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

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 < 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.

A microfluidic approach to rapid sperm recovery from heterogeneous cell suspensions

The isolation of sperm cells from background cell populations and debris is an essential step in all assisted reproductive technologies. Conventional techniques for sperm recovery from testicular sperm extractions stagnate at the sample processing stage, where it can take several hours to identify viable sperm from a background of collateral cells such as white bloods cells (WBCs), red blood cells (RBCs), epithelial cells (ECs) and in some cases cancer cells. Manual identification of sperm from contaminating cells and debris is a tedious and time-consuming operation that can be suitably addressed through inertial microfluidics. Microfluidics has proven an effective technology for high-quality sperm selection based on motility. However, motility-based selection methods cannot cater for viable, non-motile sperm often present in testicular or epididymal sperm extractions and aspirations. This study demonstrates the use of a 3D printed inertial microfluidic device for the separation of sperm cells from a mixed suspension of WBCs, RBCs, ECs, and leukemic cancer cells. This technology presents a 36-fold time improvement for the recovery of sperm cells (> 96%) by separating sperm, RBCS, WBCs, ECs and cancer cells into tight bands in less than 5 min. Furthermore, microfluidic processing of sperm has no impact on sperm parameters; vitality, motility, morphology, or DNA fragmentation of sperm. Applying inertial microfluidics for non-motile sperm recovery can greatly improve the current processing procedure of testicular sperm extractions, simplifying the fertility outcomes for severe forms of male infertility that warrant the surgery.