the need for assisted reproduction technology ( art ) procedures for the establishment of pregnancies has steadily increased worldwide . around 7% of all annual births
therefore , it is of vital importance that an efficient sperm preparation technique used for retrieval of high - quality spermatozoa contributes to the creations of high - quality embryos , with high implantation potential .
the main goal with the sperm preparation is to yield highly motile spermatozoa with good morphology and low dna fragmentation rates , criteria that support the subsequent development of high - quality embryos .
the sperm dna integrity affects fertilization , embryo development , pregnancy outcome , miscarriage rates , and abnormalities in the offspring after both in vitro fertilization ( ivf ) and intracytoplasmic sperm injection ( icsi ) . in animals ,
development of cancer and shorter lifespan in their offspring are related to the injection of spermatozoa with high dna fragmentation rates .
the recovery of a good proportion of high - quality spermatozoa depends on the quality of the semen sample , the sperm preparation method , time and storage temperature of the prepared sperm suspension .
the choice of sperm preparation medium also interferes with the recovery of high - quality spermatozoa , which affects the treatment outcome .
an effective sperm preparation method yields a high number of motile spermatozoa with good morphology and quality , which may convert more complex treatment , such as icsi , into more simple techniques of ivf or intrauterine insemination ( iui ) for a more natural and cost - effective treatments .
it is also important to evaluate the integrity of sperm dna after the sperm preparation technique since morphologically normal spermatozoa still may have dna damage .
currently , there are several techniques for detection of sperm dna fragmentation . the sperm chromatin dispersion ( scd ) assay evaluates dna fragmentation and has the same predictive values such as the sperm chromatin structure assay ( scsa ) and tdt - mediated dutp - biotin nick end labeling ( tunel ) .
in addition , this assay is easy , rapid , accurate , and not requiring sophisticated instruments .
the aim of this study was to compare the effects of three different sperm preparation media on viability , motility , and dna integrity of spermatozoa after preparation of normozoospermic samples .
the following criteria were applied for evaluation of sperm motility : rapidly progressive spermatozoa were considered as grade a. grade b spermatozoa were slowly progressive , grades c and d were dedicated to no progressive motility and immotile spermatozoa , respectively .
three culture media were selected : ( a ) sperm preparation media ( origio , mlv , denmark ) , ( b ) ham 's f10 ( biochrome , berlin , germany ) supplemented with 5 mg / ml human serum albumin , and ( c ) sperm washing medium ( vitasperm innovative biotech , tehran , iran ) , where 5 mg / ml hsa was added .
sperm motility , viability , and dna fragmentation index ( dfi ) were evaluated at 0 , 1 , 2 , and 24 h after preparation .
sperm viability and dfi were assessed after staining with eosin - nigrosin , and the scd technique , respectively .
briefly , 1.2 ml of each culture media was placed into a sterile conical tube , after which 1 ml of the semen was slowly placed under the medium at the bottom of the tube .
the swim - up was performed at 37c in an incubator at 45 angle for 45 min .
thereafter , 1 ml of the upper layer was removed , and 2 ml of the same sperm preparation media was added to the test tube .
after centrifugation at 400 g for 7 min , the pellet was resuspended in 0.5 ml preincubated media , the tubes closed tightly , and the sperm suspension analyzed at the aforementioned time intervals .
glass slides were coated with 0.65% ( w / v ) agarose and 70 l of a low - melting 1% ( w / v ) agarose was mixed with 30 l of the sperm suspension .
thereafter , 50 l of this mixture was placed on the precoated glass slides and kept at 4c for 4 min .
next , the slides were placed in a denaturation solution ( 0.08 m hcl ) for 7 min at room temperature ( rt ) in the dark .
these slides were placed in 0.4 m tris , 2-mercaptoethanol containing 1% ( w / v ) sodium dodecyl sulfate ( sds ) and 50 mm ethylenediaminetetraacetic acid ( edta ) ( ph 7.5 ) , for 10 min at rt .
after incubation , the slides were placed in a lysing solution ( 0.4 m tris , 2 m nacl , and 1% [ w / v ] sds , ph 7.5 ) for 15 min and were finally washed in a tris - borate - edta buffer ( 0.09 m tris - borate and 0.002 m edta , ph 7.5 ) for 2 min at rt .
the slides were dehydrated in ascending ethanols ( 70% , 90% , and 100% [ v / v ] ) for 2 min and left to dry at rt . for staining the slides ,
wright 's stain was mixed with a phosphate buffer solution at a ratio of 1:1 .
this mixture was layered on the slides placed in running water until the excess stains washed away and left to dry at rt .
the dna fragmentation rate was related to the halo around the sperm head and categorized into four groups : ( a ) big halo , when the halo size was more than the minor diameter of core width ( without dna fragmentation ) , ( b ) small halo , with size smaller than one - third of the minor diameter of core width , ( c ) medium halo size was considered between large and small halos , and ( d ) no halo .
large- and medium - sized halos have no dna fragmentation while small size halos or no halos have a fragmented dna [ figure 1 ]
( a ) a big halo represents no dna fragmentation , ( b ) a medium halo represents no dna fragmentation , and ( c ) no halo indicates spermatozoa with dna fragmentation the data are shown as mean  standard deviation and median ( minimum  maximum ) .
one - way analysis of variance followed by tukey as a posttest and kruskal  wallis test was applied to compare sperm parameters between media at one time point .
all hypotheses were considered two - tailed , and p < 0.05 was considered statistically significant .
the following criteria were applied for evaluation of sperm motility : rapidly progressive spermatozoa were considered as grade a. grade b spermatozoa were slowly progressive , grades c and d were dedicated to no progressive motility and immotile spermatozoa , respectively .
three culture media were selected : ( a ) sperm preparation media ( origio , mlv , denmark ) , ( b ) ham 's f10 ( biochrome , berlin , germany ) supplemented with 5 mg / ml human serum albumin , and ( c ) sperm washing medium ( vitasperm innovative biotech , tehran , iran ) , where 5 mg / ml hsa was added .
sperm motility , viability , and dna fragmentation index ( dfi ) were evaluated at 0 , 1 , 2 , and 24 h after preparation .
sperm viability and dfi were assessed after staining with eosin - nigrosin , and the scd technique , respectively .
briefly , 1.2 ml of each culture media was placed into a sterile conical tube , after which 1 ml of the semen was slowly placed under the medium at the bottom of the tube .
the swim - up was performed at 37c in an incubator at 45 angle for 45 min .
thereafter , 1 ml of the upper layer was removed , and 2 ml of the same sperm preparation media was added to the test tube .
after centrifugation at 400 g for 7 min , the pellet was resuspended in 0.5 ml preincubated media , the tubes closed tightly , and the sperm suspension analyzed at the aforementioned time intervals .
glass slides were coated with 0.65% ( w / v ) agarose and 70 l of a low - melting 1% ( w / v ) agarose was mixed with 30 l of the sperm suspension .
thereafter , 50 l of this mixture was placed on the precoated glass slides and kept at 4c for 4 min .
next , the slides were placed in a denaturation solution ( 0.08 m hcl ) for 7 min at room temperature ( rt ) in the dark .
these slides were placed in 0.4 m tris , 2-mercaptoethanol containing 1% ( w / v ) sodium dodecyl sulfate ( sds ) and 50 mm ethylenediaminetetraacetic acid ( edta ) ( ph 7.5 ) , for 10 min at rt .
after incubation , the slides were placed in a lysing solution ( 0.4 m tris , 2 m nacl , and 1% [ w / v ] sds , ph 7.5 ) for 15 min and were finally washed in a tris - borate - edta buffer ( 0.09 m tris - borate and 0.002 m edta , ph 7.5 ) for 2 min at rt .
the slides were dehydrated in ascending ethanols ( 70% , 90% , and 100% [ v / v ] ) for 2 min and left to dry at rt . for staining the slides ,
wright 's stain was mixed with a phosphate buffer solution at a ratio of 1:1 .
this mixture was layered on the slides placed in running water until the excess stains washed away and left to dry at rt .
the dna fragmentation rate was related to the halo around the sperm head and categorized into four groups : ( a ) big halo , when the halo size was more than the minor diameter of core width ( without dna fragmentation ) , ( b ) small halo , with size smaller than one - third of the minor diameter of core width , ( c ) medium halo size was considered between large and small halos , and ( d ) no halo .
large- and medium - sized halos have no dna fragmentation while small size halos or no halos have a fragmented dna [ figure 1 ]
( a ) a big halo represents no dna fragmentation , ( b ) a medium halo represents no dna fragmentation , and ( c ) no halo indicates spermatozoa with dna fragmentation
the data are shown as mean  standard deviation and median ( minimum  maximum ) .
one - way analysis of variance followed by tukey as a posttest and kruskal  wallis test was applied to compare sperm parameters between media at one time point .
all hypotheses were considered two - tailed , and p < 0.05 was considered statistically significant .
there were no significant differences in progressive motility between the different sperm preparation media after swim - up at 0 , 1 , and 24 h. however , the rate of progressive motility was significantly higher in spermatozoa prepared by sperm preparation media , when compared to vitasperm ( 68.3  11.5 vs. 58  9.8 , p = 0.03 ) [ figure 2 ] . there were insignificant differences in motility or viability between the different groups [ tables 1 and 2 ] .
in addition , there was no difference in the percentage of normal morphology between sperm preparation media , vitasperm , and ham 's f10 after swim - up ( 15.9  2.1 , 16.4  3.2 , and 15.8  2.3 , respectively ) .
the sperm dfi increased significantly 1 h after swim - up in vitasperm versus sperm preparation media ( 3  2.7 vs. 1.1  0.8 , p = 0.02 ) ; however , there was insignificant difference in comparison to ham 's f10 ( 1.7  1.1 ) . however , the dfi increased significantly over time in the vitasperm medium versus other two media [ figure 3 ] . the percentage of progressively motile spermatozoa after swim - up .
* p = 0.03 total number of motile sperm ( % ) at different time intervals after swim - up sperm viability rates ( % ) at different time intervals after swim - up using eosin - nigrosin stain the rate of sperm dna fragmentation index after swim - up of normozoospermic samples in different sperm preparation media at different time intervals .
the quality of the prepared sperm suspension affects the outcome of all types of inseminations , whether it is for iui , ivf , or icsi . after sperm preparation ,
the recovery of motile spermatozoa affects the decision of which method would be the most suitable and efficient for the couple .
if there is a high recovery of highly motile spermatozoa with good morphology and low dfi , the prepared sperm suspension is suitable for more physiological methods , such as iui or ivf . whereas , borderline or male factor samples might be more suitable for icsi .
the handling of the semen sample , choice of sperm preparation method , and storage temperature of the prepared sperm suspension affect the recovery rates of sperm , their fertilization ability , the integrity of the sperm dna , their ability to support embryo development and the implantation rate .
the effects of different sperm preparation techniques on sperm parameter values and the dna integrity have been evaluated in several studies .
it seems that the yield of morphologically normal spermatozoa with good chromatin stability is obtained after preparation of the semen sample by high - density gradient centrifugation in comparison with conventional swim - up and glass wool filtration methods . in this study
, we evaluated the efficacy of three different sperm preparation media on motility , viability , and dna integrity of human spermatozoa at different time intervals after sperm preparation .
our data showed that the nature of the sperm preparation medium affects sperm motility and the dna integrity of the prepared spermatozoa .
depending on the quality , the storage temperature , and the incubation time of the sperm suspension , as well as the preparation medium , the level of sperm dna fragmentation varies .
oxidative stress and lipid peroxidation have been suggested to affect sperm motility by inducing damages to the axoneme and depletion of intracellular adenosine triphosphate , which reduces the sperm motility .
it is thought that the main product of oxidative stress , 8-hydroxy , 2deoxyguanosine , also causes most of the damage to the sperm dna .
the extent of sperm dna fragmentation could be used as a sperm quality marker , which predicts the fertilization capability of the prepared spermatozoa .
the main causes to sperm dna fragmentation are thought to be related to failure in the chromatin remodeling during spermiogenesis or an increase in reactive oxygen species ( ros ) levels due to oxidative stress .
however , the impact of sperm dna fragmentation on fertilization has still not been confirmed .
however , a defect in sperm chromatin stability might affect the sperm cells ability to penetrate the rigid zona pellucida .
the scd test is based on the halo formation of dispersed dna loops after acid denaturation , where spermatozoa with a high dna fragmentation rate do not create a halo .
it has been shown that the scd gives similar results to that of both the scsa and the tunel assay and that it can be used as a prognostic tool for sperm dna fragmentation rate .
oxidative stress in subnormal specimens is induced by other types of contaminating cells , immature and bad morphology spermatozoa , as well as the sperm wash method .
we , therefore , used normozoospermic samples for this study , to reduce potential sources of oxidative stress to spermatozoa and for accurate interpretation of the results .
the method of choice for preparation of semen may affect the production of ros , thereby inducing oxidative stress .
however , younglai et al . showed that there was no significant difference in dna fragmentation rate in spermatozoa prepared by direct swim - up from the semen sample or after prewash of the semen sample . in this study , all samples were prepared by the direct swim - up method ; thus , the findings should only be related to the quality of the sperm preparation media .
formation of free radicals and oxidative stress also seem to play a physiological role in sperm capacitation and fusion between the spermatozoa and the oocyte .
antioxidants , such as ascorbic acid and tocopherols , seem to prevent the detrimental effects that ros have on sperm motility and dna fragmentation .
the inclusion of antioxidants in sperm preparation media might , therefore , reduce the detrimental effect that ros have on sperm during the preparation procedure .
the quality of the sperm preparation media affects the sperm recovery of high - quality spermatozoa which subsequently may influence the art outcomes .
 aim : the goal was to compare the effects of three different sperm preparation media on sperm motility , viability , and dna integrity of semen samples from normozoospermic men.methods:a total of 15 normozoospermic males were included in the study . the semen analysis ( sa ) 
 was performed in accordance with the who guidelines ( 2010 ) . 
 after sa , each sample was divided into three aliquots , and swim - up was performed with three different sperm preparation media ( sperm preparation media , origio , denmark ; ham 's f10 , biochrome , berlin , germany ; and vitasperm , innovative biotech , iran ) . 
 sperm motility , viability , and dna fragmentation were evaluated at 0 , 1 , 2 , and 24 h after swim-up.results:there were no significant differences , at any time intervals , in the total sperm motility between the different sperm preparation media . however , the rate of progressive motility was significantly higher in spermatozoa prepared using the media from origio in comparison with vitasperm ( p = 0.03 ) , whereas no significant difference was found against ham 's f10 medium . 
 no significant differences in sperm viability were seen between the media products . however , 1 h after swim - up , the extent of sperm dna fragmentation was lower in the medium from origio versus vitasperm ( p = 0.02).conclusions : the data showed that the quality of medium for preparation of semen samples from normozoospermic men significantly affects the performance of spermatozoa in assisted conception programs . 
INTRODUCTION
METHODS
Patients
Semen analysis
Sperm preparation
Sperm chromatin dispersion test
Statistical analysis
RESULTS
DISCUSSION
CONCLUSIONS
Financial support and sponsorship
Conflicts of interest