Elva I. Cortés-Gutiérrez

A dynamic assessment of sperm DNA fragmentation versus sperm viability in proven fertile human donors

OBJECTIVE To analyze any possible dynamic correlation between sperm DNA fragmentation and sperm viability. DESIGN The rate of viability loss and the rate of increase of the frequency of sperm cells with fragmented DNA were determined at 0, 1.5, 4.5, and 24.0 hours after thawing samples from donors with proven fertility. SETTING Academic biology and reproductive medicine centers. PATIENT(S) Fifteen male donors with proven fertility for a maximum of six births at the reproductive medicine center. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Sperm DNA fragmentation and viability dynamics expressed as logarithmic coefficients of change. RESULT(S) The dynamics of sperm DNA fragmentation and sperm viability adjusted to a logarithmic function with an initial highest velocity that progressively decreases. Nevertheless, the rates were not statistically significantly correlated. CONCLUSION(S) In the short term, dynamic dysfunction of membrane permeability does not result in DNA fragmentation and thus must be considered as independent parameters of sperm quality.

Micronuclei in cervical smears and peripheral blood lymphocytes from women with and without cervical uterine cancer

Cervical cancer represents the second most common malignant neoplasia in women world-wide. In Mexico, cervical cancer is the most common female malignancy. It has been recently seen an increased frequencies of micronuclei (MN) lymphocytes and cervical epithelial cells of cervical cancer patients. The aim of this hospital-based unmatched case-control study was to investigate the association between progressive stages in development of cervical cancer and frequency of micronucleated cells in the cervical epithelium and peripheral lymphocytes of 40 women, grouped by disease stage. Women at the Obstetrics and Gynecology Hospital of the Instituto Mexicano del Seguro Social (IMSS) in Monterrey, Mexico were diagnosed and classified on the bases of the Papanicolaou (PAP) smear and colposcopy/biopsy into control, low-grade squamous intraepithelial lesions (LGSIL), high-grade squamous intraepithelial lesions (HGSIL), and invasive groups. Analysis of the MN data in both cell types revealed (a) homogeneity among women within each of the four groups with regard to MN frequency, (b) in general, a correlation between MN frequency and grade of cervical lesion, and (c) a positive linear trend between the MN frequency and increased cervical cancer risk. In conclusion, we suggest that MN are a useful biomarker of cancer risk. Nonetheless, these results should be validated by other researchers.

Evaluación del daño en el DNA espermático

Infertility affects almost 20% of couples in reproductive age and the male factor being responsible of 50% of this infertility. Among the classic parameters that determine a good seminal quality such as sperm motility, sperm morphology or the quality of the of acrosomes and/or sperm membranes, the integrity of the DNA molecule is crucial to carry out a successful fertilization. Nevertheless, the study of this parameter has not been straightforward approached. This fact has shunned its incorporation, as a routine technique, within a standard seminogram. The aim of the present review is to summarize and update those technologies that are considered more successful to study sperm DNA fragmentation with special emphasis to: 1) the levels of technological complexity and the possibility of its use in laboratories of andrology, according with the equipment and the resources available, and 2) the effects and possible implications of high level of sperm DNA fragmentation for fecundation, embryo development and fertility.

New Application of the Comet Assay Chromosome–Comet Assay

The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains.

Evaluation of DNA Single and Double Strand Breaks in Women with Cervical Neoplasia Based on Alkaline and Neutral Comet Assay Techniques

A hospital-based unmatched case-control study was performed in order to determine the relation of DNA single (ssb) and double (dsb) strand breaks in women with and without cervical neoplasia. Cervical epithelial cells of 30 women: 10 with low grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 without cervical lesions were evaluated using alkaline and neutral comet assays. A significant increase in global DNA damage (ssb + dsb) and dsb was observed in patients with HG-SIL (48.90 ± 12.87 and 23.50 ± 13.91), patients with LG-SIL (33.60 ± 14.96 and 11.20 ± 5.71), and controls (21.70 ± 11.87 and 5.30 ± 5.38; resp.). Pearson correlation coefficient reveled a strong relation between the levels ssb and dsb (r2 = 0.99, P = 0.03, and r2 = 0.94, P = 0.16, resp.) and progression of neoplasia. The increase of dsb damage in patients with HG-SIL was confirmed by DNA breakage detection-FISH (DBD-FISH) on neutral comets. Our results argue in favor of a real genomic instability in women with cervical neoplasia, which was strengthened by our finding of a higher proportion of DNA dsb.

Chromosomal abnormalities and polymorphic variants in couples with repeated miscarriage in Mexico

Cytogenetic studies have an important role in the evaluation of couples with repeated miscarriages and poor obstetric history. To estimate the prevalence of chromosomal abnormalities and polymorphic variants in 158 couples with repeated miscarriages, a cross-sectional study was conducted in Monterrey, Mexico from 1995 to 2003. Peripheral blood lymphocytes were cultured for chromosomal studies using standard methods. Twelve couples showed chromosomal abnormalities (7.60%), two Robertsonian translocations (1.27%), two balanced translocations (1.27%), one inversion (0.63%), and one a novel insertion (0.63%). This insertion [46, XX, ins (15;8) (q26;p11p23)] is unique, and is the third reported in association with repeated abortion. Mosaicism was observed in six couples (3.80%, three with structural abnormalities and three with numerical abnormalities). A female to male ratio of 1.4:1 was observed. In addition to these chromosomal abnormalities, polymorphic variants in constitutive heterochromatin of the 1qh+, 9qh+, and 16qh+ chromosomes were observed in 25 couples (15.82%), of the Yqh+ chromosome in 21 couples (13.29%), and of satellite in 35 couples (22.15%). In conclusion, chromosome analysis is necessary for appropriate clinical management of these patients.

Sister chromatid exchanges in peripheral lymphocytes from women with carcinoma of the uterine cervix

Sister chromatid exchanges (SCE) are reciprocal exchanges between sister chromatids. It has been reported that in patients with cervical cancer, the frequency of SCE in peripheral lymphocytes is significantly higher than that in normal individuals; however, other studies have shown no significant difference. The aim of this unmatched case-control study was to compare the mean number of SCE per metaphase in lymphocytes from women with and without carcinoma of the cervix uteri. The SCE specimens were prepared by the fluorescence plus giemsa technique in peripheral lymphocytes from 28 women with carcinoma of cervix uteri and 28 controls. The mean number of SCE per metaphase in women with carcinoma of cervix uteri (7.80 +/- 1.05) was higher than the control group (6.98 +/- 1.13) (P < 0.05; t-test). This study had a statistical power of 0.80 and an alpha value of 0.05. This finding suggests that an increased number of SCE in peripheral lymphocytes is associated with cervical cancer. We consider that the lack of reported association of SCE and cervical cancer might be attributed to the none determination of the statistical power and sample size.

Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay

Key Concepts The two-dimensional Two-Tailed Comet assay (TT-comet) protocol is a valuable technique to differentiate between single-stranded (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. Protein lysis inherent with the TT-comet protocol accounts for differences in sperm protamine composition at a species-specific level to produce reliable visualization of sperm DNA damage. Alkaline treatment may break the sugar–phosphate backbone in abasic sites or at sites with deoxyribose damage, transforming these lesions into DNA breaks that are also converted into ssDNA. These lesions are known as Alkali Labile Sites “ALSs.” DBD–FISH permits the in situ visualization of DNA breaks, abasic sites or alkaline-sensitive DNA regions. The alkaline comet single assay reveals that all mammalian species display constitutive ALS related with the requirement of the sperm to undergo transient changes in DNA structure linked with chromatin packing. Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. The TT is a valuable tool for identifying SSBs or DSBs in sperm cells with DNA fragmentation and can be therefore used for the purposes of fertility assessment. Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. A series of methodologies to assess DNA damage in spermatozoa have been developed but most are unable to differentiate between single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. The two-dimensional Two-Tailed Comet assay (TT-comet) protocol highlighted in this review overcomes this limitation and emphasizes the importance in accounting for the difference in sperm protamine composition at a species-specific level for the appropriate preparation of the assay. The TT-comet is a modification of the original comet assay that uses a two dimensional electrophoresis to allow for the simultaneous evaluation of DSBs and SSBs in mammalian spermatozoa. Here we have compiled a retrospective overview of how the TT-comet assay has been used to investigate the structure and function of sperm DNA across a diverse range of mammalian species (eutheria, metatheria, and prototheria). When conducted as part of the TT-comet assay, we illustrate (a) how the alkaline comet single assay has been used to help understand the constitutive and transient changes in DNA structure associated with chromatin packing, (b) the capacity of the TT-comet to differentiate between the presence of SSBs and DSBs (c) and the possible implications of SSBs or DSBs for the assessment of infertility.

TRICHOMONAS VAGINALIS: IDENTIFICATION OF SOLUBLE AND MEMBRANE-ASSOCIATED PHOSPHOLIPASE A1 AND A2 ACTIVITIES WITH DIRECT AND INDIRECT HEMOLYTIC EFFECTS

A direct hemolytic activity, dependent on phospholipase A (PLA) activity, was located in the particulate subcellular fraction (P30) of Trichomonas vaginalis. We identified soluble direct and indirect hemolytic activities in the spent medium and soluble fraction (S30) of T. vaginalis strain GT-13. Spent medium showed the highest specific indirect hemolytic activity (SIHA) at pH 6.0 (91 indirect hemolytic units [HU]/mg/hr). Spent medium and P30, but not S30, showed direct hemolytic activity. PLA activity was protein dose dependent and time dependent. The highest PLA activity was observed at pH 6.0. All trichomonad preparations showed phospholipase A1 (PLA A1) and phospholipase A2 (PLA A2) activities. Indirect and direct hemolytic activity and PLA A1 and PLA A2 diminished at pH 6.0 and 8.0 with increasing concentrations of Rosenthals inhibitor. The greatest effect was observed with 80 μM at pH 6.0 on the SIHA of S30 (83% reduction) and the lowest at pH 8.0, also on the SIHA of S30 (26% reduction). In conclusion, T. vaginalis contains particulate and soluble acidic, and alkaline direct and indirect hemolytic activities, which are partially dependent on alkaline or acidic PLA A1 and PLA A2 enzymes. These could be responsible for the contact-dependent and -independent hemolytic and cytolytic activities of T. vaginalis.

TRICHOMONAS VAGINALIS: IDENTIFICATION OF A PHOSPHOLIPASE A–DEPENDENT HEMOLYTIC ACTIVITY IN A VESICULAR SUBCELLULAR FRACTION

Trichomonad total extracts (TTE), or vesicular (P30) and soluble (S30) subcellular fractions from 3 pathogenic Trichomonas vaginalis strains (GT-3, GT-13, and GT-15), lysed both human and Sprague–Dawley rat erythrocytes in a time- and dose-dependent manner. The entire hemolytic activity of TTE was located in P30, showing 2 peaks of maximum activity, one at pH 6.0 and another at pH 8.0, in the presence of 1 mM Ca2+. Hemolytic activity on rat erythrocytes was greater at pH 6.0 (6.71 ± 0.33 hemolytic units [HU]/mg/hr to 11.60 ± 0.24 HU/mg/hr) than at pH 8.0 (3.81 ± 0.30 HU/mg/hr to 5.75 ± 0.65 HU/mg/hr), and it was greater than that on human red blood cells at pH 6.0 (2.67 ± 0.19 HU/mg/hr to 4.08 ± 0.15 HU/mg/hr) or pH 8.0 (2.24 ± 0.09 HU/mg/hr to 2.81 ± 0.06 HU/mg/hr). The alkaline and acidic hemolytic activity diminished (60–93% at pH 6.0 and 78–93% at pH 8.0) by the effect of 80 μM Rosenthals inhibitor, which also inhibited 27–45% and 29–54% trichomonad alkaline and acidic phospholipase A activities, respectively. Vesicles, vacuoles, and hydrogenosomes were rich in P30. Trichomonas vaginalis has a hemolytic PLA, which could be involved in its cytopathogenic mechanism.