Phosphatidylcholine and L-acetyl-carnitine-based freezing medium can replace egg yolk and preserves human sperm function

Abstract

Background: Conventional cryopreservation methods induce chemical and mechanical damage to the sperm membranes. The cryoprotectant potential of phospholipids of vegetal origin as soybean lecithin has been investigated as a substitute for egg yolk in diluents used for the cryopreservation of human spermatozoa. Therefore, the objective of this study was comparing the efficacy of a synthetic cryoprotectant supplemented with L-α-phosphatidylcholine (PC) and L-acetyl-carnitine (ANTIOX-PC) and the standard egg-based TEST-yolk buffer (TYB) in preserving sperm motility and chromatin quality in cryopreserved semen samples.

Methods: Prospective experimental study in which semen samples from 63 men with normal sperm motility and 58 men with low sperm motility were included and analyzed both before and after cryopreservation using ANTIOX-PC or TYB freezing media. Sperm quality was evaluated by routine semen analysis and DNA fragmentation index using the Terminal deoxynucleotidyl transferase dUTP nick end labeling assay.

Results: Differences in the post-thaw progressive motility and DNA fragmentation index were not detected between TYB and ANTIOX-PC cryoprotectants in both normal and low sperm motility groups (P>0.05). However, ANTIOX-PC medium retained higher non-progressive motility and lower percentage of immotile sperm when compared to TYB medium, resulting in a greater total motile sperm count (P<0.05), regardless baseline values of motility characteristic of the normospermic or asthenozoospermic samples.

Conclusions: ANTIOX-PC medium was effective to protect human sperm during a freeze-thaw cycle compared to the TYB medium. A clinically relevant advantage in better preserving kinetic parameters as higher total motility and lower immotile post-thawed sperm from ANTIOX-PC, in normal and low motility semen samples, demonstrated the positive impact of phospholipid and antioxidant treatment on sperm cryotolerance with high potential for egg yolk lipids replacement and biosafety.

Keywords: DNA fragmentation index; L-acetyl-carnitine; Lα-Phosphatidylcholine; Sperm cryopreservation; progressive motility.

Figure 1

Terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) positive human spermatozoa obtained from men with low motility and cryopreserved in the standard egg-based TEST-yolk buffer (TYB) (A,B,C,D) or L-α-phosphatidylcholine (PC) and L-acetyl-carnitine (ANTIOX-PC) (E,F,G,H) medium. 4',6-diamidino-2-phenylindole (DAPI) (A and E), Fluorescein isothiocyanate-stained image (FITC) (B and F), merge (C and G), and phase-contrast image (D and H). TUNEL negative spermatozoa fluoresced blue (spermatozoon without fragmented DNA), whereas the TUNEL-positive spermatozoa fluoresced bright green (spermatozoon with fragmented DNA). Scale bar of 25 µm.