Introduction: Sperm cryopreservation requires in-person visits to produce an ejaculate and immediately freeze the sample. Recently, companies began offering services for mail-in semen analysis (SA) testing and cryopreservation. However, the effects of delayed cryopreservation associated with shipping times were generally understudied. Our laboratory has continued to optimize our cryopreservation technique. This study aims to define the effect of delayed cryopreservation on the recovered total motile count (TMC) compared to standard immediate cryopreservation, using our improved methods. Methods: Fresh semen samples were provided from healthy volunteers. Samples were diluted with 5mL of a proprietary preservation solution, mixed, and allowed to liquify for 30 minutes. Samples were then analyzed per World Health Organization (WHO) guidelines V5 and those with abnormal concentration, volume, or motility were excluded. Samples were then divided into 3 aliquots. Aliquot #1, T0 was immediately frozen with equal part cryoprotectant. Aliquot #2, T24 was left at room temperature for 24hrs, analyzed on the SQA (sperm quality analyzer) and then frozen with cryoprotectant. Aliquot #3, T48 was left at room temperature for 48hrs, analyzed on the SQA, and then frozen with cryoprotectant. 24hrs after the third aliquot was frozen, all aliquots were thawed in an incubator at 37C for 10 minutes and analyzed on the SQA. Results: Eight adult men provided a total of 26 semen samples. Baseline SA values were: volume 3.4ml +/- 1.2, concentration 128.2 +/-48.7 x 106, motility 49% +/-7%; TMC 211 +/- 109 million. A decline in % motility was observed after specimens sat at room temperature, as well as after freeze-thaw in most individuals. The average loss from freeze/thaw in TMC at T0 was 44.0%, at T24 was 47% relative to T24 pre-freeze, and at T48 was 65% relative to T48 pre-freeze (p=0.046) (Figure 1). Conclusions: This analysis reveals a superior freeze-thaw protocol compared to our prior methods, as we continue to refine our process, and follow best practice guidelines of experts in the field. Further investigation is needed to improve the methods needed for shipping with the goal of improving the freeze-thaw recovery rate with delayed cryopreservation. SOURCE OF Funding: Fellow Health Inc.
