ICSI with Frozen/Thawed Sperm is Less Effective than with Fresh Sperm in a Primate Model

Abstract

Objective: Comparison of fertility between frozen-thawed and fresh sperm using ICSI was done to detect the presence of occult cyro-damage. A non human primate model was used due to ethical constraints in the human for this evaluation using normal male sperm and oocytes. Design: A prospective study in a primate model at a regional primate research center. Methods: Semen collected from male rhesus monkeys by penile electroejaculation was extended with either A) 80 mM sucrose, 5% glycerol in TALP-Hepes or B) 60 mM glucose, 12% egg yolk, 8% skim milk, 3% glycerol in TEST buffer. Both groups were equilibrated 90 min at 4°C before freezing in liquid nitrogen (LN) vapors for 30 min and storage in LN for >24 hrs. Straws were thawed 5 sec in 37°C water bath, washed twice and diluted to 5 × 106/ml. ICSI was performed on mature oocytes retrieved from cycling females exposed to follicular stimulation with recombinant human hormones. Injected oocytes were co-cultured on BRL cells and observed for the presence of 2 pronuclei or cleavage as evidence of fertilization. Results: Prefreeze motilities of samples frozen with extenders A (n=8) and B (n=9) were similar (>80%). Post-thaw percent motility was higher with extender B (31.2 ± 8.3% vs 58.6 ± 7.3%, P<0.05) and with greater forward progression, however, both methods provided adequate samples for ICSI. In 4 ICSI experiments with F/T sperm from method A, oocytes fertilized at a lower rate (16 of 39; 41%) compared to 30 of 38 oocytes injected with fresh sperm (79%) (P<0.01). Similarly, in 5 experiments with F/T sperm from method B, 22 of 40 oocytes (55%) fertilized compared to 34 of 40 oocytes injected with fresh sperm (85%) (P<0.01). Development to blastocyts by day 8 was similar in all groups (50–70%). Conclusions: Reduced fertilization rates after ICSI with F/T sperm suggest a deficiency in oocyte activating ability even though good motility is recovered post-thaw. While no loss in embryonic development was observed, ICSI with F/T sperm may require superimposition of oocyte activation strategies for efficient use in banking of valuable genetic stocks. Supported by NIH RR12804, A142709, and Ares Advanced Technology, Inc.