Rapid thawing human sperm does not affect basic parameters in normozoospermic men : a double-blind prospective study

ARTICLE INFO _______________________________________________________________ _____________________

There is not a consensus protocol for thawing human semen, which can be done in several ways.Semen can be thawed at room temperature for about 5-60 minutes or using a water bath at 37º C for 5-10 minutes or with both techniques (5)(6)(7)(8)(9)(10).Accordingly to our protocol, cryogenic vial samples are removed from the liquid nitrogen and allowed to thaw at room temperature (25º ± 2º C) for 25 minutes to achieve a complete thaw before being processed (5,8).This procedure has been performed for several years with good clinical results.
We aim to investigate whether the use of a higher thawing temperature (75º C) would be harmful to human spermatozoa when compared with room temperature (25º ± 2º C) thawing regarding motility, morphology and integrity of sperm membranes, and if it would be feasible to introduce this technique in routine assisted reproduction.

MATERIALs AND METHODs
This study was carried out from September 25 to november 4, 2009, at Professor Aroldo Fernando Camargos Laboratory of Human Reproduction, Federal University of Minas Gerais.All participants signed a consent form and completed a questionnaire about reproductive history.This research has been approved by The Ethics Committee under protocol number COEP-UFMG 348/08.
Only subjects who provided semen within the standard parameters (normozoospermic, semen volume ≥ 2 mL) set by the 1999 World Health Organization were included (11).Thirtyeight men provided sperm samples.Twenty-one of them from routine infertility screening exams (infertile group) and seventeen were proven fertile volunteers with at least one child (fertile group).
Sperm was obtained by masturbation into a non-toxic sterile collector, after two to five days from last intercourse or ejaculation.Once collected, sperm was maintained at 37º C on a warm plate until total liquefaction.Samples were analyzed within 60 minutes after ejaculation for concentration, motility, morphology and functional sperm membrane integrity and basic macroscopic parameters (liquefaction, volume, color, viscosity, pH).Rapid progressive spermatozoa (type a), slow progressive spermatozoa (type b), non progressive (type c) and immotile (type d) were defined in conformity with WHO criteria (1999).All counts were done in a neubauer chamber after appropriate dilutions of semen aliquots.
Morphology was evaluated by the hematoxylin staining technique and slides were analyzed by the Kruger strict criteria (12).Morphology of two hundred randomly chosen spermatozoa were assessed using a Carl Zeiss® optical microscope with 1,000 X millimetered ocular lens under oil immersion.
Functional sperm membrane integrity was evaluated by the hypoosmotic swelling test.Briefly, a 0.1 mL semen sample was diluted into 1.0 mL of hypoosmotic solution at 37º C inside an Eppendorf® tube and incubated for one hour in a 5% CO 2 incubator, at 37º C.After homogenization, a small drop was observed under 400X magnification.One hundred spermatozoa were analyzed in each sample and the percentage of typical morphology changes was calculated.
Test Yolk Buffer with Gentamicine (TyB-G) and 12% Glycerol (Irvine Scientific, USA) freezing medium was used (proportion 1:1) for a total volume of 1 mL.Just before cryopreservation, this mixture was divided into 2 equal samples.Before storing in liquid nitrogen (-196º C), samples were maintained in nitrogen vapor for 10 minutes at a 10 cm height from liquid nitrogen surface.Cryogenic tubes were stored for 14 to 45 days.Samples were thawed at room temperature (25º ± 2º C) by a different investigator for 25 minutes (slow thaw) or in a water bath at 75º C for 20 seconds and then maintained at 37º C for three minutes in another water bath (rapid thaw) and then analyzed for motility, morphology and functional integrity of sperm membrane.The investigator who analyzed the thawed semen was blinded to the technique used for the thawing.
The minimal sample size was estimated to be 17 patients, using a standard deviation of 10%, minimum difference to be detected of 10% and a statistical power of 81%.Data were ex-pressed as mean ± standard deviation for parametric variables and analyzed by Student's ttest.non-parametric non-paired variables were expressed as median (interquartile range) and analyzed by the Mann-Whitney test.Wilcoxon test was used to analyze non-parametric paired variables.The significance level was p < 0.05.

REsuLTs
Fertile and infertile subjects had similar age and comparable fresh sperm parameters, including total fresh spermatozoa and spermatozoa per mL count, normal morphology, head or middle piece or tail morphology defects, or cytoplasmic droplets (Table -1).
Fertile and infertile men also showed similar semen results after both thaw techniques (slow and rapid thaw).Likewise, thawed sperm didn't show differences in progressive motility (type a + b), reactive sperm percentage after hy- poosmotic swelling test or strict morphology, defects in the middle piece and tail (Table -2).Fertile thawed samples showed similar percentage of normal spermatozoa morphology after thawing by both techniques, compared with fresh samples; however there was an increase of morphological head defects (p ≤ 0.01) and a decrease of cytoplasmic droplets (p ≤ 0.004), and percentages of spermatozoa reactive to the hypoosmotic swelling test (p < 0.0001) (Table -3).There was a decrease of progressive sperm motility (p < 0.0001) (Table -4).
Thawed infertile samples didn't show difference of normal spermatozoa morphology percentage regarding slow thaw technique; nevertheless rapid thaw method demonstrated a reduction (p = 0.0034).There was a significant increase of morphological head defects only for rapid thaw protocol (p = 0.0362) and a decrease in the percentages of spermatozoa reactive to the hypoosmotic swelling test (p < 0.0001) by both

DIsCussION
Many sperm thawing methods for animals use high heating temperatures (above 50º C) (2)(3)(4).In contrast, the majority human cryopreservation protocols use thawing temperatures ranging from 20 to 37º C (5-10), requiring a longer thawing time.This fact, coupled with the need to re-fine the protocols of freezing and thawing of human semen, sparked interest in an unprecedented way to check how human sperm undergoing this type of procedure reacts.
Our hypothesis was that if the sperms of many animal species recovered so well after fast thawing at higher temperatures, it could also be the same with human sperm.So we decided to investigate whether the use of higher heating curves would be as safe to human spermatozoa as conventional thawing, and if it would be feasible to introduce this technique to routine procedures for cryopreservation of human semen in assisted reproduction.This research was able to evaluate and compare the viability of human sperm after thawing at temperatures of 75º C and room temperature using the parameters of motility, morphology and integrity of sperm membranes.The temperature of thawing in a water bath at 75º C for 20 seconds followed by immersion in water bath at 37º C for three minutes was adapted from a study that has thawed equine semen at this temperature (4).
A pre-warm (37º C) was not performed in slow thaw group before sperm motility measurement.Moreover, with this lower temperature used in the slow thaw group it would be expected a higher probability of finding a difference, but our study did not demonstrate any difference.Interesting, Calamera et al. (2010) did not show any influence of room temperature (20º C) versus 40º C on sperm motility.
Sperm recovery from fertile and infertile patients submitted to a higher thawing temperature did not diverge from slow thawing protocol, suggesting that the rapid thaw protocol appears not to be harmful for the analyzed variables.Furthermore, it was performed in a shorter time (three minutes and 20 seconds vs. 25 minutes, respectively).
Even though there would be a risk of DnA-damage once a high temperature is in-volved (75º C), this study did not have the intention of evaluating sperm DnA fragmentation.As we couldn't find any study that has used 75º C for human semen thawing, more studies should be performed in this field.
Several studies have confirmed the decline in sperm motility after thawing (11,13,14).In a recent prospective study, the motility of spermatozoa thawed in water bath at 20, 37, 38, 39 and 40º C decreased significantly (15).In our study, both thawing techniques resulted in decrease of sperm progressive motility (a + b) in fertile and infertile groups compared to fresh samples (p < 0.0001) (Table-4).However there wasn't a significant difference between techniques (p = 0.2706 and p = 0.3139, respectively, Table-2).
Regarding the sperm morphology, the most commonly used staining methods for sperm morphology evaluation include hematoxylin stain, the Papanicolaou method, the Shorr method, the Spermac method or the Diff-Quik method.Some published papers have used Kruger morphological evaluation with other dyes (8,16).The best method should be the most beneficial to the laboratory as each method has limitations (17).Therefore, hematoxylin was chosen as a routine staining procedure for Kruger strict criteria sperm evaluation.The percentage of morphologically normal thawed sperm didn't reveal a significant difference when comparing fertile and infertile groups or slow and rapid thawing methods, which is in agreement with other studies (1,13).
However, there was a decrease in the percentages of morphologically normal sperm from infertile group when using the rapid thawing protocol.This decrease is in agreement with some published papers (10,14).A possible explanation for this result can be due to lower resistance of sperm from infertile men to damage caused by the cryopreservation process (18).
The hypoosmotic swelling test is widely used in various studies involving frozen semen in the veterinary field (4,19).In human reproduction, despite having a questionable validity in procedures that involve semen freezing and thawing, data was published using this test to evaluate the integrity of the sperm membrane (1,15).Therefore in our study, we also chose to include this test to evaluate this parameter before and after sperm thawing.
Cellular membrane damage could be demonstrated by a decrease in the percentage of sperm cells reactive to the hypoosmotic swelling test when comparing fresh to thawed samples (p < 0.001).This decline is similar to another study that demonstrated reduced spermatozoa membrane integrity (1).
To our knowledge, we couldn't find any study that had done the same comparison (slow versus fast human sperm thawing).This is the first study to do this experiment.Additional analogous basic research is needed to confirm our results with non normozoospermic sperm samples and in clinical research to access normal birth rates before introducing this methodology in routine assisted reproduction.

CONCLusIONs
The techniques of fast and slow thawing showed the same recovery of spermatozoa in normozospermic men.Sperm from fertile and infertile patients submitted to higher thawing temperature did not diverge from slow thawing protocol, suggesting that the rapid thaw protocol seems not to be harmful for the analyzed variables.Furthermore, it was performed in a shorter time (three minutes and 20 seconds vs. 25 minutes, respectively).Additional research is needed to confirm its safety in clinical research before introducing this methodology in routine assisted reproduction.

Table 3 -sperm parametere before and after cryopreservation.
Wilcoxon test was used to analyze non-parametric paired variables.Data are expressed as median and interquartile interval.