Sperm DNA Fragmentation Rate Research Articles

No difference in high-magnification morphology and hyaluronic acid binding in the selection of euploid spermatozoa with intact DNA

In this study, we compared conventional sperm selection with high-magnification morphology based on the motile sperm organellar morphology examination (MSOME) criteria, and hyaluronic acid (HA) binding for sperm chromosome aneuploidy and DNA fragmentation rates. Semen from 50 severe male factor cases was processed through density gradient centrifugation, and subjected to sperm selection by using the conventional method (control), high magnification at ×6650 or HA binding. Aneuploidy was detected by fluorescence in situ hybridization with probes for chromosomes 13, 18, 21, X and Y, and DNA fragmentation by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) method. Spermatozoa selected under high-magnification had a lower DNA fragmentation rate (2.6% vs. 1.7%; P=0.032), with no significant difference in aneuploidy rate (0.8% vs 0.7%; P=0.583), than those selected by the HA binding method. Spermatozoa selected by both methods had much lower aneuploidy and DNA fragmentation rate than the controls (7% aneuploidy and 26.8% DNA fragmentation rates, respectively). In the high-magnification group, the aneuploidy rate was lower when the best spermatozoa were selected than when only the second-best spermatozoa were available for selection, but the DNA fragmentation rate was not different. In conclusion, sperm selection under high magnification was more effective than under HA binding in selecting spermatozoa with low DNA fragmentation rate, but the small difference (0.9%) might not be clinically meaningful. Both methods were better than the conventional method of sperm selection.

Sperm DNA damage has a negative association with live-birth rates after IVF

Sperm DNA damage has a negative impact on pregnancy rates following assisted reproduction treatment (ART). The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage. Following IVF, couples with <25% sperm DNA fragmentation had a live-birth rate of 33%; in contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13%. Following ICSI, no significant differences in sperm DNA damage were found between any groups of patients. Sperm DNA damage was also associated with low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men with idiopathic infertility have high sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF.Sperm DNA damage has a negative impact on assisted reproduction treatment outcome, in particular, on pregnancy rates. The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage and treatment outcome. Following IVF, couples with <25% sperm DNA fragmentation had a live birth rate of 33%. In contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13% following IVF. Following ICSI, there were no significant differences in levels of sperm DNA damage between any groups of patients. Sperm DNA damage was also associated with the very low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men have high level of sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF.

Quality of Sperm Obtained by Penile Vibratory Stimulation and Percutaneous Vasal Sperm Aspiration in Men With Spinal Cord Injury

The aim of this study was to explore sperm chromosomal aneuploidy and DNA integrity in infertile patients with spinal cord injury (SCI). Semen samples were collected from 12 infertile men with SCI by percutaneous vasal sperm aspiration (PVSA) and from 14 male SCI patients by penile vibratory stimulation (PVS). These semen samples as well as samples from 16 donors were analyzed using the hypo-osmotic swelling (HOS) test, the sperm chromatin dispersion test, terminal deoxynucleotidyl transferase-mediated terminal uridine nick-end labeling assay, and multicolor fluorescence in situ hybridization with probes specific for the chromosomes 13, 18, 21, X, and Y. There were significant differences in the percentages of motile sperm, normal morphologic sperm, normal HOS/eosin staining, and sperm DNA fragmentation between the infertile men with SCI and the control group (P < .05 and P < .01). The sperm forward motility was significantly greater in the PVSA group than in the PVS group (P < .01). The number of round cells per milliliter of semen obtained from the 14 SCI patients by PVS was between 1 million and 12 million. The rate of sperm DNA fragmentation, as identified by the sperm chromatin dispersion test, was higher in the PVS group than in the PVSA group (P < .05). The aneuploidy rates for the SCI patients were 1.5- to 1.6-fold higher for chromosomes 13, 18, and 21, and were 2.3- to 2.4-fold higher for chromosomes X and Y than for patients in the control group (P < .001). These results suggest that for men with SCI, the semen quality is poorer, the prevalence of abnormal HOS/eosin staining is greater, and sperm DNA fragmentation and sperm chromosomal aneuploidies are seen at a higher rate compared with healthy, fertile, and normospermic men.

Are the annexin V microbeads, a real solution to sperm DNA fragmentation?

ObjectiveThe aim of our study was to assess the effect of annexin V microbeads on cycles with abnormal sperm DNA fragmentation (>20%), in fertilization, embryo quality and success rate.DesignAnalytic Retrospective Observational study.Materials and MethodsWe analyzed 88 cycles of Assisted Reproduction treatments.All patients had abnormal sperm DNA fragmentation and previously had undergone one or more unsuccessful cycles of IVF/ICSI.The cycles were divided into two groups:Group 1: unsuccessful cycles of IVF/ICSI.Group 2: cycles with sperm sorting with annexin V columns (36 cycles) from these same partners.The sperm DNA fragmentation was assessed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling).The embryos were evaluated according to criteria of the Spanish Association for Reproductive Biology (ASEBIR). The transfer was performed on day 3 of embryo development.ResultsNo significant differences were found between groups or age, nor in the rates of fertilization (P>0.05). However, embryo quality did show statistically significant differences, being higher the number of top quality embryos (A, P=0.004. A+B, P=0.02) in group 2.ConclusionThe results suggest that the use of annexin V columns improves embryo quality in couples with previous failure in IVF/ICSI. So it could be used, as a useful tool, in patients with previous IVF failures. The fertilization rate shows no significant differences. This may be because sperm with fragmented DNA were capable of fertilizing the egg as effectively as sperm without DNA fragmentation. ObjectiveThe aim of our study was to assess the effect of annexin V microbeads on cycles with abnormal sperm DNA fragmentation (>20%), in fertilization, embryo quality and success rate. The aim of our study was to assess the effect of annexin V microbeads on cycles with abnormal sperm DNA fragmentation (>20%), in fertilization, embryo quality and success rate. DesignAnalytic Retrospective Observational study. Analytic Retrospective Observational study. Materials and MethodsWe analyzed 88 cycles of Assisted Reproduction treatments.All patients had abnormal sperm DNA fragmentation and previously had undergone one or more unsuccessful cycles of IVF/ICSI.The cycles were divided into two groups:Group 1: unsuccessful cycles of IVF/ICSI.Group 2: cycles with sperm sorting with annexin V columns (36 cycles) from these same partners.The sperm DNA fragmentation was assessed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling).The embryos were evaluated according to criteria of the Spanish Association for Reproductive Biology (ASEBIR). The transfer was performed on day 3 of embryo development. We analyzed 88 cycles of Assisted Reproduction treatments.All patients had abnormal sperm DNA fragmentation and previously had undergone one or more unsuccessful cycles of IVF/ICSI.The cycles were divided into two groups: Group 1: unsuccessful cycles of IVF/ICSI. Group 2: cycles with sperm sorting with annexin V columns (36 cycles) from these same partners. The sperm DNA fragmentation was assessed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling).The embryos were evaluated according to criteria of the Spanish Association for Reproductive Biology (ASEBIR). The transfer was performed on day 3 of embryo development. ResultsNo significant differences were found between groups or age, nor in the rates of fertilization (P>0.05). However, embryo quality did show statistically significant differences, being higher the number of top quality embryos (A, P=0.004. A+B, P=0.02) in group 2. No significant differences were found between groups or age, nor in the rates of fertilization (P>0.05). However, embryo quality did show statistically significant differences, being higher the number of top quality embryos (A, P=0.004. A+B, P=0.02) in group 2. ConclusionThe results suggest that the use of annexin V columns improves embryo quality in couples with previous failure in IVF/ICSI. So it could be used, as a useful tool, in patients with previous IVF failures. The fertilization rate shows no significant differences. This may be because sperm with fragmented DNA were capable of fertilizing the egg as effectively as sperm without DNA fragmentation. The results suggest that the use of annexin V columns improves embryo quality in couples with previous failure in IVF/ICSI. So it could be used, as a useful tool, in patients with previous IVF failures. The fertilization rate shows no significant differences. This may be because sperm with fragmented DNA were capable of fertilizing the egg as effectively as sperm without DNA fragmentation.

The content of DNA fragmented spermatozoa in patients with the different kinds of pathospermia

Sperm with fragmented DNA could be the result of the violation of the sperm maturation process. DNA fragmented spermatozoa are able to fertilize the oocytes, but it could lead to a poor embryo quality and a low chances for embryo implantation. The aim of this work was to examine the content of sperm with fragmented DNA in ejaculate for patients with different kinds of pathospermia and normal sperm indices. Sperm examination was done for 200 men (21 patients with normozoospermia, 82 patients with asthenozoospermia, 57 patients with oligizoospermia and 40 patients with teratozoospermia). The middle age of the patients was 41±3.3 years. The content of sperm with fragmented DNA was examined by the method of sperm chromatin dispersion (SCD) (HaloSperm, Halotech, Spain). The content of DNA fragmented spermatozoa more then 20.0% was found out in 120 from 200 examined patients (60.0% of cases). The results of DNA fragmentation test including the result of standard sperm examination are mentioned in Table 1.Tabled 1High DNA-fragmented sperm content in men with normo- and pathospermia.The results of SCD-test: more then 20.0 % of DNA-fragmented spermThe results of SCD-test: more then 20.0 % of DNA-fragmented spermConclusion of sperm examinationQuantity of patients, NQuantity of patients, %Normozoospermia3 (21)14.3%Asthenozoospermia66 (82)80.5%Oligozoospermia24 (57)42.1%Teratozoospermia27 (40)67.5%The most high content of DNA fragmented sperm was found out for men with asthenozoospermia: in 80.5 % of patients with asthenozoospermia (P<0.05). Open table in a new tab The most high content of DNA fragmented sperm was found out for men with asthenozoospermia: in 80.5 % of patients with asthenozoospermia (P<0.05). The work confirms that sperm DNA fragmentation rate is significantly higher in cases of pathospermia comparing with normozoospermia. The DNA fragmentation test could have a diagnostic and predictive meaning especially in cases of asthenozoospermia. The investigation confirms the necessity of the DNA fragmentation test for men with pathospermia before the infertility treatment using the ART.

Colloidal Centrifugation of Stallion Semen Results in a Reduced Rate of Sperm DNA Fragmentation

Stallion spermatozoa recovered and examined immediately after colloidal centrifugation resulted in a higher straight-line velocity (VSL) than sperm processed using direct conventional centrifugation (p = 0.000), but there was no differences in the progressive motility or sperm DNA fragmentation (SDF) as determined by the sperm chromatin dispersion assay. However, when centrifuged spermatozoa were incubated at 37 °C for 24 h to determine the rate of SDF (r-SDF), a lower r-SDF (p = 0.0011) was observed in those sperm recovered after colloidal separation (0.5 ± 0.1%/h) compared to direct (1.2 ± 0.4%/h) or no centrifugation (r-SDF = 1.2 ± 0.3%/h). These results confirm that colloidal separation of stallion spermatozoa results in prolonged sperm DNA longevity, but these differences were only apparent following a period of incubation and dynamic assessment. Consequently, we strongly recommend the use of the dynamic form of the SDF assay for evaluating centrifugation and/or other ex vivo procedures, as a single basal assessment of SDF may inadvertently result in a false-positive evaluation of DNA quality.

Selection of normal spermatozoa with a vacuole-free head (x6300) improves selection of spermatozoa with intact DNA in patients with high sperm DNA fragmentation rates

Intracytoplasmic morphologically selected sperm injection (IMSI, 6300× magnification with Nomarski contrast) of a normal spermatozoon with a vacuole-free head could improve the embryo's ability to grow to the blastocyst stage and then implant. However, the most relevant indications for IMSI remain to be determined. To evaluate the potential value of IMSI for patients with a high degree of sperm DNA fragmentation (n=8), different types of spermatozoa were analysed in terms of DNA fragmentation. Motile normal spermatozoa with a vacuole-free head selected at 6300× magnification had a significantly lower mean DNA fragmentation rate (4.1±1.1%, n=191) than all other types of spermatozoa: non-selected spermatozoa (n=8000; 26.1±1.5% versus 4.1±1.1%; P<0.005), motile spermatozoa (n=444; 20.8±2.7% versus 4.1±1.1%; P<0.001) and motile, normal spermatozoa selected at 200× magnification (n=370; 18.7±2.7% versus 4.1±1.1%; P<0.001) and then motile, morphometrically normal spermatozoa with anterior vacuoles (n=368; 15.9±2.9% versus 4.1±1.1%; P<0.05) or posterior vacuoles (n=402; 22.5±3.6% versus 4.1±1.1%; P<0.001) selected at 6300× magnification. For patients with high sperm DNA fragmentation rates, selection of normal spermatozoa with a vacuole-free head (6300×) yields the greatest likelihood of obtaining spermatozoa with non-fragmented DNA.

Analysis of sperm DNA fragmentationin infertility patients

Using Terminal Deoxynucleotidyl Transferase dUTP Nick End Labelling (TUNEL) technique the assessment of the frequency of the spermatozoa with fragmented DNA in the ejaculate from sperm donors and men with infertility was analyzed. The DNA fragmentation rate was higher in spermatozoa of carriers of a chromosomal structural abnormality compared with the control group. There was no correlation between the sperm DNA fragmentation rate and the parameters of semen analysis. The direct linear correlation between the frequency of the spermatozoa with fragmented DNA and vacuole sperm head was found. The DNA fragmentation rate was not correlated to the frequency of the spermatozoa with bulb, amorphous heads or spermatozoa with abnormal acrosome

USE OF QUINOLONES IN BULL SEMEN EXTENDERS TO REDUCE SPERM DEOXYRIBONUCLEIC ACID DAMAGE

Cryopreserved sperm samples from Holstein bulls (n = 20) were examined for bacterial presence and Sperm DNA Fragmentation (SDF) dynamics. SDF was assessed after thawing (T0) and at 4, 24 and 48 h of incubation (37°C) and the rate of SDF (r-SDF), as an estimator of the DNA degradation over time, was calculated. Two groups of bulls were identified based on the presence or absence of bacteria: One group (n = 10) had a readily detectable bacterial presence, while the other group (n = 10) had an undetectable bacterial presence. Differences in the SDF at T0 were not observed between these two groups. However, statistically different results were found after 24 h of incubation at 37°C (Kaplan-Meier estimator; Log-Rank Matel-Cox, p0.05) were not detected between the control and the quinolone treated sample for those samples without bacteria. However, differences (p

Correlation Between Seminal Oxidative Stress Biomarkers and Antioxidants with Sperm DNA Damage in Elite Athletes and Recreationally Active Men

To evaluate the seminal plasma 8-isoprostane, reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), catalase, total antioxidant capacity (TAC), and levels of sperm DNA fragmentation in elite athletes and recreationally active men. Prospective design was used for this study. The study was performed in the Exercise Physiology Laboratory of the Urmia University. Fifty-six elite athletes and 52 recreationally active men (18-28 years) participated in this study. All subjects had a semen sampling at baseline. Total antioxidant capacity and SOD activity were measured by colorimetric assay. Levels of ROS were measured by a chemiluminescence assay. Malondialdehyde levels were measured by thiobarbituric acid reactive substance assay. Catalase activity was measured by monitoring the initial rate of disappearance of hydrogen peroxide. Concentration of free 8-isoprostane was measured by enzyme immunoassay method. Sperm DNA fragmentation was evaluated with the terminal deoxynucleotidyl transferase-mediated fluorescein dUTP nick end-labeling assay. Recreationally active men have significantly higher levels of body fat, seminal SOD, TAC, and catalase and lower levels of V[Combining Dot Above]O2max, seminal ROS, MDA, and 8-isoprostane and subsequently lower rate of sperm DNA fragmentation when compared with elite athletes (P < 0.001). Significantly negative correlation was observed between sperm DNA fragmentation with body fat, seminal SOD, catalase, and TAC levels (P < 0.001). Significantly positive correlation was observed between sperm DNA fragmentation with V[Combining Dot Above]O2max, seminal 8-isoprostane, ROS, and MDA levels (P < 0.001). Spermatozoa from recreationally active men may be less susceptible to oxidative stress-induced DNA damage and hence infertility.

Association between obesity and alteration of sperm DNA integrity and mitochondrial activity

What's known on the subject? and What does the study add? The relationship between high levels of BMI and changes in altered standard semen analysis parameters are described in the literature. However, the functional characteristics of the sperm are essential to complete the evaluation of male infertility. Thus, this study provides important information about the functionality of the sperm of men with different levels of BMI. To assess the effect of obesity on semen analysis, sperm mitochondrial activity and DNA fragmentation. A transversal study of 305 male patients, presenting for clinical evaluation, was carried out. The patients were divided into three groups according to body mass index (BMI) as follows: eutrophic (BMI < 25 kg/m(2), n = 82), overweight (BMI ≥ 25 kg/m(2) and <30, n = 187) and obese (BMI ≥ 30 kg/m(2), n = 36). The variables analysed were semen analysis, rate of sperm DNA fragmentation and sperm mitochondrial activity. Groups were compared using one-way analysis of variance followed by a least significant difference post-hoc test. A P-value of <0.05 was considered to indicate statistical significance. No differences were observed in age, ejaculatory abstinence, ejaculate volume, sperm vitality, morphology or round cell and neutrophil count among the groups. The eutrophic group had a higher percentage of sperm with progressive motility (P = 0.001). Mitochondrial activity was lower in the obese group (P = 0.037) when compared to the eutrophic, and the percentage of sperm with DNA damage was higher in the obese group (P = 0.004) than the other two groups. Increased BMI values are associated with decreased mitochondrial activity and progressive motility and increased DNA fragmentation.

The dynamics of sperm DNA stability in Asian elephant (Elephas maximus) spermatozoa before and after cryopreservation

The purpose of this study was to investigate the occurrence of sperm DNA fragmentation in Asian elephant (Elephas maximus) spermatozoa at various processing stages before and after cryopreservation. Five semen samples from four elephants were assessed at four different stages during processing; after (1) collection and reextension in TEST-egg yolk; (2) cooling to 5 °C; (3) equilibration for 1 h with glycerol; (4) thawing. An experimental approach was adopted that allowed comparisons of DNA fragmentation rates developed after the various processing stages. For this, spermatozoa were incubated in TEST-yolk media at 37 °C for 0, 4, 8, 24 and 48 h, and sperm DNA fragmentation rates were estimated using an elephant-specific Halosperm procedure. Incubation at 37 °C induced a rapid increase in DNA fragmentation, and significant differences between males were observed. The overall rate of increase over 4 h was estimated at about 5% per hour, and no significant changes to this rate were observed at the different processing stages, even, including the post-thaw samples. As semen quality of the five ejaculates was relatively poor, the basic semen parameter data were compared with nine different samples collected 11 mo earlier to see whether the tested samples were atypical or representative of the population, As there was no significant difference between the two sets of samples, it is believed that the samples tested for DNA stability were not unusually sensitive. These results suggest that Asian elephant spermatozoa are more susceptible to DNA fragmentation than spermatozoa of other mammals.

Sperm chromosomal aneuploidy and DNA integrity of infertile men with anejaculation

To explore sperm chromosomal aneuploidy, sperm membrane and DNA integrity in infertile patients with anejaculation. Semen samples were collected from 18 infertile men with spinal cord injury (SCI) by penile vibratory stimulation (PVS) and from 14 psychogenic anejaculation (PA) patients by percutaneous vasal sperm aspiration (PVSA). These semen samples as well as samples from 16 donors were analyzed using the hypo-osmotic swelling (HOS) test, the sperm chromatin dispersion (SCD) test and multi-color fluorescence in situ hybridization (FISH) with probes specific for chromosomes 13, 18, 21, X and Y. There were significant differences in the percentages of motile sperm, normal morphologic sperm and sperm DNA fragmentation between the infertile men with SCI and the control group (P < 0.05 and P < 0.01). The sperm motility was significantly greater in the PA-PVSA group than in the SCI-PVS group (P < 0.01). The number of round cells per mL of semen obtained from the 18 SCI patients by PVS was between 1 and 8 million. The rate of sperm DNA fragmentation in the SCI-PVS group was higher than that of the PA-PVSA group (P < 0.05). The aneuploidy rates for the SCI patients were 2.4-fold higher for chromosomes 13, 18 and 21 and 2.2-fold higher for chromosomes X and Y than for patients in the control group (P < 0.0001). The semen quality is poorer, sperm DNA fragmentation and sperm chromosomal aneuploidies are seen at a higher rate for SCI patients compared to healthy, fertile and normospermic men. Whether the difference in yield is due to increased scrotal temperature, genitourinary infection, or other reasons requires further study.

Sperm FISH analysis of a 46,XY,t(3;6)(p24;p21.2),inv (8)(p11;2q21.2) double chromosomal rearrangement

A complex chromosome rearrangement (CCR) can be defined as a structural chromosomal aberration that involves at least three breakpoints located on two or more chromosomes. Highly unbalanced gametes may lead to infertility or congenital malformations. Here is reported a double rearrangement considered as the simplest possible CCR and, in a sense, not a true CCR, meiotic segregation for a 46,XY,t(3;6)(p24;p21.2),inv(8)(p11;2q21.2) male patient referred after his partner had undergone three early miscarriages. Sperm fluorescence in-situ hybridization was used to screen for translocation and inversion segregation and an interchromosomal effect (ICE) for 13 chromosomes not involved in CCR. The malsegregation rates for the reciprocal translocation and pericentric inversion were 61.2% and 1.7%, respectively. ICE analysis revealed that the observed chromosome aneuploidy rates of between 0.1% and 0.8% did not differ significantly from control values. A slight increase in cumulative ICE ( P = 0.049) was observed in the patient, relative to control spermatozoa (with rates of 4.6% and 3.1%). The sperm DNA fragmentation rate differed significantly from control values (5.0%; P = 0.001). Reciprocal translocation had no impact on meiotic segregation of the pericentric inversion in this double rearrangement. No conclusion could be drawn regarding the impact of pericentric inversion on translocation. A complex chromosome rearrangement (CCR) can be defined as a structural chromosomal aberration that involves at least three breakpoints located on two or more chromosomes, with the exchange of genetic material. Highly unbalanced gametes may lead to infertility or congenital malformations. We report here the first known double rearrangement CCR meiotic segregation for a 46,XY,t(3;6)(p24;p21.2),inv(8)(p11;2q21.2) male patient referred to our hospital after his partner had undergone three early miscarriages. Sperm fluorescence in-situ hybridization was used to screen for translocation and inversion segregation and an interchromosomal effect (ICE) for 13 chromosomes not involved in CCR (chromosomes 7, 9, 11, 12, 13, 15, 16, 17, 18, 20, 21, X and Y). The malsegregation rates for the reciprocal translocation and pericentric inversion were 61.2% and 1.7%, respectively. An ICE analysis revealed that the observed chromosome aneuploidy rates between 0.1% and 0.8% did not differ significantly from control values (between 0.1% and 0.5%). A slight increase in cumulative ICE for the 13 chromosomes ( P = 0.049) was observed in the CCR patient, relative to control spermatozoa (with rates of 4.6% and 3.1%). The sperm DNA fragmentation rate was slightly higher than our laboratory’s normal cut-off value (13%) and differed significantly from controls values (5.0%; P = 0.001). In agreement with previous results for isolated inversions, we conclude that reciprocal translocation had no impact on meiotic segregation of the pericentric inversion in this double rearrangement CCR. No conclusion could be drawn regarding the impact of pericentric inversion on translocation.

Sperm Organelle Morphologic Abnormalities: Contributing Factors and Effects on Intracytoplasmic Sperm Injection Cycles Outcomes

To (1) analyze possible relationships between motile sperm organelle morphology examination (MSOME) and sperm chromatin status, aneuploidy incidence, and patient's age; (2) determine the effects of sperm morphologic abnormalities on intracytoplasmic sperm injection (ICSI) outcomes; and (3) identify the benefits of intracytoplasmic morphologically selected sperm injection (IMSI) in patients with high DNA fragmentation rate. The study was performed in 50 patients undergoing ICSI cycles. The MSOME, sperm DNA fragmentation, and sperm aneuploidy incidence were performed in 200 sperm cells of each patient. Regression models were used to assess the relationships among sperm morphology and sperm aneuploidy, sperm DNA fragmentation, patient's age, and ICSI outcomes. In cycles with patients showing a high incidence of DNA fragmentation, oocytes were split into 2 groups according to the sperm selection method: Standard-ICSI (n = 82) and IMSI (n = 79). Fertilization and high-quality embryo rates were compared between the groups. A close relationship between sperm DNA fragmentation and the presence of vacuoles in the MSOME was noted. The patient's age was correlated to the presence of vacuoles. No correlation between sperm aneuploidy and IMSI was observed. Vacuolated cells were negatively correlated with fertilization, pregnancy, and implantation. In patients with a high incidence of sperm DNA fragmentation, fertilization and high-quality embryo rates were similar when comparing IMSI and Standard-ICSI. Our data demonstrate a correlation between paternal age and the incidence of nuclear vacuoles, as well as an effect of large and small vacuoles on late embryo development.

Quinolones Abolish Bacterial Presence in Holstein Bull Sperm Samples and Indirectly Modulate the Kinetics of Sperm DNA Damage.

Commercial cryopreserved semen samples from Holstein bulls (n=47) were examined for bacterial presence and its effect on the dynamic of sperm DNA fragmentation (SDF). Commercial semen doses produced in Spain (six ejaculates per individual) were included in this analysis. SDF were assessed after thawing (T0) and at 4, 24 and 48 hours of incubation at 37°C, using the commercial variant of the sperm chromatin dispersion test for Bovine (Halomax; Halotech SL, Madrid, Spain). Two groups of individuals, based on the presence/absence of bacteria in semen samples, were created. Using fluorescence microscopy, one group (n=23) had a readily detectable bacterial presence during the incubation time while the other group (n=24) had an undetectable bacterial presence during an equivalent period of incubation time. The comparison of the dynamic loss of DNA quality was assessed using the nonparametric maximum likelihood Kaplan-Meier estimator. The log rank test statistic (Mantel-Cox) which compares estimates of the hazard functions between the two groups at each time interval was used (SPSS v.17.0 for Windows; SPSS Inc., Chicago, IL, USA).When both groups were compared, differences in the frequency of sperm with damaged DNA immediately after thawing were not observed. However, these differences became statistically different (P<0.05) after 24 hours of sperm incubation. The extenders used during the preparation of every sperm sample contained the standard antibiotics used to putatively prevent these situations. Within this scenario, a first experiment was conducted to test the direct effect of bacteria on SDF over time. To this purpose, sperm samples from 6 different bulls without bacteria were inoculated after thawing with 5µL of sperm from a sample exhibiting bacterial presence at 48 hours. Determination of SDF was conducted after 0, 4, 24, and 48 hours of incubation at 37°C. Significant differences (p<0.05) were observed between SDF dynamics from the same sample without bacteria (control) and the inoculated sample. In a second experiment, 10 semen samples from different bulls were treated with 1µg/mL of ciprofloxacin at T0 in addition to existing antibiotics in the extender media. Five samples belonged to the group of bulls that repeatedly presented bacteria in semen and the other five were samples from bulls with an undetectable presence of bacteria in sperm. After determination of SDF at 0, 4, 24, and 48 hours of incubation at 37°C for the control sample and the treated sample, significant differences in the rate of SDF (p>0.05) were not detected on samples without bacteria. However, significant differences (p<0.05) were observed when comparing the control and the quinolone-treated samples with bacterial presence. These results call attention to two main points: 1) under similar conditions of sperm handling the dynamic behaviour of SDF can be directly correlated with sperm bacterial presence and 2) sperm DNA fragmentation can be significantly reduced by incubating sperm samples with quinolones. (poster)

Seasonal changes in sperm DNA fragmentation of Murciano-Granadina goats: The compelling case for dynamic assessment

Evidence is provided of seasonal changes in the rate of DNA fragmentation dynamics of sperm collected from goats Murciano-Granadina breed), in a Spanish Breeding Centre at 41°N latitude. Results show that the initial assessment of sperm DNA fragmentation (T0) was not sufficient to differentiate sperm DNA fragmentation of bucks collected at different times of the year (Kruskal–Wallis 2.399; P=0.493). However, when thawed semen was subsequently incubated at 37°C for periods of between 2 and 48h, differences were recorded in the rate of sperm DNA fragmentation (r-SDF), that are indicative of seasonal changes in reproduction (Log Rank: Mantel–Cox; χ2=158.6; DF=3; P<0.001). Goat semen collected and cryopreserved in the Spanish winter and spring resulted in a poor post-thaw sperm DNA longevity and recorded a r-SDF of 0.8 and 1.3 per hour, after 48h of incubation respectively. The r-SDF subsequently decreased to 0.16 and 0.36 per hour during summer and autumn. It is therefore, recommended that semen samples from Murciano-Granadina goats housed above the 40°N latitude be collected and cryopreserved in the summer and early autumn. This would result in a more stable DNA molecule, which is likely to improve the reproductive success.

Dynamics of sperm DNA fragmentation in patients carrying structurally rearranged chromosomes

This investigation was conducted to assess the baseline level of sperm DNA fragmentation (SDF) in a cohort of patients presenting chromosomal rearrangements (nine reciprocal translocations and two inversions). In a separate experiment, a dynamic analysis to calculate the rate of SDF (rSDF), after a varying period of sperm storage (0 h, 1 h, 4 h, 8 h and 24 h) at 37 °C, was performed. Results were compared with eight fertile donors. Different experimental approaches to assess SDF, such as terminal transferase dUTP nick-end labelling (TUNEL), sperm chromatin structure assay (SCSA) and sperm chromatin dispersion test (SCDt), were used. No differences for the baseline level of SDF were found. Carriers of reorganized genomes showed statistically higher levels of SDF than did control donors (p = 0.025 for TUNEL; p = 0.022 for SCSA; p = 0.014 for SCDt). However, 54.5% (6/11) of the patients presented values similar to those of control donors. There was no significant difference in rSDF (p = 0.34). Nevertheless, the results suggest that a high variability for SDF and rSDF exists in these patients. Routine analysis of SDF and rSDF should be considered in patients presenting rearranged genomes to determine fertility status for assisted reproductive techniques (ART) purposes.

Bacteria in bovine semen can increase sperm DNA fragmentation rates: A kinetic experimental approach

Cryopreserved straws of semen (n=228) from Holstein bulls (n=47) were examined for bacterial presence and sperm DNA fragmentation (SDF) dynamics. Commercial semen doses (representing six ejaculates per individual) were randomly selected from a bull stud in Spain. The dynamics of SDF were assessed after thawing (T0) and at 4, 24, 48, 72 and 96h of incubation at 37°C, using the commercial variant of the sperm chromatin dispersion test for Bovine (Halomax®). One group of bulls showed a bacterial presence in semen samples between 0 and 96h of incubation (n=23, group A) while the other did not (n=24, group B). Immediate post-thaw differences in SDF were not observed when both groups were compared. However, the rate of increase in SDF (rSDF) over time, considered as an estimate of the kinetic behaviour of sperm DNA survival, was significantly higher (P<0.05) in semen samples from group A (0.7% per hour) versus group B (0.05% per hour). Polymerase Chain Reaction (PCR) assay was used for DNA amplification using primers designed for specific regions of the bacterial gene that codifies for 16S rRNA. Different species within the phyla Bacteroidetes, Firmicutes, Proteobacteria, Cyanobacteria, Fusobacteria and Actinobacteria were identified. The results show that (1) SDF at baseline (T0) may not be affected by the presence of bacteria but the rSDF can increase due to bacterial growth during incubation, (2) the increase in the rSDF is characteristic of some bulls but not for others, and (3) certain bacterial strains are repeatedly found in separate ejaculates from the same bull.

Adolescent varicocele: improved sperm function after varicocelectomy

To assess the effect of varicocelectomy on sperm function (DNA integrity and mitochondrial activity) and levels of lipid peroxidation in seminal plasma of adolescents. Prospective study. Patients recruited from a local public school. Adolescents (14-19 years old), Tanner stages IV or V with varicocele grades II or III, attending a local public school. Two semen collections with a one week interval between collections before bilateral varicocele repair using subinguinal microsurgical varicocelectomy, and two semen collections with a one week interval between collections three months after the surgery. Rate of sperm DNA fragmentation as assessed by the Comet assay and categorized as classes I (no DNA fragmentation) to IV (high DNA fragmentation). Rate of mitochondrial activity as assessed by the diaminobenzidine assay and categorized as grades I (all mitochondria active) to IV (all mitochondria inactive). Levels of lipid peroxidation in seminal plasma by a colorimetric method that quantifies a lipid peroxidation subproduct (malondialdehyde). Concerning DNA integrity, the samples after varicocelectomy showed more spermatozoa with intact nuclear DNA (grade I) and less spermatozoa with Comet grades II, III, and IV. Regarding mitochondrial activity, the samples after varicocelectomy showed less cells with inactive mitochondria (class III). No differences were observed in classes I, II, and IV. Concerning lipid peroxidation, no significant differences were observed. This study was able to demonstrate that varicocelectomy in adolescents is associated with increased sperm DNA integrity and mitochondrial activity. However, levels of seminal products of lipid degradation (malondialdehyde) are not different.