SHORT COMMUNICATION

Vitrified sperm banks: the new aseptic technique forhuman spermatozoa allows cryopreservation at �86 °CR. Sanchez1,2, J. Risopatron1,3, M. Schulz1,2, J. V. Villegas1,4, V. Isachenko5 & E. Isachenko5

1 BIOREN-CEBIOR, Universidad de La Frontera, Temuco, Chile;

2 Department of Preclinical Sciences, Faculty of Medicine, Universidad de La Frontera, Temuco, Chile;

3 Department of Basic Sciences, Faculty of Medicine, Universidad de La Frontera, Temuco, Chile;

4 Department of Internal Medicine, Faculty of Medicine, Universidad de La Frontera, Temuco, Chile;

5 Department of Obstetrics and Gynecology, University of Cologne, Cologne, Germany

Keywords

Cryopreservation—human sperm—semen

bank—sperm function—sperm vitrification

Correspondence

Raul Sanchez Gutierrez, MD, Facultad de

Medicina, Universidad de La Frontera,

Avenida Francisco Salazar 01145, Casilla

54-D, Temuco, Chile.

Tel.: +56 45 324248;

Fax: +56 45 325600;

E-mail: rsanchez@ufro.cl

Accepted: April 02, 2012

doi: 10.1111/j.1439-0272.2012.01314.x

Summary

The vitrification technique is simple, quick, cost-effective and has showed a sig-

nificantly stronger cryoprotective effect in contrast to conventional freezing.

The method is based on the rapid cooling of the cell by direct immersion in

liquid nitrogen (LN2), thereby avoiding the formation of ice crystals, due to

the lower risk of water thawing, which impairs cell function. The aim of this

study was to evaluate the effect of storage at �86 °C compared to the conven-

tional �196 °C (under LN2) on essential parameters of the functioning of

aseptically vitrified human sperm. Sperm motility, integrity of mitochondrial

membrane potential and the rate of DNA fragmentation were determined. The

comparison of �86 °C and �196 °C demonstrated no statistical difference in

sperm progressive motility (73% vs. 77%), integrity of mitochondrial mem-

brane potential (71% vs. 74%) or DNA fragmentation (3.1% vs. 2.9%). In con-

clusion, aseptically vitrified sperm can be preserved at �86 °C; eliminating the

use of LN2 simplifies and significantly reduces the costs associated with storage

in sperm banks by decreasing the time and space needed for storage, the effort

in finding stored samples, and by improving safety for the operator. However,

for prolonged storage further studies are needed.

Introduction

The conventional freezing of spermatozoa is currently used

to preserve fertility in patients with cancer or for reproduc-

tive medicine programs. However, this technique causes

important chemical–physical damage to the intracellular

structures including sperm membranes by increasing lipid

peroxidation, decreases sperm motility rates and mito-

chondrial activity, inducing various processes associated

with cell death (O’Connell et al., 2002). As an alternative,

we have recently developed a vitrification aseptic technique

that preserves the sperm cells without the need to add sem-

inal plasma and/or cryopreservants (Isachenko et al.,

2011a,b). Because spermatozoa contain large amount of

proteins, sugars and other components, the intracellular

matrix is highly viscous and compartmentalised and may

act as natural cryoprotectant. Although the spermatozoon

is osmotically fragile, it can be frozen using proteins and

sugars as nonpermeable extracellular cryoprotectants, thus

avoiding the use of permeable cryoprotectants (Koshimoto

et al., 2000). According to these data, we have also previ-

ously analysed the contribution of glucose, sucrose and tre-

halose as sperm cryoprotective agents during the

vitrification method. The best preservation of motility and

viability was obtained post-thawing with 0.25 M sucrose in

human tubular fluid medium (Schulz et al., 2006;

Isachenko et al., 2008). The vitrification technique showed

a significantly stronger cryoprotective effect as compared

to standard slow freezing. This method adequately pre-

served the sperm function with high motility, low cryoca-

pacitation, high mitochondrial membrane potential, low

DNA fragmentation, acrosome well-conserved and high

membrane integrity in comparison with standard method

(Isachenko et al., 2004, 2008, 2011a,b). Vitrification is

based on the ultra-rapid freezing of the cell by direct

immersion in liquid nitrogen (LN2), thereby avoiding the

© 2012 Blackwell Verlag GmbH 1Andrologia 2012, xx, 1–3

formation of ice crystals. This might increase the tempera-

ture needed to store sperm due to the lower risk of ice

thawing that impairs the cell function. The aim of this study

was to evaluate the effect of storage at �86 °C compared to

conventional �196 °C (under LN2), on essential parameters

of the function of aseptically vitrified human sperm.

Materials and methods

Vitrification aseptic technique

Five semen samples were obtained from five healthy

donors. The sperm suspension after swim-up was centri-

fuged, and the cells were re-suspended at 15 9

106 cells ml�1 in vitrification solution [0.25 M sucrose in

HTF medium (Quinn et al., 1985)] supplemented with

1% human serum albumin (HTF-HSA). A sterile 0.25-ml

insemination straw (Minitub, Tiefenbach, Germany) was

filled with 100 ll of the sperm suspension; these were

placed in 0.5 ml plastic straws hermetically closed by heat

at both sides and plunged into LN2. Twelve straws were

prepared for each donor. Then, half the straws were

stored at �86 °C and the other half at �196 °C for

60 days and then thawed for analysis. The warming was

performed by quickly and directly submerging one straw

in 5 ml of HTF-HSA pre-warmed at 37 °C.

Evaluation of sperm motility

Sperm motility was assessed immediately after warming

the samples. Motility was estimated under the light

microscope using 4009 magnifications.

Detection of DNA fragmentation

DNA fragmentation was evaluated by TUNEL assay (Gor-

czyca et al., 1993), using the In Situ Cell Death Detection

kit (Roche®, Mannheim, BW, Germany) according to

manufacturer’s instructions. The sperm that had incor-

porated the fluorescent label at the damaged sites of the

DNA were detected by flow cytometry (FACSCalibur;

Becton-Dickinson, Mountain View, CA, USA).

Detection of mitochondrial membrane potential

To evaluate the mitochondrial membrane potential of the

spermatozoa, 5, 5′, 6 6′–tetrachloro-1,1′,3,3′ tetraethyl-

benzimidazol-carbocyanine-iodide known as JC-1 (Smiley

et al., 1991) was used. This test was performed using the

Mitochondrial Permeability Detection kit AK-116 (MIT-

E-wTM; BIOMOL International LP, Plymouth Meeting,

PA, USA) according to the manufacturer’s instructions.

The red fluorescent sperm were detected using a FAC-

SCalibur flow cytometer (Becton-Dickinson).

Statistical analysis

The student’s t-test was used to assess the statistical sig-

nificance. Differences at P < 0.05 were considered statisti-

cally significant.

Results and discussion

Our results showed no significant difference between stor-

ing the vitrified human sperm either at �196 °C under

liquid nitrogen or at �86 °C. Table 1 shows the results

of sperm parameters evaluated in vitrified sperm after

storing at �86 °C or at �196 °C for 60 days. There was

no significant difference in sperm progressive motility

(73% vs. 77%), integrity of mitochondrial membrane

potential (71% vs. 74%) or DNA fragmentation (3.1% vs.

2.9%). These results demonstrate that it is possible to

eliminate the use of liquid nitrogen for male gamete stor-

ing. As vitrification does not use a specially developed

cooling program, it does not need to use permeable cryo-

protectants as does programmable conventional freezing.

Vitrification has been found to be simple, quick, cost-

effective and able to provide high recovery of motile sper-

matozoa after warming, as well as effective protection of

spermatozoa against the mutagenic effects of permeable

cryoprotectants (Fraga et al., 1991) and damage due to

cryopreservation (Hammadeh et al., 1999; Duru et al.,

2001; Isachenko et al., 2004; Meseguer et al., 2004). This

method, based on the ultra-rapid freezing of the cell by

direct immersion in LN2, may provide effective preserva-

tion of a very small volume �1 to 5 ll – of cell suspen-

sions (Isachenko et al., 2011a) or larger volumes – up to

500 ll – using the aseptic vitrification technique

described by Isachenko et al.(2011b). To test the effect of

storing the vitrified sperm at �86°, we evaluated sperm

motility and two methods that describe the cells’ integrity

and functionality.

Table 1 Maintenance of human sperm function after vitrification:

comparison between storing at �86 °C and �196 °C

Parameter

Vitrified human sperm

stored at

�196 °C �86 °C

Progressive (PR) motility (%) 77.0 ± 2.5 73.8 ± 2.4

Intact DΨm (%) 74.6 ± 1.6 71.7 ± 1.7

DNA fragmentation (%) 2.9 ± 0.8 3.1 ± 0.6

The vitrified human sperm were stored by 60 days, thawed and analy-

sed. The results were expressed as mean ± SD from five experiments.

The differences between both temperatures were not significant.

2 © 2012 Blackwell Verlag GmbH

Andrologia 2012, xx, 1–3

Sperm bank at �86 °C R. Sanchez et al.

We previously achieved high spermatozoa motility

(>70%) and showed that this new cryopreservation

method provides a high protection rate of cytoskeletal

structures (Schulz et al., 2006). According to recent

results from our laboratory, spermatozoa cryopreserved

by vitrification were able to produce pregnancy with a

subsequent healthy baby born after in vitro fertilisation or

intrauterine insemination (Isachenko et al., 2012; Sanchez

et al., 2011).

In summary, the aseptic vitrified sperm can be pre-

served at �86 °C, improving and simplifying the storing

process in sperm banks. The advantages of eliminating

the need for LN2 to store the cryopreserved spermatozoa

are reduction of costs, time, storage space, effort in find-

ing stored samples and increase in safety for the operator.

This method will be especially useful for oligoasthenotera-

tozoospermic patients for storing motile spermatozoa to

gather enough of them for intrauterine insemination or

prior to ICSI. However, for prolonged storage, further

studies are needed.

Acknowledgements

This work was supported by the Direccion de Investigac-

ion, Universidad de La Frontera, D10-0021.

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R. Sanchez et al. Sperm bank at �86 °C