Elevated Sperm DNA Fragmentation Research Articles

The incidence and etiology of sperm DNA fragmentation in the ejaculates of males with spinal cord injuries.

Retrospective descriptive study. To determine the incidence and probable etiology of sperm DNA fragmentation (SDF) in a sample of males with spinal cord injury (SCI). Hospital in Toledo, Spain; University-based Genetics laboratory in Madrid, Spain. Semen collected by vibro-stimulation from 27 males with various levels of spinal cord injury. Classical semen parameters, SDF, leukocytospermia and pro-oxidant capacity were assessed and compared with a cohort of normozoospermic fertile donors (n = 10). Males with SCI presented with lower semen quality compared with normozoospermic donors with respect to progressive motility (p = 0.0002), SDF (p < 0.00005), pro-oxidant capacity (p = 0.0191) and leukocytospermia (p < 0.00005). Although there was no significant correlation between semen quality and time since the lesion occurred, the period of abstinence appeared to be positively correlated with SDF (r = 0.486; p = 0.041). When the semen parameters of males with SCI were categorized based on those with cervical and thoracic lesions, sperm concentration was higher for those with cervical damage (p = 0.0257). Males with complete lesions (AIS A) had ejaculates that were lower in progressive motility (p = 0.0040) than those with incomplete injuries (AIS B-D). Ejaculates of males with SCI have excessively elevated SDF when compared with normozoospermic donors, which is likely to be associated with coincident high levels of leucocytospermia and pro-oxidant capacity. We propose that these phenomena are caused by the accumulation and degeneration of spermatozoa in the cauda epididymidis.

Extended indications for sperm retrieval: summary of current literature.

Sperm retrieval combined with intracytoplasmic sperm injection (ICSI) is the treatment of choice for couples with untreatable azoospermia-related infertility. However, an increasing body of evidence has been mounting, suggesting that ICSI with testicular sperm instead of ejaculated sperm (when both are available) increases pregnancy outcomes in some specific scenarios. This has led to the exploration of extended indications for sperm retrieval. This review summarizes the current literature concerning sperm retrieval and ICSI for non-azoospermic men with elevated sperm DNA fragmentation, oligozoospermia, and cryptozoospermia.

Paternal age is a predictor of elevated sperm DNA fragmentation in infertile men

Paternal age is a predictor of elevated sperm DNA fragmentation in infertile men

OR18-5 Elevated Semen Oxidative Stress in Male Partners as Novel Marker of Recurrent Pregnancy Loss

Background: Idiopathic recurrent pregnancy loss (RPL) is the loss of three or more consecutive pregnancies prior to 20 weeks of gestation. Female factors in RPL are well known, but the influence of sperm function on RPL has been poorly understood. Recent studies demonstrate that male partners of women with RPL have higher risk of DNA fragmentation, but the underlying reasons have not been previously studied. Determining the mechanisms of sperm damage has potential to open new therapeutic approaches for couples. Objective: To investigate the role of seminal reactive oxygen species (ROS) as a novel marker of sperm function in male partners of women with RPL. Methods: Serum levels of reproductive hormones and sperm quality of male partners with RPL (N=49) were compared with a group of unaffected controls (n=34). All participants completed a questionnaire for conditions impairing sperm quality such as testicular surgery, chronic use of medication, sexually transmitted infections, smoking and alcohol intake. Semen ROS was measured using a validated in-house chemiluminescent luminol assay. DNA fragmentation was measured using the validated Halosperm G2 kit. Results: Sperm morphology was significantly lower in the RPL group when compared with controls (% sperm with normal morphology: 4.5 ± 0.4, control; 3.4± 0.3, RPL, P<0.001). Mean seminal ROS levels were more than 4-fold higher in the RPL group when compared with controls (mean semen ROS in RLU/sec/106: 2.3 ± 0.9, control; 9.3 ± 4.2, RPL, P<0.05). Mean levels of sperm DNA fragmentation index (DFI) were more than 2-fold higher in the RPL group when compared with controls (mean DFI: 7.4 +1.2, control; 16.3+1.5, RPL, P<0.0001). Levels of serum morning testosterone (T) were non- significantly lower in the RPL group when compared with the control group (mean T in nmol/L: 17.9 ± 1.4, control; 15.8 ± 0.8, RPL, P>0.05). Conclusions: Our data suggest that male partners of women with RPL had elevated levels of semen ROS, elevated sperm DNA fragmentation and lower percentage of normal morphology when compared with controls. Our data have important implications for the management of couples with RPL as male partners of women with RPL may benefit from routine assessment of reproductive endocrine and sperm function.

The Effect of Sperm DNA Fragmentation Index on the Outcome of Intrauterine Insemination and Intracytoplasmic Sperm Injection.

Context:Elevated sperm DNA fragmentation index (DFI) is found to affect normal embryonic development, implantation and fetal development after intrauterine insemination (IUI), in vitro fertilization and intracytoplasmic sperm injection (ICSI). Estimation of DFI by terminal deoxynucleotidyl transferase-mediated fluorescent deoxy uridine nucleotide nick end labeling (TUNEL) assay was found to have a high predictive value for pregnancy after fertility treatments.Aim:This study aims to find the effect of increased sperm DNA fragmentation by TUNEL assay on reproductive outcome after IUI and ICSI.Primary Objective:To find the association of DFI and pregnancy rate in IUI and ICSI.Secondary Objectives:To find the association of DFI with fertilization and implantation in ICSI. To find the association of DFI with miscarriage rate.Settings and Design:A prospective observational study performed at a tertiary care university teaching hospital.Subjects and Methods:105 male partners of infertile couple planned for IUI and ICSI underwent estimation of sperm-DFI by TUNEL assay. The treatment outcomes were compared between the DFI-positive (≥20%) and DFI-negative (<20%) groups.Statistical Analysis Used:Statistical analysis was performed by SPSS version 17, Software. P < 0.05 was considered statistically significant.Results:The men with abnormal semen analysis were significantly higher in the DFI-positive group (77.15% vs. 22.85%). There was no significant difference in the pregnancy rate in IUI cycles (17.6% vs. 11.8%); but in the ICSI, the pregnancy rate was significantly reduced in the DFI-positive group (16.7% vs. 47.4%).Conclusions:Elevated DFI significantly affects the pregnancy rate in ICSI cycles.

Should a Couple with Failed In Vitro Fertilization or Intracytoplasmic Sperm Injection and Elevated Sperm DNA Fragmentation Use Testicular Sperm for the Next Cycle?

There is growing evidence indicating that intracytoplasmic sperm injection with testicular sperm rather than ejaculated sperm might be advantageous in achieving pregnancy for couples in which the male has high levels of sperm DNA fragmentation (SDF). Meta-analysis has shown that SDF rates are markedly lower in testicular sperm than in ejaculated sperm, and the odds of achieving pregnancy and a live birth are significantly higher.

Evaluation of sperm chromatin structure and DNA strand breaks is an important part of clinical male fertility assessment.

As reviewed by Agarwal et al. (1) and recently stated by Vu Bach and Schlegel (2), “numerous studies have attempted to assess the association between elevated sperm DNA fragmentation (SDF) and ART outcomes. Unfortunately, variations between SDF assays, protocols, and thresholds and differences in study populations have resulted in systematic reviews and meta-analyses fraught with heterogeneity and unable to come to robust conclusions”. However, these early meta-analyses were considered a near impossible task leading to vague and questionable conclusions. Recent studies have refined protocols for the Sperm Chromatin Structure Assay (SCSA) (3,4) TUNEL (5), SCD (6) and COMET (7) tests. A major consideration for standardizing a protocol is to identify the SDF thresholds. While this has often been described as a single% SDF, e.g., 30% listed as the SCSA threshold in Agarwal et al. (1), more recent SCSA studies have listed two or three thresholds depending on the method of fertilization (see below).

Sperm DNA fragmentation: the evolution of guidelines for patient testing and management.

Sperm DNA fragmentation (SDF) represents a perplexing biological phenomenon that can, at an elevated level, interfere with either initiating and/or maintaining pregnancy. Although we are in our third decade of studying this problem, few investigators have attempted to assess the value of the various available assays for SDF with the specific aim of amalgamating them into practice guidelines for managing SDF. Agarwal, in collaboration with urological experts from North and South America, has done just that, employing a number of clinical scenarios involving SDF to produce some evidence-based, practical guidelines for patient management (1). But if in our third decade of investigation, why are management guidelines so difficult to come by for patients with elevated SDF? First, it should be emphasized that SDF is an exceedingly complex process involving not one, but a number of causative mechanisms that can generate a variety of insults to the integrity of sperm DNA. Sakkas and Alvarez (2) have described six main mechanisms that can damage both sperm nuclear as well as mitochondrial DNA. Included in this list are testicular apoptotic processes during spermatogenesis, aberrations during the events of chromatin re-modeling, induction by exogenous caspases and nucleases plus damage by chemo- and radiotherapy as well as environmental toxicants. Perhaps the most robust events occur in the epididymis in response to insult by reactive oxygen species. That epididymal and ejaculated sperm have higher amounts of DNA fragmentation than testicular sperm is now well documented (3-5). What is not known are the testicular events that render sperm labile to chemical radicals traversing the epididymis. Secondly, the insults can produce single-stranded breaks, double-stranded breaks and/or nucleotide damage. Not all are detected by the same tests, the different types of damage are repaired to different degrees by processes resident in the oocyte and thus, do not have the same prognosis. Likely though, it is the tests used to detect and measure SDF, along with their varying levels of complexity, that introduce the bulk of the conflicting data and conclusions common in the SDF literature.

Clinical implication of DNA fragmentation in male infertility.

Recently, sperm DNA fragmentation (SDF) has been recognized as an important marker of sperm quality that can be used as a predictive factor for fertility in men. Although some controversies exist regarding the role of SDF in infertility, the fact remains that semen from infertile men possess a higher level of SDF than fertile controls (1,2). Elevated SDF may not only contribute to higher rates of failed fertilizations and spontaneous pregnancy loss, but it can also affect assisted reproductive techniques (ART) outcomes in terms of oocyte fertilization, embryo quality, clinical pregnancy and live birth rate. However, controversy still exists regarding the association between SDF and the outcomes of intracytoplasmic sperm injection (ICSI) (3,4).

The presence of human papillomavirus in semen does not affect the integrity of sperm DNA.

It remains unknown whether human papillomaviruses (HPVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen samples of 22 normozoospermic patients undergoing infertility treatment, nine fertile donors and seven fertile men with a risk of HPV infection (genital warts or condylomas) were included in the study. The samples were examined by an INNO-LiPA test PCR-based reverse hybridisation array that identifies 28 types of HPVs as simple or multiple infections. Sperm DNA integrity was determined by sperm chromatin dispersion assay (SCD). Our preliminary findings demonstrate an increase in HPV infection in infertile men with respect to fertile men. However, the sperm DNA fragmentation index was not increased in semen containing these viruses.

Relationship of sperm DNA fragmentation (TUNEL) to blastulation and euploidy after IVF

To determine if elevated sperm DNA fragmentation is related to embryo euploidy and blastulation rates. Retrospective cohort study. 86 IVF cycles in couples where the male partner had a TUNEL assay performed on unprocessed semen samples from 1/2011- 12/2015 were analyzed for percent blastulation and results of comprehensive chromosomal screening (CCS) by trophectoderm biopsy. Only cycles, in which the partner underwent a prior semen evaluation for DNA fragmentation with the Terminal deoxynucleotidyl transferase-mediated d-UTP Nick End Labeling (TUNEL) assay were included for analysis. Donor egg cycles were excluded. Results were analyzed according to whether TUNEL results were normal or abnormal (≥7%). Data was further stratified according to female age categories. Fisher exact t test and Chi-square were used to analyze data and p<0.05 was considered significant. Mean age of patients in the study cohort was 36.8 ± 5.1 for the female and 43.2 ± 10.2 for the male partner. Ninety-one percent of the embryos were fertilized with ejaculated sperm and the remaining with surgically retrieved sperm. Of 678 embryos evaluated, 305 reached the blastocyst stage and 93% of blastocysts underwent PGS. The mean TUNEL result in the study cohort was 8.9 ± 7.4%. Overall, the rate of euploidy in the PGS group according to age was 64% (age ≤ 37), 32.8% (age 38-40), 16.6% (age 41), and 6.8% (age ≥ 42). There was no correlation between the rate of aneuploidy and abnormal TUNEL result except for women of age ≤ 37 years, p=0.003. Overall blastulation rates were 64.7% (age ≤ 37), 52.9% (age 38-40), 48.4% (age 41), 30.6% (age ≥ 42), which did not correlate with abnormal TUNEL for any age group. Elevated sperm DNA fragmentation may correlate with decreased euploidy for younger women ≤ 37 undergoing IVF, but does not likely correlate with blastulation rates. The relationship between sperm DNA fragmentation and early IVF outcomes of embryo development and ploidy should be further evaluated in studies powered to detect a difference in outcomes. Sperm DNA fragmentation may affect pregnancy and ongoing pregnancy rates through mechanisms beyond early embryo development.Tabled 1Rates of aneuploidy and blastulation as compared with TUNEL assayAgeTunelEuploidAneuploidp-valueBlastocystNo Blastocystp-value≤ 37n=135n=76*0.003n=242n=1320.28% embryos abnormal TUNEL55.2%44.8%62.7%37.3%% embryos normal TUNEL74.7%25.3%68.1%31.9%38-40n=21n=430.14n=63n=560.51% embryos abnormal TUNEL47.1%52.9%58.1%41.9%% embryos normal TUNEL27.7%72.3%45.9%43.9%≥ 41n=8n=660.26n=68n=1170.14% embryos abnormal TUNEL16.7%83.3%54.2%45.8%% embryos normal TUNEL8.5%92.0%30.7%14.6%Total164185373305*statistical significance. Open table in a new tab

V3-11 SURGICAL MANAGEMENT OF EJACULATORY DUCT OBSTRUCTION DUE TO PROSTATIC UTRICULAR CYST

You have accessJournal of UrologyMale Voiding/Sexual Dysfunction/BPH/ Incontinence/Infection/Infertility1 Apr 2016V3-11 SURGICAL MANAGEMENT OF EJACULATORY DUCT OBSTRUCTION DUE TO PROSTATIC UTRICULAR CYST Phil Bach, Filipe Tenorio Lira Neto, Ryan Chuang, Bobby Najari, Richard Lee, Philip Li, and Marc Goldstein Phil BachPhil Bach More articles by this author , Filipe Tenorio Lira NetoFilipe Tenorio Lira Neto More articles by this author , Ryan ChuangRyan Chuang More articles by this author , Bobby NajariBobby Najari More articles by this author , Richard LeeRichard Lee More articles by this author , Philip LiPhilip Li More articles by this author , and Marc GoldsteinMarc Goldstein More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1531AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Azoospermia is present in 10-15% of infertile men and can be divided into two broad categories: obstructive azoospermia (OA) and non-obstructive azoospermia. OA accounts for approximately 40% of azoospermia cases and can be caused by a blockage anywhere along the male reproductive tract. 5% of OA cases are secondary to ejaculatory duct obstruction, which may be caused by a variety of congenital or acquired etiologies. The mainstay of management for ejaculatory duct obstruction is transurethral resection of the ejaculatory ducts (TURED). We present the case and surgical management of a man with primary infertility secondary to ejaculatory duct obstruction caused by a prostatic utricular cyst. METHODS A 38 year old man with primary infertility presented with low semen volume, severe oligospermia and highly elevated sperm DNA fragmentation. He had an unremarkable physical exam, normal hormone levels, and a midline utricular cyst on both transrectal ultrasound and MRI, suggestive of ejaculatory duct obstruction. In the operating room, we successfully retrieved abundant highly motile sperm from both testes and vasa deferens. Next, to assess patency, we injected indigo carmine into the right vasotomy site and noted the dye coming out of the left vasotomy site, suggesting that both vasa deferens emptied into a common, obstructed cavity. A vasogram with water soluble contrast subsequently revealed bilaterally patent vasa deferens emptying into an obstructing midline utricular cyst. A TURED was carefully performed using a 24-Fr resectoscope while indigo carmine was instilled into both vasa deferens. Once inside the cyst, both ejaculatory ducts were visualized and a number of small stones were flushed out. A Foley catheter was placed for 24 hours and the patient instructed to ejaculate frequently to maintain patency of the freshly resected cyst. RESULTS Following the resection, indigo carmine could be seen flowing freely from a widely patent outflow tract. The patient tolerated the procedure well and was discharged home without complications. The sperm retrieved from the testes and vasa deferens had significantly lower DNA fragmentation than the ejaculated sperm and is currently being used for in vitro fertilization. Results from a follow-up semen analysis are pending. CONCLUSIONS While uncommon, ejaculatory duct obstruction due to midline utricular cyst is highly amenable to surgical management with TURED. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e470 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Phil Bach More articles by this author Filipe Tenorio Lira Neto More articles by this author Ryan Chuang More articles by this author Bobby Najari More articles by this author Richard Lee More articles by this author Philip Li More articles by this author Marc Goldstein More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using bench top flow cytometer for evaluation of sperm DNA fragmentation in fertility laboratories: protocol, reference values, and quality control

The purpose of this study is to provide a detailed protocol and quality control steps for measuring sperm DNA fragmentation (SDF) by terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay using a new bench top flow cytometer, determine the reference value of SDF, and assess sensitivity, specificity, and distribution of SDF in infertile men and controls with proven and unproven fertility. Semen specimens from 95 controls and 261 infertile men referred to a male infertility testing laboratory were tested for SDF by TUNEL assay using Apo-Direct kit and a bench top flow cytometer. Percentage of cells positive for TUNEL was calculated. Inter- and intraobserver variability was examined. TUNEL cutoff value, sensitivity, specificity, and distribution of different cutoff values in controls and infertile patients were calculated. The reference value of SDF by TUNEL assay was 16.8% with a specificity of 91.6% and sensitivity of 32.6%. The positive and negative predictive values were 91.4 and 33.1%, respectively. The upper limit of DNA damage in infertile men was significantly higher (68.9%) than that in the controls (19.6%). TUNEL assay using flow cytometry is a reproducible and easy method to determine SDF. At a cutoff point of 16.8%, the test showed high specificity and positive predictive value. The results of this test could identify infertile men whose sperm DNA fragmentation does not contribute to their infertility and confirm that a man who tests positive is likely to be infertile due to elevated sperm DNA fragmentation.

Detecting and Minimizing Sperm DNA Damage

In recent years, with the advancement in sperm cell biology and the development of additional testing techniques, sperm DNA fragmentation has been recognized as one of the important causes of reduced fertility potential. Elevated sperm DNA fragmentation rates also significantly diminish the chance of success in assisted pregnancies. Sperm DNA damage can impair fertilization, disrupt embryonic development, and increase rates of miscarriage and poor conception rates. Newer studies suggest the possibility of an increased risk of childhood cancer when an embryo develops from DNA-damaged sperm. There is limited data from large, randomized, controlled trials to support improvement in male fertility with current interventions such as antioxidant therapy, varicocelectomy, and antibiotics treatment in genital tract infections. Nonetheless, research efforts have shown improvements in semen parameters and these interventions are low risk. Therefore, when the external risk factors are known, every effort should be made to minimize sperm DNA damage.

Increased numbers of DNA-damaged spermatozoa in samples presenting an elevated rate of numerical chromosome abnormalities

Is there a relationship between DNA damage and numerical chromosome abnormalities in the sperm of infertile patients? A strong link between DNA fragmentation and the presence of numerical chromosome abnormalities was detected in human sperm. Chromosomally abnormal spermatozoa were more likely to be affected by DNA fragmentation than those that were chromosomally normal. Several studies have described the presence of elevated levels of DNA damage or chromosome defects in the sperm of infertile or subfertile men. However, the nature of the relationship between sperm DNA damage and chromosome abnormalities is poorly understood. The fact that some assisted reproductive techniques have the potential to allow abnormal spermatozoa to achieve oocyte fertilization has led to concerns that pregnancies achieved using such methods may be at elevated risk of genetic anomalies. For this prospective study, semen samples were collected from 45 infertile men. Samples were assessed for DNA fragmentation using the Sperm Chromatin Dispersion Test (SCDt) and for chromosome abnormalities using multi-colour fluorescence in situ hybridization (FISH) with probes specific to chromosomes 13, 16, 18, 21, 22, X and Y. Additionally, both parameters were assessed simultaneously in 10 of the samples using a protocol combining SCDt and FISH. A significant correlation between the proportion of sperm with a numerical chromosome abnormality and the level of DNA fragmentation was observed (P < 0.05). Data from individual spermatozoa subjected to combined chromosome and DNA fragmentation analysis indicated that chromosomally abnormal sperm cells were more likely to display DNA damage than those that were normal for the chromosomes tested (P < 0.05). Not only was this association detected in samples with elevated levels of numerical chromosome abnormalities, but it was also evident in samples with chromosome abnormality rates in the normal range. The inability to assess the entire chromosome complement is the main limitation of all studies aimed at assessing numerical chromosome abnormalities in sperm samples. As a result, some of the sperm classified as 'chromosomally normal' may be aneuploid for chromosomes that were not tested. During spermatogenesis, apoptosis (a process that involves active DNA degradation) acts to eliminate abnormal sperm. Failure to complete apoptosis may explain the coincident detection of aneuploidy and DNA fragmentation in some spermatozoa. In addition to shedding light on the biological mechanisms involved in the processing of defective sperm, this finding may also be of clinical relevance for the identification of patients at increased risk of miscarriage or chromosomally abnormal pregnancy. In some instances, detection of elevated sperm DNA fragmentation may indicate the presence of chromosomal abnormalities. It may be worth considering preimplantation genetic screening (PGS) of embryos produced using such samples in order to minimize the risk of aneuploidy.

No increased sperm DNA fragmentation index in semen containing human papillomavirus or herpesvirus

It remains unknown whether human papillomaviruses (HPVs) or human herpesviruses (HHVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen from 76 sperm donors was examined by a PCR-based hybridization array that identifies all HHVs and 35 of the most common HPVs. Sperm DNA integrity was determined by the sperm chromatin structure assay. HPVs or HHVs, or both, were found in 57% of semen samples; however, sperm DNA fragmentation index was not increased in semen containing these viruses.

Finasteride-induced secondary infertility associated with sperm DNA damage

To report a case of low-dose finasteride-induced secondary infertility with associated elevated sperm DNA fragmentation index (DFI) and otherwise normal semen parameters. Case report. University hospital. A 48-year-old man on low-dose finasteride and his 37-year-old wife with normal menses and normal gynecologic exam. Determination of sperm DFI and discontinuation of low-dose finasteride. Sperm DFI. The sperm DFI done a year earlier was 30%. This value was unchanged when repeated 2 months later. The patient was advised to stop finasteride. Three months after discontinuing the finasteride, the DFI decreased to 21% and subsequent DFI after another 3 months improved to 16.5%. To date, there is still no documented full-term pregnancy or live birth. The significant reduction in DFI within 3 months of finasteride cessation and continued improvement suggests a causal link between finasteride and sperm DNA damage. We hypothesize that low-dose finasteride may exert a negative influence on sperm DNA integrity, resulting in increased pregnancy losses. We suggest that in infertile men using finasteride, sperm DFI should be measured in addition to semen parameters, and a trial of discontinuation of finasteride may be warranted.

Data analysis of two in vivo fertility studies using Sperm Chromatin Structure Assay–derived DNA fragmentation index vs. pregnancy outcome

The 2006 American Society for Reproductive Medicine Compendium of Practice Report found no significant effects of elevated sperm DNA fragmentation by using a 30% DNA fragmentation index (DFI) threshold for natural fertilization and SCSA data (odds ratio, 1.07; 95% confidence interval, 0.39-2.93). In contrast, it is shown in this article that these two in vivo studies showed significant odds ratios of 6.54 (95% confidence interval, 1.71, 24.91) and 7.58 (95% confidence interval, 2.54, 22.67), which resulted in the conclusion that the pregnancy (PG) rates are statistically significantly higher for the group with DFI below the thresholds of 30% and 40%, respectively. In addition, all the chi(2) statistics used to test the null hypotheses of no association between the pregnancy status and DFI for natural (normal) fertilization confirmed this conclusion that the probability of pregnancy for the group with <30% or <40% DFI was statistically significantly higher than that for the group with >30% or >40% DFI.

Significant decrease in sperm deoxyribonucleic acid fragmentation after varicocelectomy

To measure sperm DNA integrity values before and after varicocelectomy in patients with elevated preoperative levels of sperm DNA fragmentation. Retrospective. Private urology clinic. Eleven patients with grade 1, 2, or 3 varicocele. Varicocelectomy. Sperm DNA fragmentation values were assessed before and after varicocelectomy. Ninety percent of the patients showed a significant decrease in sperm DNA fragmentation levels. Although this study was small, 10 of the 11 patients with varicocele showed a significant decrease in sperm DNA fragmentation after varicocele repair. Elevated sperm DNA fragmentation has been shown to have a significant negative effect on pregnancy outcome with use of in vivo, IUI, routine IVF, and to a lesser extent intracytoplasmic sperm injection fertilization; therefore pregnancy outcome may improve after varicocelectomy.

P-619: Characterization of four sperm cell populations resolved and flow cytometry sorted by the Sperm Chromatin Structure Assay (SCSA®)

1) characterize by light microscope image analysis and neutral pH Comet analysis the four SCSA derived sperm populations consisting of: Main (no DNA damage detected), Moderate (moderately damaged sperm) and High (highly damaged sperm) DNA Fragmentation Index (% DFI) and High DNA Stainability (HDS is a measure of immature chromatin). 2) Estimate the selection frequency of the moderate DFI populations for ICSI patients in comparison to the more natural selection of sperm by the eggs for the routine IVF patients. Retrospective. ART patients undergoing routine IVF or ICSI 1) SCSA- sort the four sperm populations for: a) computer image analysis of Feulgen stained sperm for morphology and morphometry, and b) neutral pH Comet- defined DNA strand breaks. 2) Statistical frequency selection estimation of the Moderate DFI sperm population by ICSI technicians vs more natural selection of sperm by eggs in routine IVF resulting in pregnancy/miscarriage. The morphology/morphometry features of the Moderate DFI sperm population were equally normal as the Main population sperm but one-half were Comet positive. The High DFI sperm population showed cell morphology/morphometry features with considerable signs of necrosis/apoptosis and were 100% Comet positive. Semen samples derived from the routine IVF pregnancies had significantly lower % moderate DFI values in comparison to the % moderate DFI population pregnancies from the ICSI pregnancies (P<0.001). Sperm with normal morphology/morphometry may have single and double strand DNA breaks. The SCSA detects both single-strand DNA breaks (moderate DFI and neutral Comet negative) and 100% sperm with single and double strand DNA breaks (neutral Comet positive). Since one- half of the Moderate DFI sperm have DNA double strand breaks identified by the neutral pH Comet assay but with normal morphology/morphometry, the Moderate DFI population is likely to be picked out as frequently for ICSI injection as the main population without SCSA-identifiable DNA damage. The high DFI population is likely detected as abnormal and not used for ICSI. To date, six ICSI studies have shown a relationship between elevated sperm DNA fragmentation and increased miscarriage rates. These Moderate DFI sperm may be responsible for decreased pregnancy and increased spontaneous abortion rates, as well as the possibility for increased birth defects seen at birth or latent genetic defects seen in later life.