Human motile sperm recovery after cryopreservation: freezing in nitrogen vapor vs the direct plunge technique

Abstract

Objectives: The following will be tested: 1) Freezing in nitrogen vapor prior to submerging will have a higher motile sperm recovery rate compared to directly submerging in liquid nitrogen, 2) The length of time a sample spends in nitrogen vapor will not affect the motile sperm recovery, and 3) The volume of sample to be frozen will not affect the recovery of motile sperm. Design: This is a prospective study performed through a university infertility clinic. The semen samples were obtained from 20 male partners of couples being screened for in vitro fertilization. Materials and Methods: Semen was collected and washed with 5 volumes of modified Ham’s F10 medium and centrifuged at 120× g for 10 minutes. The pellet was resuspended in 5 mL of medium and centrifuged in a similar manner. The swim-up sperm was prepared with the second pellet overlaid with 1 mL of medium, containing 1% human serum albumin, and incubated for 1 hour at 37 C. Equal volumes of sperm suspension and cryoprotectant were mixed by gentle stirring. Sperm density and motility were recorded before freezing. Each vial contained 1 mL of the sperm/cryoprotectant mixture. All vials were eventually submerged in liquid nitrogen for 40 minutes and thawed at room temperature for 1 hour. The percent of motile sperm recovered was assessed. Hypothesis 1: Comparing freezing in nitrogen vapor versus direct plunge, one vial was frozen for 6 minutes in the nitrogen vapor 2 cm above the liquid surface prior to submerging. The paired vial was kept at room temperature for 6 minutes before submerging. Hypothesis 2: Two vials were frozen in vapor for 6 and 12 minutes prior to submerging them. Hypothesis 3: To assess the effect of sample volume on motile sperm recovery, three separate vials were prepared, each containing either 1.0, 0.5, or 0.25 mL of the sperm/cryoprotectant mixture. Each vial was suspended in vapor for 6 minutes prior to submerging into the liquid. Paired t test analysis was used for objectives 1 and 2, with repeated measures analysis of variance used to determine the significance of objective 3. Results: Pure motile sperm density and motility were assessed for 20 samples after the addition of cryoprotectant. The sperm density and motility ranged from 0.9 to 120 million sperm/mL (33.2, SD ± 35.0) and 78–100% (86.5, SD ± 12.4), respectively. The mean motile sperm recovered for vapor freezing prior to plunge was 54.9% (SD ± 15.4), compared to submerging directly, which was 21.5% (SD ± 10.0); this was significant with a P < .05. The initial sperm density, <20 or ≥20 million/mL, did not influence the percent of motile sperm recovered ( P > .05). Motile sperm recovered from samples in vapor for 6 and 12 minutes was 49.7% (SD ± 8.71) and 51.8% (SD ± 10.4), respectively; this was not significant. The volume of purified motile sperm frozen did not significantly alter the percent of motile sperm recovered. Conclusion: Directly submerging a sample from room temperature into liquid nitrogen has shown a reduction of motile sperm recovery by 65.0%, compared to exposing the sample to liquid nitrogen vapor prior to submerging, which only showed a reduction of 31.7%. Motility appears to be influenced by the rate at which the sample is frozen. There was no significant impact on motile sperm recovery compared to the amount of sperm preserved, length of time the sample was in liquid nitrogen vapor, or the initial sperm density. This is the first study to isolate the motile faction from semen and examine the recovery rate between the two cryopreservation techniques. The clinical application implies that increasing the recovery of motile sperm could improve pregnancy rates and this simplified technique is possible without obtaining special equipment that could impose a significant financial burden with similar results.