Cryoprotectant‐free vitrification of human spermatozoa in new artificial seminal fluid

Abstract

Vitrification is a new method that has been recently introduced in Assisted Reproduction Technique programs. The aim of this study was to design a new medium similar to normal human seminal fluid (SF), formulation artificial seminal fluid (ASF), and to compare the cryoprotective potency of this medium with SF and human tubal fluid (HTF) medium. Thirty normal ejaculates were processed with the swim-up technique and sperm suspensions were divided into four aliquots: (i) fresh sample (control); (ii) vitrification in HTF medium supplemented with 5 mg/mL human serum albumin and 0.25 mol sucrose (Vit HTF); (iii) vitrification with patients' SF (Vit SF); and (iv) vitrification in ASF (Vit ASF). After warming, sperm parameters of motility, viability, and morphology were analyzed using WHO criteria. Also, sperm pellets were fixed in 2.5% glutaraldehyde and processed for scanning electron microscopy and transmission electron microscopy observations. The results showed that progressive motility (46.09 ± 10.33 vs. 36.80 ± 13.75), grade A motility (36.59 ± 11.40 vs. 16.41 ± 11.24), and normal morphology (18.74 ± 8.35 vs. 11.85 ± 5.84) and viability (68.22 ± 10.83 vs. 60.86 ± 11.72) of spermatozoa were significantly higher in Vit ASF than in Vit HTF. All parameters were better in Vit ASF than in Vit SF, but only viability was significantly different (p = 0.006). After cryopreservation, deep invagination in cytoplasm and mechanically weak point sites and folded tail were commonly observed. But, this phenomenon was more significant in Vit HTF and Vit SF than in ASF (p < 0.05). In transmission electron microscopy evaluation, acrosome damage, plasma membrane loss, chromatin vacuolation, and disruption of mitochondria arrangement and structures were observed in all vitrified groups. Adherence of several tail sections together was also seen in all cryo groups. But this was seen more in Vit HTF and Vit SF than in ASF (p < 0.05). In conclusion, vitrification of human spermatozoa with ASF can effectively preserve the quality of sperm motility in comparison with Vit HTF.

Looking back and looking forward: contributions of electron microscopy to the structural cell biology of gametes and fertilization

“…Comparing oocytes that were in vitro maturated to in vivo matured by conventional TEM indicated that they are overall similar [64]. Successful cryopreservation of gametes and embryos is crucial for ART and thus its effect was studied extensively [55,[65][66][67][68][69][70][71]. SEM was used to show that the needle used for intracytoplasmic sperm injection (ICSI) does not damage the ZP or oolemma [72].…”

Simulated Genital Tract Fluids and Their Applicability in Drug Release/Dissolution Testing of Vaginal Dosage Forms

“…Finally, with the aim of evaluating an artificial culture medium similar to seminal plasma with regard to its effect on the fine structure of human spermatozoa postvitrification, Agha-Rahimi et al developed an artificial seminal fluid (ASF) based on the recipe of Owen and Katz (49). For their objective, they slightly modified the Owen and Katz SFS medium by adding antioxidants, changing the main buffering species, and adjusting the osmolality.…”

Cryoprotectant-free vitrification of spermatozoa: Fish as a model of human

“…In publications of Isachenko et al (), Chen et al (), Agha‐Rahimi, Khalili, Nottola, Migliett, and Moradi () authors describe effective technologies with direct dropping of sperm suspensions into liquid nitrogen.…”