Effect of leukocytospermia and processing by discontinuous density gradient on sperm nuclear DNA fragmentation and mitochondrial activity

Purpose

To assess the effect of leukocytospermia and semen processing on sperm DNA and mitochondria.

Methods

Twenty-two patients with and 41 without leukocytospermia were included. Sperm DNA fragmentation was assessed by the Comet assay, and mitochondrial activity by a colorimetric method for active mitochondria. Semen was processed using Percoll, and motility, DNA fragmentation, and mitochondrial activity were analyzed pre- and post-processing.

Results

No differences were observed in age, abstinence, volume, sperm morphology, progressive motility, concentration, and vitality (p?>?0.10). Variables were grouped according to time (pre- vs post-processing) and group (leukocytospermia vs non-leukocytospermia) because no interactions could be observed. Leukocytospermia was associated to increased DNA fragmentation, while semen processing led to a decrease in DNA fragmentation and to increased mitochondrial activity.

Conclusion

While semen processing selects sperm with higher rates of DNA integrity independent of the presence or absence of leukocytes in semen, samples without leukocytospermia present more sperm without DNA fragmentation. Semen processing also selects sperm with higher mitochondrial activity.     

The correlation between idiopathic leukocytospermia, embryo quality and outcome in the FIVET and ICSI procedures

BACKGROUND: An adverse effect of leukocytospermia in seminal fluid on motility and fertilizing power of spermatozoa has been described. This detrimental effect could be mediated by radical oxygen species (ROS). Recently, a direct effect on nuclear DNA of sperm induced by ROS has been described, although the chance of fertilization did not seem altered during ICSI procedure. Aim of this prospective case-control study was to compare the outcome of results in term of fertilization rate and embryo quality in patients with and without idiopathic leukocytospermia during IVF and ICSI cycles. METHODS: Seventy-two patients selected for a program of IVF and ICSI were admitted in the study. Fourty-two patients underwent IVF procedure, 14 with idiopathic leukocytospermia and 28 without, and thirty underwent ICSI procedure, 16 with leukocytospermia and 14 without leukocytospermia. RESULTS: Statistical significant differences on cleavage rate of embryos between leukocytospermia and control group in IVF cycles were observed. In ICSI procedure a low fertilization, cleavage rate and percentage of good embyros in the presence of leukocytospermia were evidenced. CONCLUSIONS: The presence of a significant number of leukocytes in semen, even in idiopathic condition, could affect the results of IVF and ICSI procedure. An adverse effect of lipoperoxidation process of plasma membrane and damage of nuclear chromatin of sperm, as result of leukocyte contamination could be hypothesized and future studies needed in order to verify the role of ROS on sperm functions.     

Leukocytospermia is associated with increased reactive oxygen species production by human spermatozoa

To investigate the role of increased seminal leukocytes in enhancing reactive oxygen species (ROS) production by human spermatozoa.A prospective study.Male infertility clinic.Forty-eight infertile men.Standard semen analysis. Assessment of sperm nuclear DNA damage by sperm chromatin structure assay. Incubation of spermatozoa from nonleukocytospermic samples with blood neutrophils.Spontaneous and phorbol 12-myristate 13-acetate (PMA)-induced ROS production in pure-sperm suspensions (after removal of leukocytes) as measured by a chemiluminescence assay.Levels of spontaneous and PMA-induced ROS production in pure-sperm suspensions from the infertile men with a diagnosis of leukocytospermia (n = 16) were significantly higher compared with the case of infertile men without leukocytospermia (n = 32) and with the case of a control group of healthy volunteers (n = 13). A similar pattern of increased ROS was observed when spermatozoa were incubated with blood neutrophils. Leukocytospermia was associated with a significant decrease in sperm motility and increase in DNA damage.Increased seminal leukocytes may play a role in stimulating ROS production by human spermatozoa. Such stimulation may be mediated via direct cell-cell contact or by soluble products released by leukocytes. Poor sperm quality in leukocytospermic samples may be due to leukocyte-mediated oxidative stress.     

Contemporary evidence on the physiological role of reactive oxygen species in human sperm function

Reactive oxygen species (ROS) play an important role in male fertility. Overproduction of reactive oxygen species (ROS) has been associated with a variety of male fertility complications, including leukocytospermia, varicocele and idiopathic infertility. The subsequent oxidative insult to spermatozoa can manifest as insufficient energy metabolism, lipid peroxidation and DNA damage, leading to loss of motility and viability. However, various studies have demonstrated that physiological amounts of ROS play important roles in the processes of spermatozoa maturation, capacitation, hyperactivation and acrosome reaction. It is therefore crucial to define and understand the delicate oxidative balance in male reproductive cells and tissues for a better understanding of both positive as well as negative impact of ROS production on the fertilizing ability. This review will discuss the specific physiological roles, mechanisms of action and effects that ROS have on the acquisition of structural integrity and physiological activity of spermatozoa.     

Does leukocytospermia associate with poor semen parameters and sperm functions in male infertility? The role of different seminal leukocyte concentrations

To investigate the effect of leukocytospermia on standard semen analysis and sperm function tests such as acrosome reaction, hypoosmotic swelling, antisperm antibody binding and cervical mucus penetration, a prospective clinical study was performed. Two hundred and nineteen male infertility patients undergoing investigation and treatment were included in the study. There was a significant association between acrosome reaction positivity and leukocytospermia according to WHO (World Health Organization) criteria. Increased hypoosmotic swelling test score, higher sperm concentration and enhanced acrosome reaction were closely related to leukocytospermia. When the patients were divided into subgroups according to seminal leukocyte concentrations, acrosome reaction and hypoosmotic swelling were observed to be higher in semen samples with higher leukocyte concentrations compared to those with low seminal leukocyte concentration. In addition, higher sperm concentrations were observed in semen samples with increased leukocyte levels compared to semen samples with low leukocyte levels. These results suggest that leukocytospermia may have a favorable effect on some sperm functions at seminal leukocyte concentrations between 1 and 3x10(6)/ml.     

Seminal immune response in infertile men with leukocytospermia: effect on antioxidant activity.

OBJECTIVE: To investigate the incidence of leukocytospermia and relation to T helper cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin-4 (IL-4), antisperm antibodies and antioxidant activity. Design: Semen samples from 176 infertile men and 24 fertile controls were investigated. METHODOLOGY: The protocol included tubal patency test, hysterosalpingography and laparoscopy and dye test and ovulation through mid-luteal phase progesterone for the wives. The husbands had semen analysis, cytomorphology evaluation and semen culture. Seminal TNFalpha and IL-4, antisperm antibodies, total antioxidant activity, superoxide dismutase and zinc were determined. RESULTS: Leukocytospermia occurred in 44.3% of the infertile men compared to 12.5% of the fertile men (P<0.01). Thirty-six (20.5%) men had pathogenic bacterial organisms which constituted 46.2% of those with leukocytospermia. Sperm parameters were worse with leukocytospermia in terms of sperm count (P<0.01), total motility progressive motility (P<0.01), morphology, asthenozoospermia, sperm membrane integrity and antisperm antibodies. TNFalpha and IL-4 had an inverse relationship; the expression of TNFalpha was higher with leukocytospermia and bacteriospermia (P<0.001), while IL-4 was higher in fertile controls (P<0.005). Incidence of antisperm antibodies was higher with leukocytospermia. Total antioxidant activity, superoxide dismutase and zinc were lower with leukocytospermia. CONCLUSION: Leukocytospermia impairs sperm function through reduced antioxidant activity and enhanced T helper 1 modulation.     

Effect of human oviductal in vitro secretion on human sperm DNA integrity

PURPOSE: In the female genital tract spermatozoa interact with the oviductal secretion. The aim of the study was to evaluate the effect of conditioned media (CM) from cultures of human oviductal tissue, on sperm DNA integrity. The effect of H(2)O(2) on sperm DNA integrity, before and after incubation under capacitating conditions, was also investigated. METHODS: Motile sperm obtained from normozoospermic semen samples were incubated (4 h or 22 h) in the presence or absence of CM and further exposed to H(2)O(2). DNA damage was detected by the comet assay. RESULTS: The CM significantly reduced the DNA damage associated with sperm incubation, and also decreased the effect of H(2)O(2) after 4 h incubation, compared to controls. The H(2)O(2) caused a dose-dependent deleterious effect on sperm DNA integrity both before and following 22 h of capacitation. CONCLUSION: The oviductal tissue CM increased the stabilization of the sperm DNA structure under culture conditions.     

Increased DNA damage in sperm from leukocytospermic semen samples as determined by the sperm chromatin structure assay

OBJECTIVE: To determine DNA damage as measured by the sperm chromatin structure assay (SCSA) in subsets of human spermatozoa at different stages of maturation in patients who are undergoing infertility evaluation. DESIGN: Prospective study. SETTING: Andrology laboratory at a tertiary care hospital. PATIENT(S): Fifty-six patients undergoing infertility evaluation. Patients with normal semen parameters (n = 17), abnormal semen parameters (n = 29), leukocytospermia (n = 10), and a group of healthy fertile men (n = 18) were included in the study. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The shift of green (native DNA) to red (denatured, single-stranded DNA) fluorescence was measured and quantified using the expression alpha(t) (red fluorescence/[red + green fluorescence] per cell). Sperm DNA damage was examined in subsets of spermatozoa isolated by a three-step density gradient. The DNA damage was correlated with classic semen characteristics.RESULT(S): Leukocyte concentration in semen was directly correlated with chromatin alterations in immature and mature sperm. Leukocyte concentration in semen was also directly correlated with immature germ cell concentration and the percentage of abnormal forms in semen. CONCLUSION(S): The increase in chromatin alterations and DNA damage in sperm, as defined by the sperm chromatin structure assay from leukocytospermic samples may be related to alterations in the regulation of spermatogenesis.     

Sperm DNA integrity as diagnosis and prognosis element of male fertility

Recent progress in reproductive biology has improved comprehension physiology of the spermatozoa and on the fertilization mechanisms. This new knowledge has carried out the elaboration of tests on male fertility based on sperm genomic integrity. This review presents some of these techniques and brings a reflexion element on the application and use of sperm DNA integrity in the investigation of male fertility. The single cell gel electrophoresis (COMET assay), Sperm Chromatin Structure Assay (SCSA), In Situ Nick Translation (NT: Nick Translation) and Terminal Uridine Nick-End Labelling (TUNEL assay) are actually the most currently used techniques for the measure of sperm DNA integrity in research clinic. From a certain point of view, TUNEL assay, SCSA, COMET assay and NT assay are complementary. The TUNEL and COMET can measure single and double strand breaks of DNA, the SCSA can detect the abnormalities in the chromatin compaction and the NT assay can detect the single strand breaks of DNA. The exact origin of sperm DNA fragmentation is not established yet. However, several mechanisms have been proposed: defect in the chromatin compaction during spermiogenesis; reactive oxygen species production by immature spermatozoa; apoptosis during spermatogenesis. It becomes important to consider the possible consequences of the oocyte fertilization by a spermatozoon having a high degree of DNA fragmentation. The use in routine of some of these tests must however pass by a standardization of the inter laboratory protocols and obviously, by the establishment of both in vivo and in vitro discriminating threshold values in order for these tests to present a good predictive value for pregnancy outcome.     

A simple DNA disc chip in a microarray design based on modified comparative genomic hybridization for sperm DNA analysis

OBJECTIVE: A DNA disc chip assay, based on comparative genomic hybridization, was designed to measure changes in sperm DNA intensities. The objective was to analyze the DNA integrity of hyperactive sperm cells after mild heat treatment. DESIGN: The assay based on a multiple cell comet assay was used to analyze changes in genomic DNA. Washed sperm DNA were tested on the assay and images stored in a microarray design. SETTING: Clinical and academic research environment. PATIENT(S): Frozen-thawed washed sperm from different donors (n = 7). INTERVENTION(S): Discarded sperm leftover from trial washes carried out at 37 degrees and 40 degrees C were frozen and processed for the DNA disc chip assay. MAIN OUTCOME MEASURE(S): Fluorescent intensities of DNA disc chips and sperm variables. RESULT(S): Heat treatment resulted in more than eightfold increase in sperm hyperactive motility with little degradation in DNA integrity. Sperm with low hyperactivation was associated with alterations in DNA after heat treatment. CONCLUSION(S): The DNA disc chip assay was simple, inexpensive, and permitted assisted reproduction technologies laboratories to use comparative genomic hybridization for cytogenotoxicity testing. However, the assay required manual processing, a fluorescent microscope, and computer. The data showed an association between sperm hyperactivation and DNA integrity suggesting that the hyperactivation marker may be used for selecting quality sperm for intracytoplasmic sperm injection. More studies are needed to examine temperature effects on ejaculated human sperm.     

Germ cell abnormalities in streptozotocin induced diabetic mice do not correlate with blood glucose level

Purpose

To assess the effect of streptozotocin induced hyperglycemia on germ cell integrity, DNA ploidy and methylation status for a period of two spermatogenesis cycles in adult male Swiss albino mice.

Methods

Streptozotocin injected mice were monitored for hyperglycemia at a regular interval for a period of 36 and 72 days. The DNA integrity in epididymal spermatozoa was determined by the comet assay. Flow cytometric analysis was done in germ cells to assess the DNA ploidy. The global methylation analysis in germ cells was done by 5-methyl cytosine immunostaining.

Results

Streptozotocin administration successfully resulted in hyperglycemic response which significantly affected serum testosterone level, sperm DNA integrity and DNA ploidy at the end of 36 days. However, no changes were observed in either epididymal sperm concentration or germ cell methylation status. In contrast, at the end of 76 days, although serum testosterone level, sperm DNA integrity and DNA ploidy status were unperturbed significantly in hyperglycemic group, the epididymal sperm concentration and methylation status of preleptotene/zygotene cells were significantly altered. Importantly, an attempt to find out the association between the blood glucose levels and the abnormalities in hyperglycemic group failed to demonstrate any correlation.

Conclusions

The germ cell abnormalities observed in hyperglycemic group could be interpreted as a primary effect of streptozotocin and not due to hyperglycemia. Our results call for further evaluation of streptozotocin before its application to study the hyperglycemic responses on male germ cells.     

Novel associations between specific sperm morphological defects and leukocytospermia

OBJECTIVE: To examine the relationship between leukocyte concentrations in semen and sperm morphology in a group of infertile men and healthy fertile donors. DESIGN: A prospective clinical study. SETTING: Male infertility clinic at a tertiary care teaching hospital and a reproductive medicine unit at a Women's Hospital in the United Kingdom. PATIENT(S): Fifty-six infertile men and 13 healthy fertile sperm donors (control). INTERVENTION(S): Standard semen analysis, seminal leukocyte concentration, and the assessment of sperm morphology and sperm deformity index (SDI), applying Tygerberg's strict criteria. MAIN OUTCOME MEASURE(S): Granulocyte concentrations in semen, percentages of different sperm morphological abnormalities, and SDI scores. RESULT(S): Leukocyte concentrations were statistically significantly and negatively correlated with the proportion of sperm with damaged acrosomes, cytoplasmic droplet, tail defects, and SDI scores with normal and borderline morphology. The percentage sperm motility was significantly and negatively correlated with leukocytic concentration in semen. However, the leukocytic concentration was not significantly correlated with sperm concentration. CONCLUSION(S): This is the first study to report a significant positive correlation between leukocytospermia and sperm tail defects, acrosomal damage, and high SDI scores. These observations suggest that leukocytospermia is associated with compromised sperm structural integrity.     

Leukocytospermia and function of the seminal vesicles on seminal quality.

OBJECTIVE: To determine possible relationships between number of leukocytes, function of seminal vesicles, and seminal quality. DESIGN: The study was carried out on men who consecutively attended an infertility clinic between June 1989 to June 1991. SETTING: This study was conducted in a private immunological center for infertility, a tertiary care center, The Centro Immunológico-Sección Esterilidad y Reproducción. PATIENTS: Semen samples from 280 infertility patients attending an Immunological Center for Infertility were analyzed. MAIN OUTCOME MEASURE: We evaluated the effect of leukocytospermia in the presence of normal or abnormal function of seminal vesicles on seminal quality. RESULTS: Sperm count, percent of motile sperm, and percent of sperm vitality were significantly reduced when both leukocytospermia and hypofunction of seminal vesicles were present (P less than 0.01). Leukocytospermic subjects with normal function of seminal vesicles showed similar seminal parameters to those nonleukocytspermics. The incidence of subjects with antisperm antibodies measured by direct immunobeads was significantly higher in leukocytospermic men with hypofunction of seminal vesicles. No differences in the incidence of antisperm antibodies with nonleukocytospermic samples were observed in those with both leukocytospermia and normal function of seminal vesicles. CONCLUSIONS: These data provide evidence that white blood cells were deleterious for seminal quality when seminal vesicles were also affected.     

Effect of leukocytospermia on fertilization and pregnancy rates of artificial reproductive technologies

The aim of this study was to investigate the fertilization and pregnancy rates of artificial reproductive technologies using semen samples without (<1 x 10(6)/mL) and with (> or =1 x 10(6)/mL) leukocytospermia. The overall fertilization rate was 63.4% (range, 44.4-87.5%) in nonleukocytospermic couples and 64.3% (range, 45.3-100.0%) in leukocytospermic couples, whereas the corresponding pregnancy rates were 34.5% and 50%, respectively. These results show that leukocytospermia may not necessarily have a negative effect on outcome after either in vitro fertilization or intracytoplasmic sperm injection.     

Comparative genomic hybridization analysis of sperm DNA apoptosis after exposure to heat shock

Purpose: A DNA disc chip assay based on comparative genomic hybridization (CGH) was developed to measure sperm DNA integrity. The objective was to correlate DNA integrity of heat-treated sperm with the sperm capacitation index (CI) determined from the sperm penetration assay. Methods: Basic semen and kinematic parameters were measured (N = 6). Sperm were washed in two-layer colloid suspensions and split portions incubated at either 37°C (control) or 40°C for 4 h. Single-stranded DNA of heated sperm were stained in SYBR Gold and hybridized to bisbenzimide (Hoechst 33342) stained control DNA in a membrane disc. Fluorescent intensities of the discs were measured and correlation analyses with sperm parameters performed. Results: Sperm CI was positively correlated (R = 0.737) with sperm DNA integrity. Two populations of sperm could be discerned: low capacitating sperm that initiated apoptosis and high capacitating sperm unaffected by heat shock treatment. The remaining parameters were not related to sperm DNA stability. Conclusions: Fragile DNA were found in a population of sperm associated with poor capacitation characteristics and apoptosis was observed after heat treatment. The results suggested that sperm dysfunction might be due to apoptotic sperm DNA resulting from an elevated temperature in the surroundings. The data suggested that the second population of high capacitating sperm induced chaperones such as heat shock proteins hsp 70 to protect against apoptosis.     

Differential effects of human papillomavirus DNA types on p53 tumor-suppressor gene apoptosis in sperm

OBJECTIVE: Sperm DNA undergoes apoptotic fragmentation when exposed to HPV DNA. Details of the specific gene regions targeted by HPV in sperm are lacking. The objective of this study was to determine the integrity of exons 5 and 8 of the p53 gene in sperm exposed to HPV DNA. METHODS: Washed sperm were exposed to either HLA-DQA1 (control) or HPV type 6b/11, 16, 18, 31, or 33 DNA fragments for 24 h at 37 degrees C. The integrity of sperm p53 exons 5 and 8 was assessed using a novel DNA disc chip assay based on comparative genomic hybridization. RESULTS: Fragmentation of exon 5 occurred after exposure to HPV DNA type 18. In contrast, only exon 8 was affected by HPV type 16. HPV DNA from type 31 or 33 was without effect on the p53 exons. Sperm motility but not hyperactivation was reduced in all HPV groups. CONCLUSION: The data suggest that different HPV types preferentially degrade different exons of important genes. Decreased motility but not hyperactivation in HPV-exposed sperm suggests retention of some fertilizing capacity and the possibility of transmitting virus-destabilized genes through fertilization.     

Effects of hydrogen peroxide on DNA and plasma membrane integrity of human spermatozoa

Objective: To evaluate the effects of oxidative stress on DNA and plasma membrane integrity of human spermatozoa.

Design: Prospective cohort study.

Setting: University-based, tertiary-care infertility center.

Patient(s): Men (n = 10) undergoing infertility investigation.

Intervention(s): Purified populations of sperm with high motility were separated using Percoll density gradients. Then, spermatozoa were incubated with 0, 10, 100, and 200 μM hydrogen peroxide (H₂O₂) under capacitating conditions.

Main Outcome Measure(s): Motion parameters were assessed by computer analysis. Genomic integrity was examined by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end–labeling (TUNEL) assay. Plasma membrane integrity was evaluated by the annexin V-binding assay, a measure of phosphatidylserine translocation.

Result(s): Under basal conditions, there was a significant and negative relationship between sperm motility and the percentages of sperm with DNA fragmentation and membrane translocation of phosphatidylserine. After a 2-h incubation, there was a significant, dose-dependent effect of H₂O₂ on motion parameters (decrease) and DNA fragmentation (increase). The percentage of annexin V⁻ live (normal) cells declined significantly as the level of oxidative stress increased. Although the percentages of annexin V⁺ live cells (sperm depicting translocation of phosphatidylserine) and necrotic cells increased at the highest H₂O₂ levels, these changes were not significant.

Conclusion(s): In vitro sperm incubation with H₂O₂ induces DNA fragmentation in a dose-dependent fashion. The sublethal effects of oxidative stress on motion parameters were not significantly associated with membrane translocation of phosphatidylserine.     

Prostatitis and fertility: The biologist's point of view

There are two leukocytospermia: the leukocytospermia below 10⁶ cells/ml comes from the epididymis. It is physiological, improves sperm quality and ART outcomes and therefore must be respected. Leukocytospermia above 10⁶ cells/ml are of prostatic origin and reflect a chronic prostatitis. The results of IVF and ICSI with these sperm are always surprisingly improved when compared to those obtained using semen without leukocytes at all. But this improvement is offset by a dramatic increase in the miscarriage rate. Should we treat this leukocytospermia or its cause? A clinical trial is conducted in Cochin hospital with the PHRC Sigma (Male Genital Track Inflammatory Syndrome) that will help us answer this question. It seems, a priori, that it is better to treat the cause and to respect the leukospermia.     

The influence of asymptomatic leukocytospermia on interleukin IL-2 and IL-6 levels in male semen plasma

AIM: The study presents the results of the search for a correlation between leukocytospermia and the interleukin IL-2 and IL-6 levels in male semen plasma. MATERIAL AND METHODS: Interleukin levels were assessed by immunoenzymatic method (ELISA). Study covered 3 groups of males: I--fertile (No. = 16); II--with leukocytospermia (No = 16); III--with oligoasthenozoospermia (No = 20). RESULTS: The study revealed higher levels of IL-6 in a group of males with leukocytospermia (10.01 +/- 2.57 pg/ml) and with oligoasthenozoospermia (13.53 +/- 2.78 pg/ml) compared to a group of fertile males (7.68 +/- 1.4 pg/ml) (p < 0.01), however no statistically significant differences in IL-6 levels between group II and III have been observed. Higher level of IL-2 has been noted in III group (16.68 +/- 5.86 pg/ml) compared to I and II group (9.77 +/- 3.18 pg/ml and 6.05 +/- 2.16 pg/ml, respectively) (p < 0.01). No statistically significant difference in IL-2 semen plasma level between the fertile male group and the group with leukocytospermia has been revealed. CONCLUSION: Leukocytospermia is accompanied by an increase in IL-6 semen plasma level whereas IL-2 level remains statistically unchanged.     

Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity.

OBJECTIVE: To investigate effects of cryopreservation on sperm motility and DNA integrity. DESIGN: Pre-cryopreservation and post-cryopreservation analysis of motility and DNA integrity of semen and prepared sperm samples. SETTING: A hospital andrology laboratory. PATIENT(S): Forty men attending the Regional Fertility Centre, Belfast, Northern Ireland. INTERVENTION(S): Each sample was divided, and an aliquot was frozen unprepared. Remaining aliquots were prepared by Percoll density centrifugation (95.0:47.5) or direct swim-up procedure and divided into aliquots to allow direct comparison of fresh and frozen semen and prepared sperm (frozen with or without the addition of seminal plasma) from the same ejaculate. Samples were frozen by static-phase vapor cooling and being plunged into liquid nitrogen. Thawing was carried out at room temperature. MAIN OUTCOME MEASURE(S): Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay, and motility was determined using computer-assisted semen analysis. RESULT(S): Sperm frozen unprepared in seminal fluid appeared more resistant to freezing damage than frozen prepared sperm. Further improvements can be achieved by selecting out the subpopulation of sperm with best motility and DNA integrity and freezing these sperm in seminal plasma, making this the optimal procedure. CONCLUSION(S): Freezing sperm in seminal plasma improves postthaw motility and DNA integrity.