Buccal micronucleus cytome assay of populations under chronic heavy metal and other metal exposure along the Santiago River, Mexico.

The Santiago River is one of the most contaminated rivers in Mexico, with heavy metal levels above the allowed limits. Scientific evidence indicates that chronic heavy metal exposure leads to cytogenotoxic effects. The aims of this study were to evaluate the genotoxic and cytotoxic effects of such exposure in buccal mucosa cells by micronucleus (MN) assay and to identify other nuclear abnormalities (NAs), such as nuclear buds (NBUDs), binucleated cells (BNs), pyknotic nuclei (PNs), karyorrhexis (KX), karyolysis (KL), and abnormally condensed chromatin (CC). Assays were performed on samples from four populations located alongside the Santiago River that are under chronic exposure to heavy metals and other metals (HMMs), and the results were compared with those of a population without exposure to HMMs. The exposed group showed increased frequencies of NAs (KX, CC, and KL), which are associated with cytotoxic damage, and NBUDs, which are associated with genotoxic damage. Increased frequencies of NBUDs and CC were observed in subjects from El Salto/Juanacatlán, Ocotlán, and Paso de Guadalupe, and an increase in KX frequency was observed in subjects from El Salto/Juanacatlán. Significant differences in KL frequency were observed in subjects from La Barca, El Salto/Juanacatlán, Paso de Guadalupe, and Ocotlán. Predictors for increased development of MNs and NBUDs were high concentrations of Al, Zn, and Cu. In conclusion, chronic exposure to HMMs, especially Al, Cu, and Zn, in the studied population could be related to increased frequencies of NAs, such as NBUDs, KX, CC, and KL, in the buccal mucosa cells.

Acceptable variability in external quality assessment programmes for basic semen analysis.

BACKGROUND: External quality assessment is essential in modern andrology laboratories. To assess the proficiency of laboratories participating in an external quality assessment programme (EQAP), limits for acceptable variability must be determined. Limits currently specified largely depend on criteria set by the organizers of individual EQAP schemes. The objective of this study was to evaluate the different criteria described in ISO 13528: 2005 for calculating acceptable variability in EQAP when applied to basic semen analysis parameters. METHODS AND RESULTS: The data used in this study were the means and standard deviations obtained for independent samples from two EQAPs, one national (Spanish) and one international (European). The acceptable variability according to ISO 13528: 2005 was calculated using four types of criteria: (i) ± 3 standard deviations of the results of all participating laboratories; (ii) ± 3 standard deviations of the results of expert laboratories; (iii) quality specifications based on biological variability, state-of-the-art and clinicians' opinions and (iv) the same quality specifications adjusted for the uncertainty of the assigned value. The first two strategies resulted in very wide ranges of acceptable variability. Conversely, the strategy based only on quality specifications resulted in very narrow ranges. For the fourth strategy, acceptable ranges were intermediate between the results produced with the other strategies. The third and fourth strategies did not produce observable differences in acceptable ranges when the model used for calculating the specifications of analytical quality was changed. CONCLUSIONS: It is essential that EQAPs for semen parameters should determine the ranges for acceptable variability in results. Moreover, these ranges must be clinically useful, i.e. the variability should have a minimal negative impact on clinical decisions. The exact definition of 'expert laboratory' is more important than the model chosen for estimating analytical quality specifications in an EQAP for semen parameters in basic semen analysis.

Comparison of methods to determine the assigned value in an external quality control programme for embryo evaluation.

This study set out to establish adequate assigned values for a National External Quality Control Programme of embryo evaluation. The results obtained by Spanish laboratories in this programme are compared with those of a group of national experts in embryo quality. Image-based embryo evaluation consists not only of classifying embryos as being of optimal, moderate or poor quality, but also of specifying the clinical decision to be taken regarding each embryo (transfer, cryopreservation or rejection). The proportion of embryos for which there was a high degree of agreement among the experts was 98.3% for embryo classification and 93.3% for clinical decision; for the laboratories, the respective values were 44.2 and 42.5%. With respect to the interobserver agreement among laboratories and experts, kappa coefficients were lower than 0.6 both for classification and for clinical decision. The experts recommended cryopreservation of a higher percentage of embryos classified as poor quality than did the laboratories (28 versus 4%, P = 0.05). The data obtained show that the agreement among laboratories is lower than among experts, and that the concordance among experts and laboratories is moderate. Therefore, it is recommended that an assigned value from external quality control programmes is established based on the consensus values obtained from experts.

Solubilized human zona pellucida competes with a fucosylated neoglycoprotein for binding sites on the human sperm surface.

OBJECTIVE: To evaluate the implication of fucose binding sites on the sperm surface in the sperm-zona pellucida (ZP) binding by examining the ability of a fucosylated neoglycoprotein to compete with solubilized zona proteins for sperm binding. DESIGN: The binding of a fucosylated neoglycoprotein to spermatozoa was analyzed in relation to sperm capacitation and the actual acrosomal status. The effect of solubilized ZP material on the binding was examined. SETTING: Private hospital, public research center, and university department. PATIENTS, PARTICIPANTS: Healthy volunteers with normal sperm parameters. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Fluorescence signal detected by fluorescence-activated cell sorting or fluorescence microscopy on spermatozoa incubated with fluorescein isothiocyanate-labeled fucosylated neoglycoprotein. RESULTS: There was a small subpopulation of spermatozoa that showed a strong binding activity for the fucosylated neoglycoprotein, and the size of this subpopulation increased with the time of exposure to capacitating conditions. The binding sites were located on the head (mainly on the acrosomal region) of acrosome-intact spermatozoa. The neoglycoprotein binding was competitively inhibited by solubilized ZP. CONCLUSIONS: The data show that a commercially available fucosylated neoglycoprotein competes with solubilized ZP proteins for binding sites on the sperm surface. Further research into the relationship between the neoglycoprotein binding and zona binding is warranted to evaluate the potential diagnostic usefulness of this neoglycoprotein.

Follicular fluid alpha-fetoprotein, carcinoembryonic antigen, and CA-125 levels in relation to in vitro fertilization and gonadotropin and steroid hormone concentrations.

OBJECTIVE: To investigate the possible role of alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and CA-125 in the ovarian follicle. DESIGN: alpha-Fetoprotein, CEA, and CA-125 were measured in human follicular fluid (FF) and correlated to IVF outcome and gonadotropin and gonadal steroid hormones. SETTING: The In Vitro Fertilization Program at Virgen de las Nieves Hospital, Granada, Spain. PATIENTS: Thirty-six FF from 12 infertile women with irreparable tubal damage who underwent treatment for ovarian stimulation with clomiphene citrate, hMG, and hCG. INTERVENTIONS: Laparoscopic follicular aspiration followed 48 hours later by ET. MAIN OUTCOME MEASURE: Serum and FF AFP, CEA, and CA-125 levels. RESULTS: alpha-Fetoprotein, CEA, and CA-125 were detected in all samples of serum and FF. There were no significant differences between the serum and FF levels. Follicular fluid AFP, CEA, and CA-125 of fertilized oocytes were similar to those in the unfertilized oocytes group. There were no significant correlations between the FF AFP, CEA, and CA 125 levels and gonadotropin and gonadal steroid hormone levels. CONCLUSIONS: alpha-Fetoprotein, CEA, and CA-125 are present in FF after ovarian stimulation, but the mean intrafollicular levels do not differ significantly regardless of the outcome of oocyte IVF.

Crisis treatment in a day hospital. Impact on medical care-seeking.

The cost-effectiveness of a day hospital program is documented by examining the utilization of medical and psychiatric services before and after a time-limited following crisis-presentations. Patients in crisis have been shown to have increased need for services following a crisis. In contrast, medical-care events decreased for the population treated in the day hospital. The results, with respect to reduction of medical care were greatest for those patients judged to have benefited from the psychiatric interventions.

Genetic testing of sperm donors for cystic fibrosis and spinal muscular atrophy: evaluation of clinical utility.

OBJECTIVE: To evaluate the clinical utility of genetic testing for cystic fibrosis (CF) and spinal muscular atrophy (SMA) in sperm donors. STUDY DESIGN: We studied the results of the genetic tests for CF and SMA applied to 372 sperm donor candidates. The CF carrier screening test analysed 32 mutations on the CFTR gene. Regarding SMA, the carrier test studied possible deletions of SMN1/2 by Multiplex Ligation-dependent Probe Amplification (MLPA) methodology. RESULTS: The carrier frequency obtained was greater for SMA than for CF. After adjusting the results obtained for the sensitivity of the tests, and taking into account the prevalence of female carriers in our population, the probability of transmission of the disease to the child from a donor with a negative genetic test was about five times lower in the case of SMA than in CF, although this difference was not statistically significant. The number of donors needed to screen (NNS) to avoid the occurrence of a child being affected by CF and SMA in our population was similar in both cases (1591 vs. 1536). CONCLUSIONS: This study demonstrates the need to include SMA among the diseases for which genetic screening is performed in the process of sperm donor selection. We believe that testing donors for SMA is as important and as useful as doing so for CF.

Quality specifications for seminal parameters based on clinicians' opinions.

BACKGROUND: The aim of this study was to identify analytical quality specifications for seminal parameters based on clinicians' opinions, and to compare with those based on biological variability and state of the art. MATERIAL AND METHODS: Two questionnaires with case histories were sent to laboratories participating in the Spanish programme of external quality on semen analysis for distribution to as many specialist clinicians as possible. Our intention was to determine the critical difference (CD), defined as the difference needed between two consecutive results obtained from semen analysis to be 95 % confident that the two results actually are different. Subsequently, we calculated the specifications of analytical quality in accordance with the clinicians' opinions. RESULTS: The CDs obtained from the median value of the differences between the initial value and that given in the clinicians' replies were similar in clinical situations of improvement or worsening in the infertile normozoospermic male, and also in worsening situations for male patients presenting a significant alteration in seminal parameters. For improvement in this latter case, the CD cited as necessary in the clinicians' opinion was much higher than that for the other clinical situations. At a desirable level of quality, for concentration and total motility the coefficients of variation in the clinicians' opinion were below those based on biological variability and the state of the art. However, for type "a+b" motility, type "a" motility, morphology and vitality the coefficients of variation based on the clinicians' opinions were higher than those based on biological variability and lower than those based on the state of the art. CONCLUSIONS: Quality specifications for seminal parameters based on clinicians' opinions depend to a large extent on the clinical situation and on the seminal parameter being analysed.

External quality control program for semen analysis: Spanish experience.

PURPOSE: Results from an external quality control programme for semen analysis carried out in Spain are analysed. METHODS: Quality control materials were distributed and the following seminal parameters were determined: concentration, total motility, progressive motility, rapid progressive motility, morphology and sperm vitality. The between-laboratories coefficients of variation were assessed on different types of quality control material. RESULTS: The majority of participating laboratories utilised manual versus computer-assisted semen analysis methods. Some between-laboratories coefficients of variation ranges were: 20.8-33.8% for concentration (semen pool suspension); 13.9-19.2% for total motility (videotapes); 54.2-70.2% for sperm morphology (strict criteria using stained smears); and 9.8-41.1% for sperm vitality (stained smears). There was an inverse relation between mean percentage of sperm and coefficients of variation between laboratories for sperm motility, morphology and vitality. CONCLUSIONS: These data highlight the urgent need for improvement in the overall quality of andrology testing.

Sperm plasma membrane integrity measurement: a combined method.

Sperm plasma membrane characteristics were measured by a combined method consisting of the hypo-osmotic swelling test and staining with either the eosine Y (HOS-eosine test) or propidium iodide dye (HOS-propidium test). Sperm samples were washed and resuspended in BWW medium (fraction I). Aliquots of the washed spermatozoa were treated by a swim-up technique to select motile spermatozoa (fraction II). After separation of motile cells, residual sperm pellets were treated separately (fraction III). These three fractions were subjected to the hypo-osmotic swelling test, lipid peroxidation measurement, and the HOS-eosine and HOS-propidium tests. The HOS-eosine test makes it possible to distinguish 4 types of spermatozoa: type 1: HOS+/eosine-; type 2: HOS-/eosine-; type 3: HOS-/eosine+ and type 4: HOS+/eosine+ (Fig. 1). HOS-propidium test shows equal results as HOS-eosine test. Fraction I spermatozoa showed 55.2 +/- 3.6% type 1; 12.6 +/- 1.0 type 2; 28.0 +/- 2.9 type 3; and 4.2 +/- 0.6 type 4 cells. Fraction II spermatozoa were characterized by high percentages of type 1 cells, low percentages of types 3 and 4, and very low values of lipid peroxidation (5 times smaller than fraction I). Fraction III showed a low percentage of type 1, a high percentages of the other types, and an enhanced value of lipid peroxidation (2 times higher than fraction I). The prognostic value of the HOS-eosine test was evaluated in an IVF programme. Preliminary results show that a high incidence of types 2 and 4 spermatozoa is often associated with fertilization failure.

Sperm plasma membrane integrity in fertile and infertile men.

Sperm plasma membrane characteristics were analysed by a combined method, the HOS-eosine test (HOS-E test), that consists of the hypoosmotic swelling test (HOS test), and the eosine-Y staining. Semen samples were categorized into four groups (Normo-, Oligo-, Astheno-, and Oligoasthenozoospermic) and subjected to the standard analysis (spermiogram), HOS test, eosine-nigrosine test (reflecting sperm viability); and HOS-E test. HOS-E test makes it possible to distinguish four groups of spermatozoa: type 1, HOS+/eosine-; type 2, HOS-/eosine-; type 3, HOS-/eosine+; and type 4, HOS+/eosine+. Normozoospermic samples showed 61.2 +/- 1.4% type 1, 9.2 +/- 0.8% type 2, 22.6 +/- 1.1% type 3, and 6.8 +/- 0.6% type 4 spermatozoa. Oligozoospermic samples showed no significant differences in these values, whereas asthenozoospermic samples showed a higher percentage of types 3 and 4 and a lower percentage of type 1. Oligoasthenozoospermic samples showed high percentages of types 2, 3, and 4 and a low percentage of type 1. Sperm plasma membrane integrity is a necessary condition for motility and fertilization. So, it is not surprising that semen samples with abnormal motility showed a HOS-E result indicative of a defective plasma membrane.

Biological variation of seminal parameters in healthy subjects.

BACKGROUND: A study was undertaken to assess the components of biological variation of seminal parameters in healthy subjects. METHODS: Twenty donor candidates were included in a 10-week follow-up study. Within- and between-subject biological variation, indices of individuality and heterogeneity, coefficient of reliability, critical differences, analytical goals and the lowest value observed with a <5% probability of having a true value less than the World Health Organization (1999) reference value were estimated for the following seminal parameters: concentration, total motility (WHO grades a + b + c), progressive motility (grades a + b), rapid progressive motility (grade a), sperm morphology and vitality. All analysis was performed by a single technician according to WHO 1999 guidelines for routine semen analysis. Analytical variation was assessed on different types of quality control material (frozen straws, sperm suspension, videotape, and slides) and at different (low, medium, high) quality levels. RESULTS: The analytical variation observed depended on the quality control material used and the level of semen quality. Concentration was the semen parameter with highest within- and between-subject variation, and vitality the lowest. Indices of individuality were all <0.7, and coefficients of reliability were high (0.68-0.84). The critical difference for sequential values significant at P < 0.05 for vitality, progressive motility and morphology (34.4, 49.2 and 58.0% respectively) were lower than for concentration (77.8%). CONCLUSIONS: The study results showed that conventional reference values for seminal parameters have little diagnostic value because of their marked individuality, though seminal parameters can be useful for assessing differences in an individual's serial results, in particular of progressive motility, morphology and vitality.

Adrenal hormones in human follicular fluid.

Considerable evidence indicates that adrenal hormones may affect gonadal function. To assess the role of some adrenal hormones in human follicular fluid and their relationship with the ability of the oocyte to be fertilized and then to cleave in vitro, cortisol and dehydroepiandrosterone sulfate were measured in follicular fluid obtained at the time of oocyte recovery for in vitro fertilization from cycles stimulated by clomiphene citrate, human menopausal gonadotropin and human chorionic gonadotropin. Thirty-six follicular fluid containing mature oocyte-corona-cumulus complexes and free of visible blood contamination were included in this study. There was no significant difference in follicular fluid dehydroepiandrosterone sulfate concentration between follicles with oocytes which did or did not fertilize (5.1 +/- 1.1 vs 5.8 +/- 2.0 mumol/l). However, follicular fluid from follicles whose oocytes were not fertilized had levels of cortisol significantly higher than those in follicular fluid from follicles containing successfully fertilized oocytes (406.0 +/- 75.9 vs 339.2 +/- 37.0 nmol/l; p < 0.005). No significant correlations were found between rates of embryo cleavage and cortisol and dehydroepiandrosterone levels in follicular fluid. We conclude that cortisol levels in follicular fluid may provide an index of fertilization outcome, at least in stimulated cycles by clomiphene citrate, human menopausal gonadotropin and human chorionic gonadotropin.

Insulin and insulin-like growth factor I in follicular fluid after induction of ovulation in women undergoing in vitro fertilization.

This study was undertaken to evaluate the relationship between concentrations of insulin and insulin-like growth factor I (IGF-I) in follicular fluid and fertilization and cleavage of human oocytes fertilized in vitro. The concentration of oestradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, testosterone, insulin and IGF-I was determined in 36 follicular fluids, free of visible blood contamination and containing mature oocyte-corona-cumulus complexes, obtained from 12 women undergoing in vitro fertilization. Follicular development was induced by clomiphene citrate and human menopausal gonadotrophin, and follicular aspiration was performed 35 h after an ovulatory dose of human chorionic gonadotrophin. Concentrations of IGF-I were significantly higher in follicular fluids associated with mature oocytes that fertilized and cleaved, than in follicular fluid associated with mature oocytes that did not fertilize (P < 0.001). There was no difference in the concentration of insulin between follicular fluids from which fertilized oocytes were obtained and those with oocytes that remained unfertilized. No significant correlations were found between rates of embryo cleavage, concentrations of insulin and IGF-I. Multiple linear regression analysis demonstrated that the concentrations of IGF-I in follicular fluid were predicted statistically by a negative regression coefficient for the concentration of testosterone, and by a positive regression coefficient for the concentration of progesterone in follicular fluid. No candidate variable was included in the model to predict concentrations of insulin. These data suggest an important role for IGF-I in the mature follicle.

Distribution of free amino acids in human preovulatory follicles.

The purpose of the present study was to investigate the possible role of intrafollicular amino acids in the human ovarian follicle. Follicular fluid (FF) and oocytes were obtained from 12 women for in vitro fertilization. Follicular development was induced with clomiphene citrate and human menopausal gonadotropin. Thirty-six FF samples, free of visible blood contamination and containing mature oocytes were used to measure free amino acids, steroids and gonadotropins. The FF were divided into three groups: (1) follicles yielding oocytes that were unfertilized (n = 12); (B) follicles containing oocytes that were fertilized and cleaved to less than four cells (n = 12); (C) follicles yielding oocytes that were fertilized and cleaved to four or more cells (n = 12). The concentrations of histidine, phenylalanine and asparagine in FF were significantly greater than those in plasma at the time of follicle aspiration. However, amino acid concentrations in FF did not differ significantly between the three groups studied. No significant relationships were found between intrafollicular levels of amino acids and those of LH, FSH, estradiol, progesterone and testosterone. These results suggest that intrafollicular amino acids are not involved in the regulation of human preovulatory follicle.

In silico analysis of protein neoplastic biomarkers for cervix and uterine cancer

Worldwide, cervical and uterine cancers are the most deadly cancers in women, with high prevalences, especially in developing countries. The Human Protein Atlas (HPA) portal was explored for proteins expressed in a tissue- or cervix and uterine cancer-specific manner. The group of proteins differentially expressed and with enhanced expression in the glandular and surface epithelial (squamous) cells retrieved from HPA were further explored using the Protein Information and Knowledge Extractor (PIKE) portal to compile biological information that is found in different databases, and repositories on the Internet. Thus, the lists of candidate proteins found in HPA, and PIKE portals may be used as a starting point for the discovery and validation of biomarkers for cervix and uterine cancer employing proteomics approaches as described in the present article (AU)

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