); SP2, rapid and progressive spermatozoa (43.7 %); and SP3, rapid spermatozoa but non-linear movement (37.2 %). While SLC has been implemented for sperm separation in suboptimal and/or low concentrated sperm samples, this trial demonstrates that SLC is not efficient to separate different sperm populations in normospermic ram sperm samples containing high concentrations of spermatozoa.

INNOVATIVE ULTRA-RAPID VITRIFICATION METHOD FOR FIVE OR FEWER TESTICULAR SPERMATOZOA FROM NON-OBSTRUCTIVE AZOOSPERMIC MEN AFTER MICROSURGICAL TESTICULAR SPERM EXTRACTION (MICRO-TESE)

Clinical outcomes of ICSI when using only five or fewer testicular spermatozoa after cryopreservation have been unsuccessful and are considered to be inferior to those using testicular fresh spermatozoa from Micro-TESE. A possible cause of these poor results has been the lack of a successful freezing technique. In these cases, repeated Micro-TESE and simultaneous oocyte pick up has been the only available treatment. We developed an innovative ultra-rapid vitrification method for five or fewer testicular spermatozoa from non-obstructive azoospermic men after Micro-TESE. This study included 113 non-obstructive azoospermic men using Micro-TESE, it was conducted retrospectively at St. Mother Clinic between 2011 and 2018. Each motile spermatozoon was carefully aspirated tail first into the pipette, put into a 2-μl microdroplet media of the vitrification medium near the tip of the Cryotop (Kitazato Corporation, Tokyo, Japan) submerged in liquid nitrogen vapor for 2 min and then immediately plunged into liquid nitrogen. The vitrified spermatozoa were warmed by dipping them into a droplet media. Successfully recovered motile sperm were selected and used for ICSI. Number of patients, transfer cycles and collected sperms were 113, 192 and 560. Mean age of patients and their wives were 32.0±3.7y and 28.4±5.8y. Clinical pregnancy rate, miscarriage rate, live birth rate and number of live offspring were 24.0% (46/192), 19.6% (9/46), 19.3% (37/192) and 37 (Male: Female = 17: 20). Sperm recovery rate and survival rate were 90.3% (506/560) and 70.4% (356/506). Fertilization rate and mean number of transferred embryos were 51.6% (99/192) and 1.73 (1-2). Mean gestational weeks and mean body weight at birth were 39.23±5.27w and 2852.31±314.28g. No congenital anomalies were observed in any of the babies. This method for cryopreserving five or fewer spermatozoa from non-obstructive azoospermic men showed a recovery rate above 90% and a survival rate of about 70% which is the highest data ever been reported. The clinical pregnancy rate was 24.0% and 37 healthy babies were born.

P–126 A comparison of the efficacy of sperm freezing using high security tubes versus high security straws

Abstract Study question Are there any advantages in using High security tubes rather than High Security straws for conventional slow sperm freezing? Summary answer Freezing sperm in High Security tubes (HST) improved post-thaw recovery rate and motility, and also reduced processing and handling compared to High Security straws (HSS). What is known already The use of High Security freezing consumables (HSFC) in an IVF setting is a safe and effective way of eliminating concerns related to viral cross-contamination during storage. The lower diameter of HSS does make them susceptible to warming during handling. The HSFC used in this study is the only CE marked products that are made of resin, leak-proof and shatter-proof in all cryogenic temperatures even in LN2. No previous studies have compared the use of HST with HSS for conventional human sperm freezing. This study sets out to investigate the performance of HST compared to HSS. Study design, size, duration The study was designed as a controlled split-sample study with blind post-thaw analysis. Following the routine WHO analysis of 20 semen samples, the remainder of each of the samples was evenly divided and cryopreserved by conventional slow freezing in each of the two different HSFC. The freeze was conducted simultaneously by the same practitioner, employing the same freezing protocol and cryoprotectant. The pre-freeze and post-thaw concentration, total and progressive sperm motility were recorded. Participants/materials, setting, methods At one IVF clinic, semen samples with sperm density ≥15million/ml, ≥40% motility, ≥1.5ml were included. Cryoprotectant (SpermFreeze, Fertipro) was added dropwise to unprepared semen and kept at room temperature for 10 minutes before loading into HSFC (0.5ml CBS™HSS; CBS™HST). HSFC were heat-sealed (SYMS; SYMSIII sealers) and placed in vapour for 30 minutes before plunging into LN2. Samples were thawed by immersion in a 37Cº water bath for 5 minutes and analysed using WHO methods. Main results and the role of chance Paired-t test was used to compare the percentage motility between the different HSFC. All analysis was considered statistically significant when p &amp;lt; 0.01. We demonstrated that the sperm recovery rate (Percentage total motility post-thaw/ Percentage total motility pre-freeze) in HST was 66.63 ± 14.94 (mean ± standard deviation) compared to 40.80 ± 14.69 in HSS. In the HSS, the percentage post-thaw total motility was 19.99 ± 7.21 and the percentage post-thaw progressive motility was 12.26 ± 2.59. In the HST, the percentage post-thaw total motility was 32.57 ± 8.33 and the percentage post-thaw progressive motility was 23.08 ± 5.53. The overall improvement when using HST against HSS was 12.53 ± 5.69, 10.44 ± 5.29 for the total motility and the progressive motility respectively. Comments were recorded regarding the handling and the condition of the HSS and HST for each freeze event. Neither device displayed any leakage of LN2 or any explosion during the warming. The freezing process was easier and faster using HST rather than HSS. It was also noted that the entire sample can be recovered from the HST, unlike the HSS. Limitations, reasons for caution The study looked at sperm recovery in terms of motility only. DNA damage was not considered as a parameter of sperm quality. Also, fertilization, pregnancy rates, live birth rates and the use of poorer quality sperm samples have not been investigated. Wider implications of the findings: For conventional sperm freezing, the use of HST resulted in improved sperm motility and progression post-thaw, when compared to HSS. This finding supports the use of HST to improve the post thaw quality of sperm, benefitting patients with own frozen samples, recipients of donor sperm and donor sperm banks. Trial registration number Not applicable.

P–023 Innovative ultra-rapid vitrification method for five or fewer testicular spermatozoa from non-obstructive azoospermic men after microsurgical testicular sperm extraction (Micro-TESE)

Abstract Study question What technique can be used to successfully cryopreserve five or fewer testicular spermatozoa from non-obstructive azoospermic men? Summary answer This method for cryopreserving five or fewer spermatozoa from non-obstructive azoospermic men showed a recovery rate above 90% and a survival rate of about 70%. What is known already Clinical outcomes of ICSI when using only five or fewer testicular spermatozoa after cryopreservation have been unsuccessful and are considered to be inferior to those using testicular fresh spermatozoa from Micro-TESE. A possible cause of these poor results has been the lack of a successful freezing technique. In these cases, repeated Micro-TESE and simultaneous oocyte pick up has been the only available treatment. Study design, size, duration Evaluation of the efficiency of cryopreservation by modified permeable cryoprotectant-free vitrification method (HTF supplemented with 0.1M sucrose and 10% SPS) for five or fewer testicular spermatozoa from 113 non-obstructive azoospermic men using Micro-TESE was conducted retrospectively at St. Mother Clinic between 2011 and 2018. Participants/materials, setting, methods This study included 113 non-obstructive azoospermic men. Each motile spermatozoon was carefully aspirated tail first into the pipette, put into a 2-μl microdroplet media of the vitrification medium near the tip of the Cryotop (Kitazato Corporation, Tokyo, Japan) submerged in liquid nitrogen vapor for 2 min and then immediately plunged in liquid nitrogen. The vitrified spermatozoa were warmed by dipping them into a droplet media. Successfully recovered motile sperm were selected and used for ICSI. Main results and the role of chance Number of patients, transfer cycles and collected sperms were 113, 192 and 560. Mean age of patients and their wives were 32.0±3.7y and 28.4±5.8y. Clinical pregnancy rate, miscarriage rate, live birth rate and number of live offspring were 24.0% (46/192), 19.6% (9/46), 19.3% (37/192) and 37 (Male: Female = 17: 20). Sperm recovery rate and survival rate were 90.3% (506/560) and 70.4% (356/506). Fertilization rate and mean number of transferred embryos were 51.6% (99/192) and 1.73 (1–2). Mean gestational weeks and mean body weight at birth were 39.23±5.27w and 2852.31±314.28g. No congenital anomalies were observed in any of the babies. Limitations, reasons for caution The maximum number of spermatozoa to which this method can be applied successfully is about 10. When the number of aspirated spermatozoa is over 10, some of them change direction and reach the mineral oil, and once this happens, they cannot be expelled out of the pipette. Wider implications of the findings: This technique is very useful for the cryopreservation of very small numbers of testicular spermatozoa (fewer than 10) in order to avoid or reduce Micro-TESE interventions. Trial registration number N/A

P–038 Clinical outcomes of 77 testicular sperm extraction treatment cycles in non-mosaic Klinefelter syndrome patients

Abstract Study question What are the clinical results of patients with azoospermia and nonmosaic Klinefelter syndrome, using fresh and cryopreserved sperm? Summary answer The results showed a recovery rate of testicular sperm in the order of 40% and a life newborn rate of 52% when using fresh sperm What is known already In Klinefelter syndrome (KS), the rates of successful testicular sperm retrieval were shown to be similar either using conventional TESE or micro-TESE (Corona et al., 2017), which highlights that the variability observed between studies is due to differences in patient characteristics. There are a few works with a large number of KS patients elucidating the clinical outcomes using fresh and cryopreserved testicular sperm. However, these studies revealed contradictory outcomes, either revealing better (Greco et al., 2013; Vicdan et al., 2016) or worst (Madureira et al., 2014) results with cryopreserved testicular sperm, or finding no differences (Chen et al, 2019). Study design, size, duration This study includes all patients up to 2019 presenting azoospermia due to non-mosaic Klinefelter syndrome (n = 76) that went for infertility consultations in a private fertility clinic. Patients were evaluated by the same Urologist. The genetic analysis of the patients was performed at an academic institution. At examination patients did not refer other complaints besides infertility, and referred to have not received any hormone replacement therapy in the past. Participants/materials, setting, methods The 76 azoospermic patients with non-mosaic Klinefelter syndrome (KS) were treated by testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI), using fresh and cryopreserved testicular sperm. Most patients used fresh testicular sperm, where others preferred to postpone ICSI treatment cycles and used cryopreserved testicular sperm. Aneuploidy screening in children was performed by prenatal diagnosis and MLPA (Multiplex ligation-dependent probe amplification). Full embryological, clinical and newborn outcomes are provided. Main results and the role of chance Of the 76 patients with non-mosaic Klinefelter syndrome, one repeated the testicular sperm extraction (TESE) procedure. Testicular sperm were recovered in 31/77 (40.3%) of the cases. Comparisons between the 31 cases with successful sperm recover (group–1) and the 46 cases without a successful TESE (group–2) revealed no significant differences regarding age, time of infertility, testicular volume, serum levels of FSH, LH and testosterone, total number of testicular fragments analyzed, and time of search in samples. The mean male age was 34 years. In most of the cases, the testicular volume was reduced (96.1%), the levels of FSH (98.3%) and LH (94.1%) were increased, and the levels of testosterone were normal (77.6%). There were 25 intracytoplasmic sperm injection (ICSI) treatment cycles using fresh testicular sperm and 22 ICSI treatment cycles using frozen testicular sperm. The rates of fertilization (63.5% fresh sperm vs 41.6% frozen sperm), implantation (37% fresh sperm vs 13.2% frozen sperm), clinical pregnancy (60.9% fresh sperm vs 19% frozen sperm), live birth delivery (52.2% fresh sperm vs 19% frozen sperm) and newborn (65.2% fresh sperm vs 23.8% frozen sperm) were higher in the group using fresh testicular sperm. Chromosome analysis of the 21 newborn was normal. Limitations, reasons for caution Although presenting a high number of cases with azoospermic non-mosaic Klinefelter syndrome treated with testicular sperm extraction and intracytoplasmic sperm injection, future studies are needed with a higher number of cycles using frozen testicular sperm, in order to confirm or rebut that the freezing methodology affects negatively the clinical outcomes. Wider implications of the findings: Data adds further information regarding testicular sperm retrieval rates and use of fresh or frozen testicular sperm in Klinefelter syndrome (KS) patients. High newborn rates were obtained only with fresh testicular sperm. Results also reassure KS patients about the safety relative to any abnormal chromosomal transmission to the born children. Trial registration number Not applicable

Vitrification with microinjection of single seminiferous tubules: an efficient cryopreservation approach for limited testicular tissue

Research questionIs vitrification with microinjection of single seminiferous tubules an efficient cryopreservation approach for limited testicular tissue? DesignTesticular tissue from 10 patients with normal spermatogenesis were assigned to a fresh control group or one of the following cryopreservation procedures: uncontrolled slow freezing (USF) using either 1.5 or 2.1 M DMSO combined with sucrose and vitrification with or without single seminiferous tubules microinjection. ResultsSingle seminiferous tubules microinjected with cryoprotective agents (CPA) enhanced the penetration of CPA compared with CPA-treated testicular tissue fragments. Microinjection of seminiferous tubules (VLP) maintained tubule structural integrity and germ cell numbers, and reduced spermatogonial apoptosis after cryopreservation compared with vitrification without microinjection (apoptosis rate: VLP versus vitrification without microinjection, P = 0.047; VLP versus USF, P= 0.049). Freezing of single seminiferous tubules using 0.25-ml straws and traditional sperm freezing methods protected sperm retrieval and recovery rates, and the progressive motility index. ConclusionsVitrification of single seminiferous tubule with microinjection of low CPA concentration is an effective approach to testicular cryopreservation.

Comparative study of agarose-gel microcapsules and Cryotop in cryopreservation of extremely small numbers of human spermatozoa

ABSTRACT We investigated the feasibility of agarose-gel microcapsules to cryopreserve extremely small numbers of sperm for assisted reproductive technology. Semen samples were collected from 16 patients attending the center for reproductive medicine male infertility clinic at a university hospital. We used agarose microcapsules to cryopreserve extremely small numbers of sperm from 16 patients with male infertility (10 with sperm concentration ≥1 million/mL; 6 with sperm concentration <1 million/mL). Six spermatozoa were injected into agarose-gel microcapsules and cryopreserved in a liquid nitrogen tank for 7 days. The Crytop method was used for cryopreservation as a control. After thawing, spermatozoa were recovered. Sperm recovery rates, motility and viability, and recovery time were compared. The post-thawing recovery rate, motility rate, and viability rate were higher whereas the recovery time was shorter in samples preserved using the agarose-gel microcapsule method compared to samples preserved using the Cryotop method in both the group with sperm concentrations of 1 million/mL or above and the group with sperm concentrations of less than 1 million/mL. This study demonstrated that using the agarose-gel microcapsule method increased post-thawing sperm recovery rate, sperm motility rate, and sperm viability rate, and reduced sperm recovery time compared with the conventional Cryotop method when cryopreserving samples with low sperm count. Although requiring further study, the agarose-gel microcapsule method shows much promise as a new option for freezing sperm.