Comparison of postthawing sperm motility recovery between cryopreserved with and without cryoprotective agent using 4 different cryopreservationmethods

Abstract

Frozen-thawed human spermatozoa are routinely used for many assisted reproduction program.
However, cryopreserved spermatozoa was reported to yield lower pregnancy rates compared to
fresh semen in both intra uterine inseminations and in vitro fertilization/intracytoplasmic sperm
injection (IVF/ICSI) due to the reduction of sperm motility and viability induced by cryopreservation
procedures. This study was aimed to evaluate the influence of cryoprotective agent (CPA) and
cryopreservation methods on human sperm motility. This was a quasi experimental study. Thirty
seven normozoospermic semen samples collected in Permata Hati Infertility Clinics of Dr. Sardjito
General Hospital, Yogyakarta were recruited. Four different cryopreservation methods were applied
using and without CPA (TEST-yolk buffer). In simple two steps freezing, cryostraw were gradually
frozen from 8 to -4°C. In simple graduated freezing, cryostraus were directly frozen at -4°C. In
vapor phase freezing method, the samples in cryostraw were placed 1 cm above liquid nitrogen.
In the last method, the samples were directly submerged into liquid nitrogen. Thawing was
conducted by incubation at 37°C for 5 minutes. The sperm motility recovery after cryopreservation
in the 4 different cryopreservation methods was evaluated and analyzed by analysis of variance
(ANOVA). The fresh sperm motility before cryopreservation was 52.9 ± 4.50%. The recovery of
motile sperms was 17.00 ± 7.83%, 20.96 ± 5.81%, 15.06 ± 8.55% and 15.68 ± 8.3%, when
using CPA and 5.63 ± 4.63%, 5.47 ± 3.95%, 4.45 ± 4.46% and 6.08 ± 5.06% when without
CPA following direct plunge to liquid nitrogen freezing, vapor liquid nitrogen freezing, simple
graduated freezing and simple 2-steps freezing, respectively. Among methods using CPA, the
vapor phase method resulted in highest sperm motility recovery. In methods without CPA, no
significant difference of sperm motility recovery was observed among the 4 different
cryopreservation methods. In conclusion, the use of CPA for cryopreservation improves sperm
motility recovery.