Embryologists' health: a nationwide online questionnaire.

PURPOSE: The present study evaluates health status and its relation with occupational characteristics and with burnout syndrome among embryologists. METHODS: A cross-sectional design was used to conduct an online self-assessment survey, sent to all members of the Spanish Association of Clinical Embryologists. The questionnaire contained occupational questions and two standard instruments: 'Short Form-12 Health Survey' as a measure of physical (PCS-12) and mental (MCS-12) health and the Maslach Burnout Inventory-General Survey (MBI-GS) to evaluate the degree of burnout. RESULTS: The PCS-12 obtained for the Spanish embryologists was higher than that for the reference population. However, the total MCS-12 was significantly lower than that observed in non-institutionalised males and females representative of the general Spanish population aged 35-44 years. In the linear regression model, the dependent variable PCS-12 was related indirectly with the variables number of hours worked per week, BMI, back pain, leg pain and visual discomfort. In the linear regression model, the dependent variable MCS-12 was indirectly related to the gender (male reference; female coefficient regression: -3.23), exhaustion and cynicism dimensions of the MBI-GS. A total of 87 (36.3%) embryologists presented a high score on at least one of the MBI-GS dimensions. CONCLUSION: In this sample of Spanish embryologists, a norm measure (SF-12) showed their physical health to be better than the average for the general population, but that their mental health was poorer. A significant indirect relation was observed between mental health and burnout syndrome. Strategies to reduce occupational stress and problems should form part of the training provided for clinical embryologists.

Adherence to reporting guidelines in observational studies concerning exposure to persistent organic pollutants and effects on semen parameters.

STUDY QUESTION: Are studies on semen quality in men exposed to persistent pesticides reported according to the 'strengthening the reporting of observational studies in epidemiology' (STROBE) recommendations and the guidelines for the appraisal of semen quality studies (SEMQUA)? SUMMARY ANSWER: Most studies of the impact of pesticides on semen quality do not follow the STROBE and SEMQUA guidelines, thus adherence is low, especially in methodological aspects. WHAT IS KNOWN ALREADY: Much of the controversy about reduced semen quality in recent decades arises from a lack of standardization in the methodology applied, despite the existence of several validated instruments for evaluating the quality of reporting. Indeed, SEMQUA was purpose-designed for the particular characteristics of semen quality studies. STUDY DESIGN, SIZE, DURATION: A structured literature search identified eligible articles reporting on persistent pesticides and human semen quality, published in English before 1 September 2012. Opinion articles and reviews were excluded. We assessed the adherence to reporting guidelines of the articles, using and comparing the STROBE statement and the SEMQUA guidelines, in both cases with indicators relevant to observational studies of semen quality. PARTICIPANTS/MATERIALS, SETTING, METHODS: A comprehensive bibliographic search in various electronic literature databases using the key words 'sperm' and 'pesticide' obtained 1179 papers, of which 46 were valid for our purposes. The papers examined occupational (26) and environmental exposure (20). Two of the present authors independently piloted the data extraction form for this review. The articles were then evaluated by two researchers using the STROBE and SEMQUA checklists. MAIN RESULTS AND THE ROLE OF CHANGE: Although no significant differences were found between the overall degree of compliance with STROBE and SEMQUA (47.0 ± 18.5% versus 43.1 ± 11.6%), there were significant differences when only methodological aspects were considered (48.4 ± 21.0% versus 39.5 ± 17.4%; P < 0.001). We observed an increase over time in the degree of compliance, for SEMQUA (r = 0.61 and P < 0.001) and STROBE (r = 0.45 and P < 0.01). The papers that reported a negative effect of exposure to persistent pesticides on sperm concentration presented a lower level of compliance to SEMQUA (42.1 ± 18.3% versus 57.6 ± 14.2%; P < 0.01) and STROBE (40.2 ± 10.3% versus 49.5 ± 11.6%; P < 0.05) than those which recorded no such influence. The year of publication and the observed effect on sperm concentration were the only candidate variables included in the model of stepwise multiple regression model for the 'degree of compliance' variables of SEMQUA and STROBE. LIMITATIONS, REASONS FOR CAUTION: Other characteristics of reporting quality, such as legibility, were not evaluated. WIDER IMPLICATIONS OF THE FINDINGS: The low degree of compliance observed is consistent with that observed in other studies of reproductive medicine and highlights the need to improve the design of studies of semen quality. SEMQUA proved to be a more specific tool than STROBE for the field of semen quality. Editors, reviewers and authors should be aware of SEMQUA and apply it when assessing papers on semen quality. STUDY FUNDING/COMPETING INTEREST(S): No research funding was received and none of the authors have any conflict of interests.

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.

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.

Sperm chromatin structure assay and classical semen parameters: systematic review.

The present study is based on a PubMed search and compares the clinical validity of classical semen parameters (CSP) and the sperm chromatin structure assay (SCSA) in different clinical contexts. The PubMed database was searched using keywords on the sperm diagnostic test for pregnancy in three clinical scenarios: (i) couples attempting to conceive; (ii) couples who had been attempting to conceive for 12months without success; and (iii) couples treated with intrauterine insemination (IUI). There was a considerable heterogeneity among the studies included. For couples attempting to conceive following a SCSA that produced an abnormal result, the likelihood of male factor infertility ranged from a pre-test value of 7.5% to a post-test value of 32.1% [95% confidence interval (CI) 15.7-54.5], while after CSP with an abnormal result, the post-test probability was 17.3% (95% CI 11.8-24.5). For a pre-test prevalence of male factor infertility of 50%, the post-test probability of male factor infertility after an abnormal test is very similar for both SCSA and CSP. In couples treated with IUI, the clinical validity of SCSA is higher than that of sperm morphology alone, but not enough to introduce SCSA as a test in male infertility work-up.

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.

Influence of analytical and biological variation on the clinical interpretation of seminal parameters.

Quality assurance in semen analysis has been questioned recently in this journal. Based on the limited capacity of seminal parameter in the determination of fertility, the authors advocated abandoning methods of quality assurance in semen analysis for clinical situations. In this article, we explore arguments as to why quality assurance in semen analysis for clinical use is not 'a waste of time'. Imprecision and within-subject biological variations are the two major components involved in the dispersion of seminal parameter results obtained by analysis of a semen sample from an individual. As within-subject biological variation is constant across geography, time and population, imprecision is a very important factor in the quality of laboratory test results. We analyse this influence on various seminal parameters and observe that there is an amount of error that can be tolerated without invalidating the medical usefulness of seminal parameter determination. However, there is a maximum allowable analytical error above which the medical usefulness of seminal parameter results is invalidated. The level of performance required to facilitate clinical decision-making is termed quality specification. We comment on different strategies to define the maximum allowable analytical error.

Quality specifications for seminal parameters based on the state of the art.

BACKGROUND: The aim of this study was to calculate the analytical goal for seminal parameters based on the state of the art, and then to compare these specifications with those previously obtained by our group based on biological variation. METHODS: All data used for analysis were derived from the Spanish programme of external quality control on semen analysis. Over 90 laboratories participated from 1999 to 2003. Using graphs of the state of the art, we also determined the numbers of laboratories that achieved quality specifications. RESULTS: The total allowable error calculated using state of the art graphs is similar to that calculated using biological variation for concentration and total motility. However, it is much higher for morphology and rapidly progressive motility. Over 80% of the laboratories achieved the minimum quality specification based on biological variation for concentration, total and progressive motility. However, only approximately 30% of the laboratories achieved the minimum quality specification based on biological variation for morphology and rapidly progressive motility. CONCLUSIONS: The study enabled us to identify the state of the art of analytical performance for seminal parameters, and revealed the difficulty inherent in meeting the quality specifications based on biological variation.

Estudio de calidad seminal en población joven del sudeste español / Study of semen quality in a young population from the southest of Spain

En los últimos 15 años han surgido varios trabajos en los que se advierte un posible descenso en la calidad seminal en el hombre. Estos estudios han sido criticados por el sesgo que pueden presentar en cuanto a la selección de las poblaciones de estudio y en cuanto a la metodología analítica empleada. En España, los trabajos realizados en este campo parten de poblaciones procedentes de bancos de donantes de semen y de clínicas de fertilidad. En el presente estudio partimos de una muestra de 271 jóvenes del sudeste de España, con edades entre 18 y 23 años, sin conocimiento previo sobre su salud reproductiva. Los jóvenes fueron reclutados para nuestro estudio durante un período continuo de 14 meses gracias a un plan de captación que fue llevado a cabo con la colaboración de la Universidad de Almería. A los jóvenes que accedieron a colaborar se les realizó un seminograma siguiendo la metodología de análisis estándar indicada por la OMS. Los valores de la media obtenidos para el volumen del eyaculado, concentración espermática y movilidad total fueron de 3,1 ml, 76 millones de espermatozoides/ml y 59,8 por ciento respectivamente, siendo estos valores similares a los encontrados en otros estudios europeos. Con estos datos se pretende crear una base de partida a la hora de hacer estudios comparativos. A su vez, establecemos un sistema de captación de voluntarios que puede ser de gran utilidad para futuros estudios en calidad seminal (AU)

Distribution profiles of paraoxonase and cholinesterase phenotypes in a Spanish population.

The paraoxonase/arylesterase phenotype was measured in a Spanish population as previous studies have reported that the polymorphic variation in serum paraoxonase activity may affect the metabolism of organophosphates in individuals at risk of chronic intoxication. The prevalence of congenital deficiency in serum cholinesterase was also established in order to ascertain whether individuals with a congenital defect would be at a higher risk against a potential organophosphate exposure. We consider it useful to incorporate these two biomarkers into the health programme of agricultural workers with the purpose of monitoring workers who spray organophosphate pesticides, as they provide reliable indications of early-stage effects related to biochemical alterations that might precede overt clinical pictures.

Human liver paraoxonase (PON1): subcellular distribution and characterization.

The subcellular localization and different biochemical properties of a human hepatic microsomal enzyme that hydrolyses paraoxon (paraoxonase, PON1) were studied and compared to the paraoxon hydrolase activity found in human plasma as well as in rat liver and plasma. Having evaluated the influence of the postmortem interval by a parallel experiment performed in rats, we conclude that the paraoxonase activity was preferentially localized in the microsomal fraction. The enzyme reaction was optimized according to temperature, pH, buffer, ionic strength, substrate concentration, and enzyme protein concentration. The characterization of human liver paraoxonase included the study of optimum pH, pH stability, heat inactivation assays, and kinetic parameters (K(m) and Vmax). In addition, the enzyme activity showed an absolute requirement for exogenous calcium. The activity was lost after incubation with EDTA and partially restored by the addition of calcium; however, other metals assayed were not able to activate the human liver enzyme as did calcium. Our results support the possible identity between human plasma and liver paraoxonases. In spite of the technical difficulties of this study and the possible interference of the postmortem changes in the results, this article represents the first systematic approach to the characterization of human liver paraoxonase.

Inhibition of paraoxonase activity in human liver microsomes by exposure to EDTA, metals and mercurials.

Inhibition of paraoxon hydrolase (paraoxonase) activity by 'in vitro' exposure to EDTA, Mg2+, Co2+, Ba2+, La3+, Zn2+, Cu2+, Hg2+, p-hydroxymercuribenzoate (p-OH-MB) and phenyl mercuric acetate (PMA) was investigated in human liver microsomes. Enzyme activity was totally inhibited by 1 mM EDTA in a time-dependent manner, in contrast to previous data obtained in rat liver where an EDTA-resistant fraction was detected. The possible influence of postmortem changes in these results was checked in a parallel experiment using rat livers with different postmortem intervals. From our results the existence in human liver of an EDTA-resistant fraction cannot be discarded. Ba, La and PMA showed immediate inhibition. By contrast the other compounds tested were time-dependent inhibitors. Ba and Zn showed the highest IC50 values. Cu and mercurials (Hg, p-OH-MB, PMA) were the most potent inhibitors of human liver paraoxonase. Kinetic analysis (Lineweaver-Burk and Dixon plots) indicated that different inhibitors exhibit different inhibition patterns: competitive (EDTA, Ba, La, Cu, p-OH-MB and PMA), non competitive (Zn) and mixed (Hg). The pretreatment of sample with dithiothreitol (DTT) protects against the inhibitory effect of mercurials. Furthermore after inhibition by mercurials the activity was restored by DTT. These results confirmed the essential role of the -SH groups to maintain the catalytic activity of paraoxonase and suggest the existence of two types of -SH groups that could differ in their localization.

Divergent effects of classical inducers on rat plasma and microsomal fraction paraoxonase and arylesterase.

The effects of three different enzyme-inducing agents (phenobarbital, 3-methylcholanthrene and rifampicin) on plasma and liver microsomal fraction paraoxonase and arylesterase were studied in rats. Although phenobarbital and 3-methylcholanthrene each increased the esterase activities in microsomal fraction, only 3-methylcholanthrene was capable to increase them in plasma. By contrast, the administration of rifampicin decreased both enzyme activities in liver and plasma. The results indicate that at least there exists two esterase activities in rat liver microsomes which hydrolyse both paraoxon and phenylacetate, but only one of them is released into the blood.

Differences in the kinetic properties, effect of calcium and sensitivity to inhibitors of paraoxon hydrolase activity in rat plasma and microsomal fraction from rat liver.

The properties of a rat hepatic microsomal enzyme that hydrolyses O,O-diethyl-p-nitrophenylphosphate (paraoxon) were studied and compared to the paraoxon hydrolase activity found in rat plasma. The pH stability for both enzyme activities was optimum between pH 6.0 and 9.0. An overall analysis of the data showed that the microsomal fraction was less resistant to the effect of the pH than plasma. The kinetic constants for heat inactivation evaluated for paraoxonase in rat plasma and liver microsomal fraction indicate that paraoxonase tends to inactivate faster in rat liver microsomes than in rat plasma. The apparent activation energies of the heat inactivation process were 77.7 and 61.1 kcal/mol for rat plasma and microsomal fraction, respectively. Enzyme activity was lost after both dialysis and incubation with EDTA and partially restored by the addition of calcium. In rat plasma samples the requirement for calcium was absolute (essential activator) while in the microsomal fraction the reaction may occur, to a minimum extent, in the absence of the activator (non-essential activator). Calcium restored 85% activity when added immediately after EDTA; restored activity decreased when the time interval between addition of EDTA and calcium was increased. Other metals were not able to restore activity previously inhibited by EDTA or dialysis. The response to several inhibitors (EDTA, Mn, Co, Zn, Ba, Mg, Cu, La, Hg and p-hydroxy-mercuribenzoate) of rat plasma and microsomal fraction was studied, determining the type of inhibition and the inhibition constants. Plasma enzyme was always more resistant than liver sample to the effect of the inhibitors and showed different types of inhibition than the liver microsomal fraction. In general we found more differences than analogies between the rat plasma and liver enzyme which suggests the presence of two enzymes or two different forms of the same enzyme. Furthermore the existence of an EDTA-resistant fraction in rat liver microsomes suggests that more than one enzyme capable of hydrolysing paraoxon is present in the microsomal fraction of rat liver.

Rat liver paraoxonase: subcellular distribution and characterization.

The subcellular localization and some biochemical properties of rat liver paraoxonase have been studied in order to establish a correlation with plasma enzyme. The whole paraoxonase activity was found in the microsomal fraction. Rat plasma and liver paraoxonase showed similar optimum pH (8.5), Km (0.4 mM) and calcium requirement, but differed in the response to several inhibitors.

Partial purification of paraoxonase from rat liver.

A method for the partial purification of rat liver paraoxonase is presented. The method consists of the following steps: preparation of microsomes, solubilization with Triton X-100, adsorption on hydroxylapatite and chromatography on DEAE-52 cellulose. A partially purified preparation of rat liver paraoxonase has been obtained, showing a specific activity of 422 mU/mg with a yield of about 22% and a purification factor of 77-fold.