A new media without animal component for sperm cryopreservation: motility and various attributes affecting paternal contribution of sperm

Abstract

Purpose: Our aim was the development of a safe sperm cryopreservation New Media (NM), composed of consistent and reproducible components devoid of any animal origin, and evaluation of NM in terms of its effect on sperm structure and function as compared to regularly used yolk media (TYM) (Irvine Scientific).

Methods: We evaluated patient semen samples and cryopreserved them in duplicates in either NM or TYM. The samples were cryopreserved for either a short term of 1 week or long term of 1 month prior to thawing. The parameters investigated include sperm motility via computer-assisted semen analysis (CASA), sperm concentration, and sperm biomarkers that promote paternal contribution of spermatozoa to fertilization including hyaluronic acid binding, chromatin maturity, apoptotic markers, cytoplasmic retention, and sperm DNA integrity.

Results: As compared to TYM, NM was equally capable of sperm cryopreservation with both short-term and long-term storage in media, and after freeze-thaw and gradient processing of sperm. HA binding of sperm was comparable post thaw in both NM and yolk media. There are also no differences observed between the samples cryopreserved in NM or TYM in terms of their aniline blue staining, CK immunocytochemistry, caspase 3 immunostaining, or DNA nick translation.

Conclusions: NM has the advantage of being xeno-free, yet in preservation of sperm motility and other sperm attributes, the NM is as effective as the TYM.

Keywords: Biochemical markers; Cryopreservation; HA binding; Male fertility; Sperm maturity; Sperm motility.

Fig. 1

The experimental design illustrates the assessment of sperm samples obtained from Yale Sperm Physiology Laboratory. Samples were collected in four sets (9, 13, 11, and 18) in duplicates. The sperm samples were first evaluated post liquefaction using computer-assisted semen analysis (CASA) for sperm motility, concentration, motility, and morphology. The samples were then diluted 1:1 in new media and subjected to freezing using liquid nitrogen vapor for 20 min followed by liquid nitrogen immersion. The samples were frozen for either short term (1 week) or long term (1 month) and then thawed at 37 °C for 10 min. Sample smears were used to evaluate sperm biomarkers via aniline blue staining, creatine kinase immunohistochemistry, hyaluronic acid binding, and DNA nick translation. Sample sets 11 and 18 were used to determine post thaw sperm motility and concentration at 0 min, 1 h, and 4 h post thaw. The sample sets 11 and 18 were also subjected to gradient fractionation protocol similar to the one used for in vitro fertilization procedure. The samples were then evaluated for sperm motility and concentration at 0 min, 1 h, and 4 h post gradient. Data for sample set 11 is shown for all experiments

Fig. 2

Comparison of the effects of incubation in NM and TYM media on sperm motility post thaw and cryopreservation media for short term (1 week) or long term (1 month) post gradient (p value <0.05), N = 11. a Sperm motility with short-term incubation. b Sperm motility with long-term incubation

Fig. 3

Comparison of the effects of incubation in NM and TYM media on sperm concentration post thaw and cryopreservation in media for short-term (1 week) or long-term (1 month) post gradient (p value <0.05); N = 11. a Short-term incubation. b Long-term incubation

Fig. 4

The effects of short-term or long-term cryopreservation in NM or TYM media on the percentage of light and dark aniline blue stained sperm cells. a Short term, N = 11. b Long term, N = 11

Fig. 5

The effects of short-term or long-term cryopreservation in NM or TYM media on the percentage of light and dark creatine kinase immunocytochemistry stained sperm cells. a Short term, N = 11. b Long term, N = 11

Fig. 6

The effects of short-term or long-term cryopreservation in NM or TYM media on HA binding capacity of sperm cells. p values are included in figure (p < 0.01). a Short term, N = 11. b Long term, N = 11

Fig. 7

The effects of short-term or long-term cryopreservation in NM or TYM media on the percentage of light and dark caspase 3 immunocytochemistry stained sperm cells. a Short term, N = 11. b Long term, N = 11

Fig. 8

The effects of short-term or long-term cryopreservation in NM or TYM media on the percentage of light and dark sperm cells post DNA nick translation. a Short term, N = 11. b Long term, N = 11