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

• Published in: Primary care update for Ob/Gyns 1998-07, Vol.5 (4), p.170-170
• Main Authors: Ziegler, William F., Chapitis, Jane
• Format: Article
• Language: English
• ISSN: 1068-607X; 1878-4283
• DOI: 10.1016/S1068-607X(98)00072-9

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