Smit, M.

Clinical Application of Sperm Chromatin Structure Assessment in Andrology Patients (Doctoral Thesis)

2011-03-30T00:00:00Z

Infertility, defined as the inability to conceive spontaneously within one year, is a common medical problem. Traditionally, fertility investigations initially focus on the evaluation of ovulation and tubal patency in females, and on assessment of sperm quantity and quality in males. In about one third of couples with infertility abnormalities in classic semen parameters are found, like sperm concentration, motility and morphology. In another one third of patients a combination of infertility-related female and male factors are seen (WHO 1999). Although sperm quality parameters derived from classic semen analysis are frequently used to categorise male infertile patients, the prognostic and diagnostic information they provide is limited with a predictive power that is highest at the lower ranges of the spectrum (Barratt et al. 2010).

Sperm chromatin structure is associated with the quality of spermatogenesis in infertile patients (Article)

2010-10-01T00:00:00Z

Objective: To establish the diagnostic value of sperm chromatin structure assessment for the evaluation of male factor infertility, in addition to conventional andrological workup. Design: Cross-sectional controlled study. Setting: A tertiary referral andrology clinic. Patient(s): Two hundred seventy-nine male partners of infertile couples. Intervention(s): None. Main Outcome Measure(s): The DNA fragmentation index (DFI) determined by the sperm chromatin structure assay (SCSA), semen parameters, serum levels of reproductive hormones, and World Health Organization (WHO) classification of male factor subfertility. Result(s): In all patient categories, except those including patients with hypogonadotrophic hypogonadism, sperm antibodies, or normospermia, DFI was significantly higher compared with in proven fertile controls. After classification of the quality of spermatogenesis based on mean testicular volume (<10 ml vs. >15 ml), follicle stimulating hormone (FSH; > 10 U/L vs. <5 U/L), and inhibin-B (<100 nmol/L vs. >150 nmol/L), the DFI was significantly higher in patients with poor spermatogenesis (35.9%) than in patients with normal spermatogenesis (25.9%). In a multiple regression analysis, the teratozoospermia index, sperm vitality, and FSH were significant determinants of the DFI level. Male age was associated with DFI, but leukocytospermia, body mass index, and smoking were not confounders of DFI. Conclusion(s): Impaired spermatogenesis, irrespective of the WHO classification of male factor subfertility, is generally associated with an increase of sperm DNA damage. Copyright

Sperm DNA integrity in cancer patients before and after cytotoxic treatment (Article)

2010-08-01T00:00:00Z

Background: We assessed sperm DNA fragmentation index (DFI) in cancer patients before and after treatment to evaluate if sperm DNA integrity is compromised by cancer itself or its treatment. Methods: In a prospective study, DFI was assessed in 127 patients diagnosed with testicular germ cell tumours (TGCT), Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL) and various malignancies. The severity of cancer and tumour markers at diagnosis was recorded. Follow-up DFI after treatment was available in 52 patients who were mostly less severely affected. Results: In patients diagnosed with TGCT, HL and various malignancies, pretreatment DFI levels were not significantly different from that of proven fertile controls, but in patients with NHL an increased DFI was found. An overall significant decrease in post-treatment DFI (13.2 range 5.0-70.5) compared with pretreatment values (17.1 range 5.1-66.6) was found (P = 0.040). In TGCT patients, post-treatment DFI was significantly higher in patients who were treated with radiotherapy (16.9 range 11.5-39.9) compared with that in patients treated with chemotherapy (CT) alone (10.9 range 5.5-39.9) (P = 0.037). In HL patients, the type of treatment or number of CT cycles was not associated with DFI. Overall, post-treatment DFI in cancer patients was not significantly different from that of proven fertile controls. Conclusion: SIn this study, the presence of cancer does not seem to negatively affect the sperm DNA integrity in TGCT and HL patients; only NHL patients showed increased DFI at the time of diagnosis compared with healthy controls. Our Results: confirm previous reports that DFI decreases significantly following various anti-cancer treatments. In contrast, radiotherapy in TGCT patients is associated with an increase in DFI compared with CT treatment alone.

Reply of the Authors: Acridine orange binding to RNA interferes DNA fragmentation index calculation in sperm chromatin structure assay (Article)

2010-06-01T00:00:00Z

Increased Sperm DNA Fragmentation in Patients With Vasectomy Reversal Has No Prognostic Value for Pregnancy Rate (Article)

2010-02-01T00:00:00Z

Purpose: We evaluated sperm DNA fragmentation in patients with vasectomy reversal and its prognostic value to determine spontaneous and assisted reproductive technique pregnancy rates. Materials and Methods: We prospectively assessed DNA fragmentation with the sperm chromatin structure assay in postoperative semen samples of 70 patients with vasectomy reversal. At a median ± SD followup of 4.3 ± 0.5 years pregnancy rates were recorded. Results: DNA fragmentation in patients with vasectomy reversal was significantly increased vs that in proven fertile controls (30.2% ± 20.1% vs 15.3% ± 5.4%, p <0.001). Significant negative correlations were found between DNA fragmentation index and total sperm count, progressive motility, total number of progressive sperm, normal morphology and sperm vitality (-0.325

Decreased Sperm DNA Fragmentation After Surgical Varicocelectomy is Associated With Increased Pregnancy Rate (Article)

2010-01-01T00:00:00Z

Purpose: We prospectively evaluated changes in sperm chromatin structure in infertile patients before and after surgical repair of varicocele, and the impact on the pregnancy rate. Materials and Methods: Included in the study were 49 men with at least a 1-year history of infertility, a palpable varicocele and oligospermia. World Health Organization semen analysis and sperm DNA damage expressed as the DNA fragmentation index using the sperm chromatin structure assay were assessed preoperatively and postoperatively. Pregnancy (spontaneous and after assisted reproductive technique) was recorded 2 years after surgery. Results: Mean sperm count, sperm concentration and sperm progressive motility improved significantly after varicocelectomy from 18.3 × 106to 44.4 × 106, 4.8 × 106/ml to 14.3 × 106/ml and 16.7% to 26.6%, respectively (p <0.001). The DNA fragmentation index decreased significantly after surgery from 35.2% to 30.2% (p = 0.019). When the definition of greater than 50% improvement in sperm concentration after varicocelectomy was applied, 31 of 49 patients (63%) responded to varicocelectomy. After varicocelectomy 37% of the couples conceived spontaneously and 24% achieved pregnancy with assisted reproductive technique. The mean postoperative DNA fragmentation index was significantly higher in couples who did not conceive spontaneously or with assisted reproductive technique (p = 0.033). Conclusions: After varicocelectomy sperm parameters significantly improved and sperm DNA fragmentation was significantly decreased. Low DNA fragmentation index values are associated with a higher pregnancy rate (spontaneous and with assisted reproductive technique). We suggest that varicocelectomy should be considered in infertile men with palpable varicocele, abnormal semen analysis and no major female factors.

Low folate in seminal plasma is associated with increased sperm DNA damage (Article)

2008-01-01T00:00:00Z

Objective: To determine associations between vitamin B status, homocysteine (tHcy), semen parameters, and sperm DNA damage. Design: Observational study. Setting: A tertiary referral fertility clinic. Patient(s): Two hundred fifty-one men of couples undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatment, with subgroups of fertile (n = 70) and subfertile men (n = 63) defined according to semen concentration and proven fertility. Intervention(s): None. Main Outcome Measure(s): The DNA fragmentation index (DFI) as marker of sperm DNA damage determined using the sperm chromatin structure assay (SCSA), and semen parameters assessed according to World Health Organization criteria; tHcy, folate, cobalamin, and pyridoxine concentrations determined in seminal plasma and blood. Result(s): In the total group of fertile and subfertile men, all biomarkers in blood were statistically significantly correlated with those in seminal plasma. No correlation was found between the biomarkers in blood and the semen parameters. In seminal plasma, both tHcy and cobalamin positively correlated with sperm count. Folate, cobalamin, and pyridoxine were inversely correlated with ejaculate volume. In fertile men, seminal plasma folate showed an inverse correlation with the DNA fragmentation index. Conclusion(s): Low concentrations of folate in seminal plasma may be detrimental for sperm DNA stability.

Inhibin B is superior to FSH as a serum marker for spermatogenesis in men treated for Hodgkin's lymphoma with chemotherapy during childhood (Article)

2007-12-01T00:00:00Z

BACKGROUND: The aim of this study was to evaluate the long-term gonadal sequelae after treatment for childhood Hodgkin's lymphoma with combination chemotherapy, using up to date fertility parameters and andrological evaluation, including for the first time inhibin B. METHODS: There were 56 male patients treated from 1974-1998 for childhood Hodgkin's lymphoma with combination chemotherapy ABVD or EBVD (adriamycin/epirubicin, bleomycin, vinblastine, dacarbazine) with or without MOPP (mechlorethamine, vincristin, prednisone, procarbazine) with the intention to avoid radiotherapy. These men were studied 15.5 years (range 5.6-30.2 years) after cessation of therapy. Serum follicle stimulating hormone (FSH), luteinizing hormone (LH), inhibin B, testosterone, sex hormone-binding globulin (SHBG), sperm concentration and sperm DNA integrity were determined. RESULTS: In men treated with MOPP, median FSH and LH were significantly increased (P < 0.001) and inhibin B (17.5 versus 143 ng/l; P < 0.001) and sperm concentration (1.05 versus 49.5 × 106/ml; P < 0.05) were significantly decreased compared with patients treated without MOPP. The number of MOPP courses was significantly correlated with FSH and inhibin B levels. Only inhibin B showed an independent correlation with sperm concentration (r = 0.86; P < 0.001). CONCLUSIONS: The use of MOPP chemotherapy causes permanent gonadal damage in the far majority of male survivors of childhood Hodgkin's lymphoma and inhibin B is the most valuable serum marker for gonadal function.

Seminal plasma cobalamin significantly correlates with sperm concentration in men undergoing IVF or ICSI procedures (Article)

2007-07-01T00:00:00Z

Mild hyperhomocysteinemia is caused by B vitamin deficiencies. We hypothesize that these biochemical derangements detrimentally affect spermatogenesis. Therefore, the aim of this study was to investigate the folate, cobalamin, pyridoxine, and homocysteine concentrations in blood and seminal plasma and the associations between these biomarkers and semen parameters in men participating in an in vitro fertilization or intracytoplasmic sperm injection program. From 73 men (median age [range]: 37 years [28-53]), blood and semen samples were obtained for the determination of serum and red blood cell (RBC) folate, serum total cobalamin, whole-blood pyridoxal-5′-phosphate, plasma total homocysteine (tHcy), and serum total testosterone. Semen analysis included sperm concentration, motility, and morphology according to World Health Organization criteria. The B vitamins and tHcy concentrations were significantly correlated in blood but not in seminal plasma. The serum and RBC folate concentrations were significantly correlated also with the total folate concentration in seminal plasma (r = .44; P < .001 and r = .39; P < .001, respectively). Likewise, the total cobalamin concentration in serum and seminal plasma was significantly correlated (r = .55; P = .001). Of interest is that the total cobalamin concentration in seminal plasma was significantly correlated with the sperm concentration (r= .42; P< .001). This is in contrast to the absence of significant associations between the other vitamins and tHcy in blood and seminal plasma and any of the semen parameters. These findings suggest that folate and cobalamin are transferred from the blood to the male reproductive organs and emphasize the role of cobalamin in spermatogenesis in human. Copyright

Can inhibin-B predict the outcome of microsurgical epididymal sperm aspiration in patients with suspected primary obstructive azoospermia? (Article)

2007-05-01T00:00:00Z

Aim: To evaluate whether inhibin-B can predict the outcome of a microsurgical epidymal sperm aspiration (MESA) procedure in patients with suspected primary obstructive azoospermia (OA) and if inhibin-B can replace testicular biopsy in the diagnostic work-up of these patients. Methods: Inhibin-B levels and testicular biopsy scores were related to the outcome of MESA in 43 patients with suspected primary OA. MESA was considered to be successful when epididymal sperm could be identified during the procedure. Results: Spermatozoa were present in the epididymal aspirate in 28 out of the 43 patients (65%). Inhibin-B values were not significantly different in patients with successful or unsuccessful MESA. The modified Johnsen score, however, was significantly lower in patients with unsuccessful MESA (P=0.003). Arete testis obstruction or epididymal malfunctioning was found in 15% of patients with suspected primary OA, reflected by unsuccessful MESA despite normal inhibin-B levels and normal testicular histology. Conclusion: Inhibin-B cannot replace testicular biopsy as a diagnostic tool in the work-up of patients with suspected primary OA. Testicular biopsy is useful in identifying patients with spermatogenic arrest, who might have normal inhibin-B values.

Clinical correlates of the biological variation of sperm DNA fragmentation in infertile men attending an andrology outpatient clinic (Article)

2007-02-01T00:00:00Z

Determination of sperm DNA fragmentation, as assessed by the sperm chromatin structure assay (SCSA), has become an important tool for the evaluation of semen quality. The aim of the present study was to describe the biological variation of sperm DNA fragmentation in men attending an andrology clinic and to identify clinical correlates of the biological variation of sperm DNA fragmentation. For this study, two consecutive semen samples from 100 patients attending our andrology outpatient clinic were subjected to semen analysis, performed in parallel according to WHO guidelines and by SCSA. A good agreement between pairs of samples was found for SCSA-derived variables, as indicated by a significantly lower median coefficient of variation (CV) of the DNA Fragmentation Index (DFI) and the high DNA stainability (HDS) compared with WHO semen parameters. In half of the men attending our andrology clinic, however, the individual biological variation of DFI and HDS, expressed as CV of two samples, exceeded 10%. Dysregulation of spermatogenesis, as seen as testicular insufficiency or varicocele, was not associated with increased variability of DFI or HDS. A backward multiple linear regression analysis, however, indicated that the biological variation of DFI may be more profound in men with characteristics of normal spermatogenesis. In conclusion, we confirm previous reports that sperm DNA fragmentation has a lower biological variability than classical semen parameters. We hypothesize that the sperm chromatin structure may be more influenced in patients with normal spermatogenesis, whereas in men with disturbed spermatogenesis, the chromatin structure may be already so impaired that the effect of unidentified factors leading to variability of sperm DNA fragmentation in time may not be as profound.

Automated counting of white blood cells in synovial fluid. (Article)

2004-02-01T00:00:00Z

OBJECTIVES: To evaluate the performance of automated leucocyte (white blood cell; WBC) counting by comparison with manual counting. METHODS: The number of WBC was determined in heparinized synovial fluid samples by the use of (i) a standard urine cytometer (Kova) and a microscope (reference method) and (ii) a haematology analyser (Sysmex XE-2100; WBC/BASO and DIFF channels). Imprecision within and between days was determined by replicate analysis of a low (level A; WBC approximately 0.560 x 10(9)/l) and a high (level B; WBC approximately 1.081 x 10(9)/l) dedicated synovial fluid control (Quantimetrix). RESULTS: The WBC count of the DIFF channel was highly correlated with the WBC count of the microscopic reference method (r = 0.99; WBC analyser = 0.870 x WBC reference method + 0.413). In contrast, no agreement existed between WBC counts generated by the WBC/BASO channel of the analyser and the reference method (r = 0.52; WBC analyser = 0.008 x WBC reference method + 0.079). Within-day imprecision (4-7%) and between-day imprecision (10%) of the haematology analyser were smaller than the within-day imprecision (12%) and the between-day imprecision (20-22%) of the manual reference method. For manual counting, inter-observer coefficients of variation were 35.9% (control level A) and 21.0% (control level B). CONCLUSIONS: The WBC count in synovial fluid can be reliably determined using the DIFF channel of the Sysmex XE-2100. Automated counting of WBC in synovial fluid offers more precise and faster results than manual counting.