R. Mahfouz

SEMEN CHARACTERISTICS AND SPERM DNA FRAGMENTATION IN INFERTILE MEN WITH LOW AND HIGH LEVELS OF SEMINAL REACTIVE OXYGEN SPECIES

OBJECTIVE To examine sperm motility, total antioxidant level (TAC), DNA fragmentation, and medical history in infertile men with high seminal high reactive oxygen species (ROS). DESIGN Prospective study. SETTING(S) Tertiary care hospital. PATIENT(S) Infertile men (n=101). INTERVENTION(S) Group I (n=57) included men with seminal ROS (<250 relative light units/sec/×10(6) sperm) while group II (n=44) included men with seminal ROS levels (≥250 relative light units/sec/×10(6) sperm). MAIN OUTCOME MEASURE(S) Seminal ROS, TAC, sperm DNA fragmentation, ROS/TAC score were measured. RESULT(S) Group II had a high incidence of sperm DNA fragmentation than group I. The odds ratio of 1.25 for elevated ROS levels corresponded to >10% greater DNA fragmentation in our patients (95% confidence interval 1.01-1.53). Group II showed poor motility, a higher incidence of leukocytospermia, and higher ROS-TAC scores compared with group I. ROS was negatively correlated with sperm curvilinear velocity (r=-.24), linearity (r=-.24), and sperm motility (r=-.31). Sperm motility was correlated with %TUNEL(+ve) sperm (r=-.39). CONCLUSION(S) An increase in seminal ROS levels by 25% was associated with a 10% increase in sperm DNA fragmentation. Sperm motility was affected by seminal ROS and sperm DNA fragmentation.

TUNEL as a Test for Sperm DNA Damage in the Evaluation of Male Infertility

OBJECTIVES To standardize the TUNEL assay by establishing inter- and intraobserver variability, interassay variability, cutoff values, sensitivity and specificity of the assay, and studying the distribution of the DNA damage in a population of infertile men referred to a clinical andrology laboratory. METHODS Seminal ejaculates from 25 healthy male volunteers (controls) and 194 infertile men (with male factor infertility) referred to an andrology laboratory were examined for DNA damage by TUNEL assay using flow cytometric analysis. RESULTS Both the inter- and intraobserver variability and interassay variability was small (<10%). DNA damage in the controls was 11.9 ± 6.8% vs. 29.5 ± 18.7% in patients (P <.001). The cut-off value of 19.25% maximized the observed sensitivity (64.9%) and specificity (100%) of the assay. The distribution of DNA damage in the patients was as follows: 14.9% (29 of 194) with DNA damage between 0% and 10%; 22.7% (44 of 194) between 10% and 20%; 8.8% (17 of 194) between 20% and 30%; and 17.5% (34 of 194) between 30% and 40%. Finally, 27.3% (53 of 194) had TUNEL values >40%. CONCLUSIONS We report a detailed standardization of the TUNEL assay for clinical use, as well as reference ranges for DNA damage in normal healthy donors and infertile men. A cut-off of 19.25% with observed 100% specificity established in our program can differentiate infertile men with DNA damage from healthy men. This test can be offered to infertile patients who are idiopathic, have severe oxidative stress-related abnormal semen quality, and contribute to the infertility problem of the couple who are considering assisted reproductive techniques.

Diagnostic value of the total antioxidant capacity (TAC) in human seminal plasma

OBJECTIVE To establish cutoff value, sensitivity, specificity and intra- and interobserver variability of total antioxidant capacity (TAC) in seminal plasma from healthy donors (controls) and infertile patients. DESIGN Seminal plasma from proven fertile donors (n = 55), nonproven fertile donors (n = 45), and infertile patients (n = 42) were examined for TAC level. SETTING Reproductive research center in a tertiary care hospital. PATIENT(S) Infertile patients from male infertility clinic of various diagnoses. INTERVENTION(S) Seminal plasma TAC measurement by a colorimetric assay using the TAC assay kit, receiver operating characteristics curve. MAIN OUTCOME MEASURE(S) Seminal plasma TAC levels, cutoff value, sensitivity, and specificity. RESULT(S) Proven fertile donors showed higher TAC values (median and range): 1700 (1440-2290 microM); compared with the infertile patients: 1310 (1040-1600 microM). The best cutoff to distinguish between fertile controls and infertile men was 1420 microM. At this threshold, specificity was 64% and sensitivity 76%. CONCLUSION(S) Total antioxidant capacity of the seminal plasma as measured by the colorimetric assay is a reliable and simple test for the diagnosis and management of male infertility.

Evaluation of chemiluminescence and flow cytometry as tools in assessing production of hydrogen peroxide and superoxide anion in human spermatozoa

OBJECTIVE To examine simultaneously the levels of hydrogen peroxide (H(2)O(2)) and superoxide (O(2)(-*)) using chemiluminescence and flow cytometry. DESIGN Prospective laboratory study. SETTING Reproductive research lab in a tertiary hospital. PATIENT(S) Semen samples from 18 healthy male volunteers. INTERVENTION(S) Sperm preparation and measurement of reactive oxygen species (ROS) by chemiluminescence using luminol and lucigenin before and after H(2)O(2) exposure and by flow cytometry using dichlorofluorescin diacetate (DCFH-DA) for H(2)O(2) and dihydroethidium (DHE) for O(2)(-*). MAIN OUTCOME MEASURE(S) Sperm count, motility, viability, and ROS levels. RESULT(S) Immature sperm fractions showed significantly higher levels of ROS measured by either luminol or lucigenin compared with the neat and mature fraction. ROS levels were detectable by flow cytometry in chemiluminescence-negative samples. Both mature and immature sperm fractions had a significantly higher percentage of cells positive for H(2)O(2) compared with neat semen. On the other hand, the percentage of O(2)(-*)-positive cells in neat semen was significantly higher compared with the percentage found in mature fractions but significantly lower than that in the immature sperm fractions. CONCLUSION(S) We recommend ROS measurement by flow cytometry on the basis that it requires a lower sperm count, is comparable to chemiluminescence, and has higher specificity for intracellular ROS in viable spermatozoa. Samples tested negative by chemiluminescence still may have high intracellular H(2)O(2) generation that can be detected by flow cytometry.

Multiple mechanisms deregulate EZH2 and histone H3 lysine 27 epigenetic changes in myeloid malignancies

Polycomb repressive complex 2 (PRC2) is involved in trimethylation of histone H3 lysine 27 (H3K27), chromatin condensation and transcriptional repression. The silencing function of PRC2 complex is mostly attributed to its intrinsic activity for methylating H3K27. Unlike in B-cell lymphomas, enhancer of zeste homolog 2 (EZH2) mutations in myeloid malignancies are inactivating/hypomorphic. When we assessed the mutational status in myeloid malignancies (N=469 cases examined), we found EZH2 and EED/SUZ12 mutations in 8% and 3.3% of cases, respectively. In addition to mutant cases, reduced EZH2 expression was also found in 78% cases with hemizygous deletion (−7/del7q cases involving EZH2 locus) and 41% of cases with diploid chromosome 7, most interestingly cases with spliceosomal mutations (U2AF1/SRSF2 mutations; 63% of cases). EZH2 mutations were characterized by decreased H3K27 trimethylation and increased chromatin relaxation at specific gene loci accompanied by higher transcriptional activity. One of the major downstream target is HOX gene family, involved in the regulation of stem cell self-renewal. HOXA9 was found to be overexpressed in cases with decreased EZH2 expression either by EZH2/spliceosomal mutations or because of −7/del7q. In summary, our results suggest that loss of gene repression through a variety of mutations resulting in reduced H3K27 trimethylation may contribute to leukemogenesis.

L-Carnitine decreases DNA damage and improves the in vitro blastocyst development rate in mouse embryos

OBJECTIVE To optimize the L-carnitine (LC) concentration as a supplement in embryo culture medium and to investigate the effect of LC on developing embryos. DESIGN Experimental study. SETTING Reproductive research center at a tertiary hospital. INTERVENTION(S) To optimize the LC concentration, 420 mouse embryos were divided into seven groups and incubated with different LC concentrations (0, 0.3, 0.6, 1.2, 2.5, 5.0, and 10 mg/mL). To investigate the effect of LC on the developing embryos, 500 mouse embryos were divided into three groups and incubated with either actinomycin-D (AD; 0.005 microg/mL), hydrogen peroxide (H(2)O(2); 500 micromol/L), or tumor necrosis factor alpha (TNF-alpha; 500 ng) with and without LC 0.3 or 0.6 mg/mL. Blastocyst development rate (%BDR) and DNA damage were examined for all groups. MAIN OUTCOME MEASURE(S) Effect of LC on embryogenesis. RESULT(S) Significant improvement in %BDR was seen at LC 0.3 mg/mL compared with the control (p = 0.006). L-Carnitine at 0.3 and 0.6 mg/mL significantly reduced the blocking effect of AD, H(2)O(2), and TNF-alpha and significantly decreased the level of DNA damage. CONCLUSION(S) Embryo culture medium supplementation with LC may offer a novel and a cost-effective technique to improve the embryogenesis of cultured embryos. This may be beneficial in improving IVF outcomes.

Sperm viability, apoptosis, and intracellular reactive oxygen species levels in human spermatozoa before and after induction of oxidative stress

OBJECTIVE To investigate sperm viability, incidence of apoptosis, and intracellular basal and induced reactive oxygen species (ROS) in sperm fractions. DESIGN Prospective controlled study. SETTING Center for Reproductive Medicine at a tertiary care hospital. METHOD(S) Liquefied seminal ejaculates (n = 12) prepared by density gradient centrifugation were reconstituted to 2 mL with phosphate-buffered saline. Oxidative stress was induced by hydrogen peroxide (H(2)O(2), 100 muM). Sperm viability, intracellular ROS, and incidence of apoptosis/necrosis in neat, immature, and mature sperm fractions were assessed. RESULT(S) Before H(2)O(2) exposure, mature spermatozoa fractions showed a significantly lower incidence of apoptotic sperm and intracellular O(2)(-*) levels but higher amounts of intracellular H(2)O(2) compared with neat semen. Higher levels of intracellular H(2)O(2) were demonstrated in immature sperm fractions compared with neat or mature fractions. In all sperm fractions, intracellular H(2)O(2) levels correlated with the intracellular concentration of O(2)(-*). After H(2)O(2) exposure, neat semen showed a significantly higher percentage of apoptosis compared with the prepared mature spermatozoa. However, no differences were observed in the incidence of apoptosis between immature and mature sperm fractions. CONCLUSION(S) There is a differential shift of both intracellular H(2)O(2) and O(2)(-*) in each sperm fraction that may affect sperm quality. Sperm apoptosis is related to intracellular H(2)O(2) levels, which in turn are affected by intracellular O(-*) levels. Oxidative stress was not associated with an increased incidence of apoptosis in immature or mature sperm fractions.

Increased CDA Expression/Activity in Males Contributes to Decreased Cytidine Analog Half-Life and Likely Contributes to Worse Outcomes with 5-Azacytidine or Decitabine Therapy

Purpose: The cytidine analogs 5-azacytidine and decitabine, used to treat myelodysplastic syndromes (MDS), produce a molecular epigenetic effect, depletion of DNA-methyltransferase 1 (DNMT1). This action is S-phase dependent. Hence, genetic factors that decrease the half-lives of these drugs could impact efficacy. Documentation of such impact, and elucidation of underlying mechanisms, could lead to improved clinical application. Experimental design: Cytidine deaminase (CDA) rapidly inactivates 5-azacytidine/decitabine. The effect of CDA SNP A79C and gender on CDA expression, enzyme activity, and drug pharmacokinetics/pharmacodynamics was examined in mice and humans, and the impact on overall survival (OS) was evaluated in 5-azacytidine/decitabine-treated patients with MDS (n = 90) and cytarabine-treated patients with acute myeloid leukemia (AML) (n = 76). Results: By high-performance liquid chromatography (HPLC), plasma CDA activity was decreased as expected in individuals with the SNP A79C. Interestingly and significantly, there was an even larger decrease in females than in males. Explaining this decrease, liver CDA expression was significantly lower in female versus male mice. As expected, decitabine plasma levels, measured by mass spectrometry, were significantly higher in females. In mathematical modeling, the detrimental impact of shorter drug half-life (e.g., in males) was greater in low compared with high S-phase fraction disease (e.g., MDS vs. AML), because in high S-phase fraction disease, even a short exposure treats a major portion of cells. Accordingly, in multivariate analysis, OS was significantly worse in male versus female patients with MDS treated with 5-azacytidine/decitabine. Conclusions: Increased CDA expression/activity in males contributes to decreased cytidine analog half-life and likely contributes to worse outcomes with 5-azacytidine or decitabine therapy. Clin Cancer Res; 19(4); 938–48. ©2012 AACR.

Potential biological role of poly (ADP-ribose) polymerase (PARP) in male gametes

Maintaining the integrity of sperm DNA is vital to reproduction and male fertility. Sperm contain a number of molecules and pathways for the repair of base excision, base mismatches and DNA strand breaks. The presence of Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues has recently been shown in male germ cells, specifically during stage VII of spermatogenesis. High PARP expression has been reported in mature spermatozoa and in proven fertile men. Whenever there are strand breaks in sperm DNA due to oxidative stress, chromatin remodeling or cell death, PARP is activated. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARPs DNA-repairing abilities. Therefore, cleaved PARP (cPARP) may be considered a marker of apoptosis. The presence of higher levels of cPARP in sperm of infertile men adds a new proof for the correlation between apoptosis and male infertility. This review describes the possible biological significance of PARP in mammalian cells with the focus on male reproduction. The review elaborates on the role played by PARP during spermatogenesis, sperm maturation in ejaculated spermatozoa and the potential role of PARP as new marker of sperm damage. PARP could provide new strategies to preserve fertility in cancer patients subjected to genotoxic stresses and may be a key to better male reproductive health.

p53 Independent epigenetic-differentiation treatment in xenotransplant models of acute myeloid leukemia

Suppression of apoptosis by TP53 mutation contributes to resistance of acute myeloid leukemia (AML) to conventional cytotoxic treatment. Using differentiation to induce irreversible cell cycle exit in AML cells could be a p53-independent treatment alternative, however, this possibility requires evaluation. In vitro and in vivo regimens of the deoxycytidine analogue decitabine that deplete the chromatin-modifying enzyme DNA methyl-transferase 1 without phosphorylating p53 or inducing early apoptosis were determined. These decitabine regimens but not equimolar DNA-damaging cytarabine upregulated the key late differentiation factors CCAAT enhancer-binding protein ɛ and p27/cyclin dependent kinase inhibitor 1B (CDKN1B), induced cellular differentiation and terminated AML cell cycle, even in cytarabine-resistant p53- and p16/CDKN2A-null AML cells. Leukemia initiation by xenotransplanted AML cells was abrogated but normal hematopoietic stem cell engraftment was preserved. In vivo, the low toxicity allowed frequent drug administration to increase exposure, an important consideration for S phase specific decitabine therapy. In xenotransplant models of p53-null and relapsed/refractory AML, the non-cytotoxic regimen significantly extended survival compared with conventional cytotoxic cytarabine. Modifying in vivo dose and schedule to emphasize this pathway of decitabine action can bypass a mechanism of resistance to standard therapy.