The influence of cryoprotective media and processing procedures on motility and migration of frozen-thawed human sperm

Linda S. McGonagle¹, Michelle Goldstein¹, Joseph Feldschuh², Robert H. Foote¹

Asian J Androl  2002 Jun; 4:  137-141             

Keywords: crypreservation; thaw temperature; sperm motility; sperm migration

Abstract

Aim: The study was designed to examine the effects of cryoprotective media, and glycerolating and thawing procedures on human sperm motility and gel penetrating ability. Methods: Fifteen unselected donors provided semen varying in quality that was distributed in a factorial design across three cryoprotectants (glycerol, egg yolk-citrate-glucose-glycerol and egg yolk-tris-glucose-glycerol). Also, glycerol was added at room temperature versus at 4. Two thaw temperatures were tested (laboratory air temperature for 10 min versus a 65 waterbath for 4 seconds). The proportion of total and progressively motile sperm was estimated immediately after thawing and following incubation at 35 for 2 h. Migration of sperm for 30 min at 37 through polyacrylamide gel was tested. Results: Donors differed greatly, with post-thaw total motility of sperm ranging from 9 to 44 % (P<0.05). Egg yolk-citrate-glucose-glycerol and egg yolk-tris-glucose-glycerol were superior to glycerol alone (post-thaw values of 35, 37 and 21 %, respectively, P<0.05). This was due primarily to poor sperm survival when semen was cooled to 4 without glycerol or egg yolk. The two thaw temperatures gave similar results. Sperm migration tests paralleled the motility results, but were more sensitive in detecting differences. Conclusion: Egg yolk, particularly in a tris-based medium that is widely used in domestic animals, improved the cryopreservation of both good and poor quality human semen.

1 Introduction

The survival of spermatozoa during freezing and thawing is affected by many factors. These include type and concentration of cryoprotectant, temperature at which the cryoprotectant is added, composition of the preserving medium, freezing rate and thawing tempera-ture, and many interactions [1-7].

Glycerol is the cryoprotectant most widely used for human semen following procedures introduced by Sherman [1]. Later, glycerol in egg yolk-citrate media, modified from a widely used semen extender for cattle [1], and TEST-egg yolk [5,8] were used for cryopre-servation of human sperm. Variable results have been reported using different method of freezing, but the inclusion of egg yolk in the protective medium usually is beneficial. No studies have been reported with human semen using a tris-buffered egg yolk cryoprotectant medium widely used successfully for many animal species [4,9].

The purpose of this study was to compare the addition of glycerol alone with glycerol in tris-egg yolk-glucose and citrate-egg yolk-glucose protective media, the temperature at which glycerol was added to the semen, and the thaw temperature, evaluated by the post-thaw motility and gel-penetrating ability of human sperm.

2 Materials and methods

2.1 Collection and examination of semen

Human semen was obtained via masturbation from 15 donors who were either regular semen donors or men who requested a routine semen analysis. One ejaculate from each donor was obtained after an abstinence period of 3 days. All samples were weighed to estimate volume and allowed to liquify at room temperature. Upon liquefaction each sample was evaluated microscopically by two observers using brightfield illumination at 200 to estimate the initial percentage of total motile sperm. Ten microscopic fields were averaged to arrive at this subjective estimate. Sperm cell concentration was determined with a Coulter counter. Means and standard errors for % motile sperm, volume, sperm concentration and total sperm per ejaculate, respectively, were 64 % 1.5 %, 4.10.4 mL, 89.612.010⁶ sperm/mL and 3685710⁶ total sperm. The ranges for these 15 sam-ples, respectively, were 55 % to 75 %, 2.5 to 6.9 mL, 32 to 23410⁶ sperm/mL, and 141 to 78410⁶ sperm per specimen.

Three extenders were used. One consisted of adding only 7 % glycerol (v/v) to the semen. A second extender contained 2.42 g of Tris, 1.36 g of citric acid monohydrate, and 1 g of glucose in 80 mL of double dis-tilled water, with 20 ml of egg yolk added [9]. A third extender contained 1.9 g of sodium citrate dihydrate and 1 g of glucose in 80 mL of double distilled water, with 20 mL of egg yolk added. These extenders were made up in two parts, one containing no glycerol and a second part containing 14 % glycerol (v/v), replacing an equivalent volume of water. Equal combinations of the two parts resulted in a final concentration of 7 % glycerol. These extenders were prepared in advance of the experiment, frozen at -20 in 50 mL beakers and thawed as needed for the experiment.

Sufficient straws were prepared so that semen in the three extender treatments had glycerol added at room temperature versus after cooling to 4, followed by freezing, and later thawing at two temperatures. Whole semen had 7 % (v/v) of glycerol added directly at the two temperatures, one at about 25, and the other after cooling semen for 35 min to 4. Immediately after adding glycerol, 0.25 mL straws were filled, placed in a mechanical freezer at -68 for 30 min and transferred to liquid nitrogen at -196.

Semen treatments involving the two egg yolk extenders and two temperatures of adding glycerol were processed similar to the ones with glycerol alone. Semen that was to have glycerol added at 4 was pre-extended with egg yolk buffer solution (citrate or tris) containing no glycerol and was allowed to cool to 4 for 35 min. Subsequently, 14 % glycerolated extender was added in equal volume in one step. The semen was packaged in straws and placed in a freezer at -68 for 30 min before being transferred to a liquid nitrogen tank. The semen that was exposed to glycerol at room temperature was added directly to egg yolk extenders containing 7 % (v/v) glycerol. Straws were filled immediately, placed in a -68 freezer for 30 min, and transferred to liquid nitrogen. All straws with egg yolk were processed with 2010⁶ sperm per mL.

Straws were stored for 2 months before examination. They were thawed at two temperatures, ambient temperature (about 25) and at 65. The straws thawed at room temperature were held at 25 for 10 min followed by placement in a 37 waterbath before exami-nation. Other straws were thawed in 65 water for 4 seconds [2], and then placed in the waterbath at 37.

Thawed semen was dispensed into small culture tubes, and sperm were evaluated at 0 h for the proportion that were motile (total) and that were progressively motile. Two slides were prepared per straw and two eva-luators examined at least 10 fields per slide held on a microscope stage warmer at 37. Culture tubes were capped and placed in a 35 waterbath for re-examination of the percentage of motile sperm after 2 h.

An additional treatment was included in the 2-h incubation test because semen frozen only with glycerol might require additional buffering during incubation. At the time of the initial semen evaluation after thawing one-half of the semen frozen only with glycerol was mixed with warm egg yolk-tris-glucose extender containing 7 % glycerol so as to give 2010⁶ sperm/mL. These tubes were incubated along with the other treated samples.

The viability of the frozen-thawed sperm also was evaluated by measuring their penetration into synthetic polyacrylamide gel [10,11]. Semen frozen with glycerol only was diluted to 2010⁶ sperm/mL with a phospate-buffered saline containing glucose. Sperm migration was measured after incubation at 37 for 30 min. There were insufficient sperm cells from some ejaculates to run the migration tests (see number of replicates under results) but the tests were replicated with at least 10 capillary tubes per treatment.

Semen from 15 donors was aliquotted in a factorial across three semen extenders, two temperatures of adding glycerol and two thaw temperatures. An additional treatment was incubation of semen frozen-thawed with glycerol only followed by incubation with the tris-based extender. Donors were considered random and treatments as fixed in the analysis of variance (SAS, Statistical Analysis Systems Institute, Cary, NC, 1985). All main effects and interactions were analyzed, but most of the variation was associated with the main effects, and so only these effects are reported. Two observers who evaluated sperm motility obtained results that did not differ statistically. These data were averaged for the analysis. Probabilities of P<0.05 were considered to be statistically significant.

Total motile and progressively motile sperm estimates paralleled each other, so only the total values are included. The percentages of total motile sperm (meanSEM) at 0 and 2 h after thawing were 311.0 and 160.6. The largest contributor to variation in sperm cryosur-vival was donors (P<0.01). The donor ranges in post-thaw percentages of motile sperm at 0 and 2 h were 91.6 to 442.5 and 50.9 to 301.5, respectively. Generally the samples with the highest proportion of motile sperm immediately after thawing were the samples with more motile sperm after 2 h.

The percentage of motile sperm associated with all main effects in the experiment are summarized in Table 1. Extenders and temperature at which glycerol was added differed at 0 h (P<0.05), but only the extender effect was significant after 2 h (P<0.05).

Table 1. The percentage of motile frozen-thawed spermatozoa for all main variables other than donors.

Many of the interactions among the several variables were statistically significant, but the only one with a substantial effect was the interaction of extenders with the temperature at which glycerol was added. At 0 h post-thaw in all treatments where glycerol additions had been made at room temperature, the percentages of motile sperm in the glycerol, tris-based egg yolk and citrate-based egg yolk extenders averaged 301.7, 361.9 and 351.6, respectively. Corresponding percentages for sperm first exposed to glycerol at 4 were 131.5, 372.2, and 382.1 (P<0.05). The lack of both glycerol and egg yolk during cooling the sperm to 4 had a deleterious effect on sperm survival. All other interactions, where glycerol alone was excluded, differed by only a few percentage points, so they are not presented. The results of adding the egg yolk-tris-glucose-glycerol extender after thawing the semen frozen only with glycerol did not imrove sperm survival during 2 h of incubation (Table 1).

The migration of spermatozoa after 30 min of incubation in the gel is summarized in Table 2. Migration rate of the cryopreserved sperm was lower than we had previously observed [10], perhaps due to the use of 2010⁶ sperm per ml. This relatively low number of sperm was chosen to allow all ejaculates of semen to be divided among all treatments. While this sperm concentration was satisfactory for making motility estimates, it is suboptimal for sperm migration tests.

Table 2. Migration in polyacrylamide gel at 37 of frozen-thawed sperm which had been glycerolated at room temperature versus 4.

The migration trends paralleled the motility results, as sperm motility and migration distances were higher when buffered egg yolk was included (P<0.01). There was considerable variation in the estimated sperm motility and sperm migration distance from straw to straw and most correlations between these two variables were low. However, the correlations between the two traits for sperm processed with glycerol added to egg yolk-tris-glucose-glycerol at room temperature and at 4 were, respectively, 0.83 and 0.80 (P<0.01).

All 15 samples of semen donated were used for cryopreservation to provide a wide spectrum relative to initial semen quality. This also was reflected in the range of sperm surviving freezing, consistent with the numerous reports in the literature on preserving sperm from fertile versus infertile patients.

In addition to differences among donors, methods of processing the semen for cryopreservation differ wide-ly with varying results. Glycerol was the first substance used as a cryoprotectant for human sperm [1]. Later an extender found to protect frozen bull sperm for use in cattle artificial insemination, egg yolk citrate-glucose-glycerol, was adopted in several laboratories with various modifications, and this continues to be used [12-14]. Graham et al [8] tested zwitterion buffers for semen cryopreservation. This lead to TEST-egg yolk used with human sperm [5, 15] along with HSPM [7, 16, 17] developed by Mahadevan and Trounson [18]. The zwitterion ion buffer tris (hydroxymethyl) aminomethane (Tris) combined with egg yolk, glucose and glycerol [9] has become the most widely used extender for the cryo-preservation of sperm from many mammalian species[4]. As no published experiments were found using this extender for cryopreserving human sperm this medium was tested here. It is a simple medium and was compared with other commonly used procedures involving citrate-buffered egg yolk or glycerol alone. All media included glycerol.

The effect of adding glycerol to warm and cooled human sperm was compared because in animal studies glycerol is toxic to sperm when added at ambient temperatures in some media and not in others [4]. For example, glycerol is toxic to bull sperm in the citrate-based egg yolk extender when added at room temperature, but is not toxic to bull sperm when added to a warm tris-based egg yolk extender.

Finally, a very rapid thawing temperature was included in comparison with the usually slow ambient air thaw procedure for human sperm [1, 16, 19-21]. Leffler and Walters [14] reported higher post-thaw motility of human sperm thawed at 37. Srisombat et al [6] thawed semen at 42. In animal studies very rapid thawing at 65 for 7.5 seconds provided optimal preservation of sperm motility and acrosome integrity [2,4]. A similar procedure was included here.

Many tests have been used to evaluate damage to sperm resulting from cooling, freezing and thawing. The most widely used test is the motility and progressive mo-vement of sperm [1], with many components of motility facilitated by the use of computer-assisted sperm analysis [16,22]. Membrane integrity and the hypoosmotic swelling test [5,23] are positively correlated with sperm motility and fertility. Other tests of sperm integrity include lipid peroxidation, DNA and chromatin changes[17,20] and migration of sperm through mucus or gel [10,11].

We chose total motile and progressively motile sperm as criteria that could be easily duplicated in any laboratory. Sperm penetration through gel was another test that could be duplicated easily. The gel is easily prepared. It is uniform and stabile during storage, thus having several advantages over cervical mucus, particularly when making interlaboratory comparisons. The present experiment was designed to test several novel components under conditions also reflecting cooling and freezing conditions currently in use for cryopreservation of human sperm. The novel components were the inclusion of a tris-based medium widely used for freezing mammalian sperm, the interaction between media and glycerol added at two temperatures, and a very rapid thawing procedure.

Both of the buffered egg yolk extenders were superior to glycerol alone in preserving motile sperm during freezing and thawing. This beneficial effect apparently was primarily due to the protection by egg yolk during cooling. When glycerol was added at room temperature it also protected sperm. This is interpreted to indicate that glycerol is effective as an anticoldshock agent for human sperm as well as a cryoprotectant. Egg yolk protects sperm during cooling, so glycerol can be added to semen in these extenders at 4 or 25. Once sperm are damaged during freezing, adding the tris-based egg yolk did not improve sperm survival during incubation after thawing.

Leffler and Walters [14] reported that thawing human sperm at 37 for 10 min was superior to room temperature for 30 min. In the present studies different thawing temperatures had no detectable effect. This may be due to the relatively low concentration of glycerol included. Pilikan et al [24] recommended 12 % glycerol as optimal for human sperm in a citrate-based egg yolk medium. The rapid thaw at 65 was more effective for bull sperm when glycerol concentrations were increased [2]. The sperm migration results followed a pattern similar to that described for the percentage of motile sperm. However, this test was more sensitive in detecting differences in favor of the egg yolk media over glycerol alone, regardless of the temperature at which glycerol was added.

These results indicate that egg yolk-tris-glucose-glycerol provides good protection for cryopreservation of human sperm. Further studies are needed to optimize cooling, freezing and thawing rates, along with glycerol concentration.

Acknowledgements

The authors gratefully acknowledge the technical assistance of the staff at IDANT, by Robert Warren and Theodore Rounsaville, and with the manuscript preparation by Elisa Tumino van Amburgh.

References

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Correspondence to: Dr. Robert H. Foote, Department of Animal Science Cornell University, Ithaca, NY 14853-4801, USA.
Tel: +1-607-255-2866 Fax: +1-607-255-9829
E-mail: rhf4@cornell edu
Received 2002-02-19      Accepted 2002-05-10