Proposal of guidelines for the appraisal of SEMen QUAlity studies (SEMQUA).

STUDY QUESTION: Is there a need for a specific guide addressing studies of seminal quality? SUMMARY ANSWER: The proposed guidelines for the appraisal of SEMinal QUAlity studies (SEMQUA) reflect the need for improvement in methodology and research on semen quality. WHAT IS KNOWN ALREADY: From an examination of other instruments used to assess the quality of diagnostic studies, there was no guideline on studies of seminal quality. STUDY DESIGN, SIZE AND DURATION: Through systematic bibliographic search, potential items were identified and grouped into four blocks: participants, analytical methods, statistical methods and results. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Our findings were presented to a panel of experts who were asked to identify opportunities for improvement. Then, a checklist was designed containing the questions generated by the items that summarize the essential points that need to be considered for the successful outcome of a SEMQUA. MAIN RESULTS AND THE ROLE OF CHANCE: Eighteen items were identified, from which 19 questions, grouped into four blocks, were generated to constitute the final checklist. An explanation for the inclusion of each item was provided and some examples found in the bibliographic search were cited. LIMITATIONS AND REASONS FOR CAUTION: We consider that not all items are equally applicable to all study designs, and so the hypothetical results are not comparable. For that reason, a score would not be fair to critically appraise a study. This checklist is presented as an instrument for appraising SEMQUAs and therefore remains open to constructive criticism. It will be further developed in the future, in parallel with the continuing evolution of SEMQUAs. WIDER IMPLICATIONS OF THE FINDINGS: The final configuration of the SEMQUA is in the form of a checklist, and includes the items generally considered to be essential for the proper development of a SEMQUA. The final checklist produced has various areas of application; for example, it would be useful for designing and constructing a SEMQUA, for reviewing a paper on the question, for educational purposes or as an instrument for appraising the quality of research articles in this field. STUDY FUNDING/COMPETING INTEREST(S): None.

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.

ESHRE special interest group for andrology basic semen analysis course: a continued focus on accuracy, quality, efficiency and clinical relevance.

ESHRE has been running courses for basic semen analysis since 1994 and course material has been updated regularly in response to new findings and publications. Following publication of the 5th edition of the WHO laboratory manual, entitled WHO Laboratory Manual for the Examination and Processing of Human Semen (WHO5), the Subcommittee for training of the ESHRE Special Interest Group for Andrology evaluated potential amendments to its course. In respect of the updated ESHRE course, there are eight particular areas of discourse that are reviewed (i) maintaining the four-class differential motility count allowing distinction between rapid and slow progressive sperm for assisted reproduction technology. (ii) Maintaining the four-category assessment for sperm morphology with calculation of the teratozoospermic index. (iii) Continuing to advocate the use of three categories of results: 'normal', 'borderline' and 'abnormal' with respect to the clinical interpretation of the data. (iv) Presenting clear and unequivocal methods for performing assessments e.g. morphology. (v) Correcting the inconsistencies in WHO5, some of which are actually erroneous. (vi) Reducing the requirements for substantial extra work for what are unestablished improvements in accuracy and/or precision in the final results. (vii) Presentation of logical methods of sperm preparation. (viii) Discussion of the suddenly changed limits between fertile and subfertile men.

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.

Development of a novel home sperm test.

BACKGROUND: The majority of men find the production of a semen sample an embarrassing and stressful experience. Consequently, the availability of an over-the-counter home sperm test, which would reliably and accurately allow the patient to obtain an assessment of fertility potential at their convenience, would be a major benefit. Our objective was to develop and evaluate a home sperm test that provides a visual estimate of the concentration of progressively motile sperm in a semen sample. METHODS: Three particular challenges are described (i) developing a visualization system; (ii) optimization of the detection limit; and (iii) controlling variation due to changes in ambient temperature. The accuracy of the device was tested against two reference methods: computer-assisted sperm analysis (CASA) and a hyaluronate migration test (HMT). RESULTS: In 129 semen samples, where both reference methods agreed (positive or negative), the accuracy of the device was 95%. The observed likelihood ratio of 8.8 indicated that a sample showing a red line in the device was over eight times more likely to have a positive (normal) result in CASA and HMT than a sample without a red line. CONCLUSIONS: The final device provides a visual estimate of the concentration of progressively motile sperm in a semen sample using a test that is completed within approximately 1 h of production of the sample and can be used by the man in the comfort of his own home.

Loss of an intrinsic capacity for human sperm chromatin decondensation.

Chromatin decondensation of human ejaculated spermatozoa was studied in vitro, at various points of time after ejaculation, by sperm exposure to the detergent sodium dodecyl sulphate (SDS) containing zinc chelating EDTA. Within 5 min after ejaculation EDTA revealed a capacity for decondensation in 90% of the spermatozoa. This sperm capacity decreased rapidly upon storage. The results support the concept that the capacity to decondense is a normal property of freshly ejaculated spermatozoa and that this property may be rapidly lost. The loss is most probably due to an inability of thiol groups to take part in a thiol-disulphide exchange in the sperm chromatin. A loss of functional thiols may hinder a capacity for chromatin decondensation inherent to the spermatozoon. A loss of thiols due to oxidation, that is, surplus S-S bridge formation, may also delay hypothetical extrinsic S-S cleaving factors in the ooplasm. In either case, the complete and non-delayed sperm chromatin decondensation in the ovum may be hindered. This may result in the abnormal embryonic development observed in ova fertilized with aged spermatozoa.

Zinc preserves an inherent capacity for human sperm chromatin decondensation.

The effect of zinc on the nuclear chromatin decondensation ability of human, ejaculated spermatozoa was studied by exposing washed spermatozoa to the detergent sodium dodecyl sulphate (SDS) before and after sperm storage. Treatment with EDTA increased the proportion of decondensing spermatozoa before storage. Zinc supplementation before storage reversibly inhibited spontaneous decondensation as well as EDTA-enhanced decondensation. Treatment with EDTA before storage decreased the proportion of spermatozoa decondensing after storage. Zinc supplementation during storage reduced the decrease in the proportion of spermatozoa decondensing after storage. Two effects of zinc were observed: one immediate effect (reversible inhibition of the decondensation) and one long-term effect (protection of the intrinsic capacity for decondensation during storage). Both effects may be explained by a zinc-thiol interaction in the chromatin. Spermatozoal zinc is suggested to protect an inherent capacity for decondensation, thereby helping to extend the functional life-span of the ejaculated spermatozoon.

Influence of seminal vesicular fluid on the zinc content of human sperm chromatin.

Chromatin zinc was studied using X-ray microanalysis of spermatozoa obtained from split-ejaculate fractions. Chromatin zinc, expressed as intensity ratio between zinc and sulphur (Zn/S), was unrelated to seminal zinc concentration, but was related inversely to markers of seminal vesicular secretion (fructose concentration and the proportion of zinc bound to ligands of seminal vesicular origin). It is concluded that the content of zinc in sperm chromatin can be reduced by the action of zinc ligands of seminal vesicular origin. An abnormally high contribution of seminal vesicular fluid to sperm-rich fractions of the ejaculate thus creates a risk of depleting chromatin zinc and thereby impairing zinc-dependent chromatin stability.

Importance of zinc for human sperm head-tail connection.

Head-tail detachment of ejaculated human spermatozoa was studied with phase contrast microscopy. The frequency of head-tail detachment was assessed after sperm exposure to the anionic detergent sodium dodecyl sulfate (SDS) for 60 min at 22 degrees C. Decapitation was enhanced by EDTA. Zinc reversibly inhibited native as well as EDTA induced head-tail disconnection. Still a certain proportion of spermatozoa were resistant to EDTA-treatment and this proportion significantly increased upon 24 h of saline storage. The development of EDTA resistance was enhanced by EDTA treatment before storage. A physiological role for zinc as a preserver of an inherent mechanism for head-tail detachment is suggested.

Ejaculatory sequence in men with low sperm chromatin-zinc.

The composition of seminal plasma in the sperm-rich split ejaculate fraction was studied in a group of men with a low zinc content in their sperm chromatin, to evaluate the availability of zinc at ejaculation. Men with low-chromatin zinc had, in the sperm-rich split-ejaculate fraction, high amounts of seminal-vesicular fluid, a low zinc:fructose molar ratio, and a high percentage of zinc bound to high molecular weight ligands of seminal vesicular origin (HMW-Zn). This indicates premature admixture of vesicular fluid at ejaculation. It is suggested that the zinc:fructose molar ratio and HMW-Zn in the sperm-rich fractions could be used as a measure of the availability of zinc in seminal plasma.

Sperm chromatin stability and zinc binding properties in semen from men in barren unions.

Sperm chromatin stability and zinc binding properties were studied in semen samples from 115 men living in barren unions. Of these men, 26% had a high proportion of swelling sperm, i.e. less than 80% sperm with stable chromatin after exposure to the detergent sodium dodecyl sulphate. From 2-67% of seminal zinc was bound to high molecular weight ligands of vesicular origin (HMW). This shows that, among infertile men, liquefied seminal plasma has huge variations in zinc chelating properties. The relationship between prostatic palpatory status, the proportion of abnormal sperm, the percentage zinc bound to HMW (HMW-Zn), the time between ejaculation and analysis and chromatin stability were studied. Samples with low chromatin stability were found more frequently in men with low HMW-Zn levels in semen. The proportion of stable sperm decreased in samples with prolonged exposure to seminal plasma. Neither the proportion of stable sperm heads nor the percentage zinc bound to HMW could be used to predict the future chances of the infertile men fathering children when studied 15-180 min after ejaculation. To differentiate between initial zinc-dependent stability and superstability developed in seminal plasma, other more sensitive methods must be developed.

Evaluation of the one-step eosin-nigrosin staining technique for human sperm vitality assessment.

BACKGROUND: The one-step eosin-nigrosin staining technique for assessment of sperm vitality was developed in the 1950s for various mammalian species. Although commonly used on human sperm in semen, a validation for this use has not previously been published. METHODS: The technique was evaluated on 1235 consecutive semen samples. RESULTS: The one-step eosin-nigrosin staining technique gave valid results when evaluated with sperm motility data obtained according to World Health Organization standard (1992, 1999). The mean for the sums of stained (i.e. supposedly dead) and motile sperm using the one-step eosin-nigrosin technique was 91% (SD +/- 10%). The distribution of sums for percentage stained and percentage motile sperm was similar, regardless of whether the samples had many or few dead sperm. CONCLUSIONS: Standardization and quality control of basic semen analysis demands robust, reliable and simple techniques that are easy to learn, and easy to continue to perform in the same way. The one-step eosin-nigrosin technique does not need negative phase contrast optics but can be run with ordinary bright-field microscopy. Since it also includes fewer methodological steps to control, it seems preferable in terms of standardization and quality control management. It should therefore be recommended in the basic semen analysis when sperm vitality is to be assessed.

Sperm nuclear zinc, chromatin stability, and male fertility.

Zinc excreted from the human prostate secures a high content of zinc in the sperm nucleus and contributes to the stability of the quaternary structure of the chromatin. After ejaculation, in vitro, a second type of stability, most probably involving disulfide-bridge crosslinks, supersedes the zinc-dependent stability. Normally, the nucleus of the ejaculated spermatozoon remains stable, i.e., it does not decondense when exposed to a detergent (e.g., sodium dodecyl sulfate - SDS), whereas a spermatozoon which has been exposed to a zinc-chelating medium becomes destabilized and decondenses in SDS. Spontaneous decondensation in SDS, i.e., without prior treatment with zinc-chelators, occurs among many spermatozoa from some infertile men, especially men with impaired secretory function of the prostate. This indicates that spontaneously decondensing spermatozoa have an inadequate content of zinc at ejaculation. Here, zinc in the sperm nucleus and chromatin stability was studied in semen samples from a group of men living in marriages with hitherto unexplained cause for infertility, and a group of fertile donors, who participated in an insemination program. Sperm nuclear zinc was studied with X-ray microanalysis and chromatin stability was assessed as percentage spermatozoa with stable sperm heads after exposure to SDS. Fertile donors had higher content of zinc in the sperm nuclei and had also higher proportions spermatozoa with a stabilized chromatin, than had the men living in infertile marriages.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear zinc in human epididymal and ejaculated spermatozoa.

Sperm nuclear zinc content (expressed as zinc/phosphorus and zinc/sulphur ratios) was determined with X-ray microanalysis in individual, air-dried, epididymal spermatozoa from elderly men, and in ejaculated spermatozoa from healthy donors. Ejaculated sperm nuclei contained (also after treatment with sodium dodecyl sulphate) more zinc than epididymal spermatozoa. The results indicate that the human spermatozoon accumulates zinc from the prostatic fluid upon ejaculation.

The human sperm nucleus takes up zinc at ejaculation.

Ejaculated and vasal sperm were obtained from men referred for vasectomy, and sperm nuclear elements were determined by X-ray microanalysis. Sperm head zinc concentrations, expressed as the ratio Zinc to Sulphur, were significantly higher in ejaculated than in vasal sperm. A physiological sperm nuclear zinc uptake is discussed in relation to sperm chromatin decondensation.

Semen analysis and external quality control schemes for semen analysis need global standardization.

One semen analysis laboratory [the Institute of Reproductive Medicine (IRM), Münster, Germany] was enrolled in three external quality assurance programmes in Europe (United Kingdom External Quality Assurance Scheme, European Academy of Andrology, European Society of Human Reproduction) that control for the assessment of sperm concentration, sperm motility and sperm morphology. Agreement between the participating laboratory and the sperm concentrations designated by all three programmes was good. Disagreement between two quality control (QC) programmes providing motility assessment was particularly noticeable in the distinction between motility grades a and b. For the identification of normal sperm morphology, marked differences between the standards set by different QC programmes were apparent. One programme was stricter in its categorization of normal forms, such that an overestimation of normal forms was diagnosed at IRM, whereas agreement with the other programmes was achieved. Variation of results from technicians in the IRM internal QC programme was <13%. The discrepancies between external quality control (EQC) centres demonstrated here are challenging problems to be overcome partly by the andrology laboratories and partly by the providers of EQC services. The introduction of objective, computer-aided sperm assessment in providing designated values may help to rectify this situation. Until this is achieved, EQC programmes should develop an internal programme to monitor their materials and methods for assessment.

Removal of zinc from subcellular regions of human spermatozoa by EDTA treatment studied by X-ray microanalysis.

The distribution of zinc in the head, neck and mid-piece of human ejaculated spermatozoa was investigated with energy-dispersive X-ray microanalysis. EDTA treatment within 1 h after ejaculation caused removal of 80-90% of the zinc in these regions. Storage for 24 h in own seminal plasma did not alter sperm zinc content. However, these aged spermatozoa only lost 35% of their zinc content on EDTA treatment. The stronger binding of zinc, occurring upon storage in seminal plasma, developed in all 3 parts of the cell investigated. These results are discussed in the light of the role of zinc in nuclear chromatin decondensation ability and sperm head-tail connection.

Cadmium interacts with the zinc-dependent stability of the human sperm chromatin.

Zinc normally participates in the stabilization of the chromatin of human spermatozoa, which have a high content of zinc after ejaculation. Sperm chromatin, depleted of zinc with EDTA, regained stability in the detergent SDS after exposure to Cd2+ in vitro. This effect was reversible with EDTA, but albumin could not reverse the stabilization caused by Cd2+ to the same extent as it reversed the stabilization caused by Zn2+.

Seminal fluid from men with agenesis of the Wolffian ducts: zinc-binding properties and effects on sperm chromatin stability.

Zinc-binding properties were studied in 'prostatic fluid', i.e. in seminal plasma from patients with agenesis of the Wolffian ducts, and in split-ejaculate fractions dominated by seminal vesicular fluid. The effect of seminal fluid, with different zinc-binding properties, on the stability of zinc-dependent sperm chromatin was assessed by exposing sperm to 1% sodium dodecyl sulphate (SDS) for 60 min. Citrate was the only zinc ligand in 'prostatic fluid', as revealed by gel chromatography. Zinc in this fluid enhanced the stability of sperm chromatin. In contrast, the stability of sperm chromatin was decreased in seminal plasma dominated by vesicular fluid. These results are in accordance with the concept that prostatic fluid ensures the appropriate zinc content and stability of sperm chromatin, whereas abundance of vesicular fluid may jeopardize chromatin stability by reducing chromatin zinc content.