Lars Björndahl

Sperm DNA: organization, protection and vulnerability: from basic science to clinical applications—a position report

This article reports the results of the most recent in a series of EHSRE workshops designed to synthesize the current state of the field in Andrology and provide recommendations for future work (for details see Appendix). Its focus is on methods for detecting sperm DNA damage and potential application of new knowledge about sperm chromatin organization, vulnerability and repair to improve the diagnosis and treatment of clinical infertility associated with that damage. Equally important is the use and reliability of these tests to identify the extent to which environmental contaminants or pharmaceutical agents may contribute to the incidence of sperm DNA damage and male fertility problems. A working group (for workshop details, see Appendix) under the auspices of ESHRE met in May 2009 to assess the current knowledgebase and suggest future basic and clinical research directions. This document presents a synthesis of the working groups understanding of the recent literature and collective discussions on the current state of knowledge of sperm chromatin structure and function during fertilization. It highlights the biological, assay and clinical uncertainties that require further research and ends with a series of 5 key recommendations.

Human sperm chromatin stabilization: a proposed model including zinc bridges

The primary focus of this review is to challenge the current concepts on sperm chromatin stability. The observations (i) that zinc depletion at ejaculation allows a rapid and total sperm chromatin decondensation without the addition of exogenous disulfide cleaving agents and (ii) that the human sperm chromatin contains one zinc for every protamine for every turn of the DNA helix suggest an alternative model for sperm chromatin structure may be plausible. An alternative model is therefore proposed, that the human spermatozoon could at ejaculation have a rapidly reversible zinc dependent chromatin stability: Zn(2+) stabilizes the structure and prevents the formation of excess disulfide bridges by a single mechanism, the formation of zinc bridges with protamine thiols of cysteine and potentially imidazole groups of histidine. Extraction of zinc enables two biologically totally different outcomes: immediate decondensation if chromatin fibers are concomitantly induced to repel (e.g. by phosphorylation in the ooplasm); otherwise freed thiols become committed into disulfide bridges creating a superstabilized chromatin. Spermatozoa in the zinc rich prostatic fluid (normally the first expelled ejaculate fraction) represent the physiological situation. Extraction of chromatin zinc can be accomplished by the seminal vesicular fluid. Collection of the ejaculate in one single container causes abnormal contact between spermatozoa and seminal vesicular fluid affecting the sperm chromatin stability. There are men in infertile couples with low content of sperm chromatin zinc due to loss of zinc during ejaculation and liquefaction. Tests for sperm DNA integrity may give false negative results due to decreased access for the assay to the DNA in superstabilized chromatin.

Sequence of ejaculation affects the spermatozoon as a carrier and its message

It is a widespread misunderstanding that the human ejaculation produces a homogeneous fluid with constant environment for the spermatozoa. On the contrary, spermatozoa are mainly expelled together with the zinc-rich prostatic fluid in the first ejaculation fractions. In nature, these spermatozoa most likely enter the cervical mucus before any significant contact with the later, sperm hostile fractions takes place. Spermatozoa collected in the laboratory for diagnosis, treatment or research purposes face challenges in the form of light, high oxygen concentration, low carbon dioxide concentration, increased pH, extreme variations in osmolarity, and changed bioavailability of zinc and calcium in the man-made laboratory product called seminal plasma. Possible future implications of these unphysiological conditions are discussed in relation to assisted reproduction and sperm research.

Zinc in Sperm Chromatin and Chromatin Stability in Fertile Men and Men in Barren Unions

The stability and the content of zinc of the chromatin were studied in spermatozoa from ten men with unexplained infertility, and in spermatozoa from five fertile donors. A positive relation was found between zinc in sperm nuclei (X-ray microanalysis) and the resistance of the chromatin to decondense in sodium dodecylsulfate (SDS). The infertile men had lower degree of sperm chromatin stability and lower sperm zinc content than the fertile donors. A subgroup of the infertile men, which all had minor clinical signs of prostatic inflammatory reaction, had the lowest content of zinc in the chromatin and the lowest degree of chromatin stability. A low content of nuclear zinc would impair the structural stability of the chromatin and thereby increase the vulnerability of the male genome. This mechanism may be one explanation for the reduced fertility of the men with minor inflammation of the prostate.

‘How to count sperm properly’: checklist for acceptability of studies based on human semen analysis

STUDY QUESTION Can a tool be developed for authors, reviewers and editors of the ESHRE Journals to improve the quality of published studies which rely on semen analysis data? SUMMARY ANSWER A basic checklist for authors, reviewers and editors has been developed and is presented. WHAT IS KNOWN ALREADY Laboratory work which includes semen analysis is burdened by a lack of standardization. This has significant negative effects on the quality of scientific and epidemiological studies, potential misclassification of patients and the potential to impair clinical treatments/diagnoses that rely on accurate semen quality information. Robust methods are available to reduce laboratory error in semen analysis, inducing adherence to World Health Organization techniques, participation in an external quality control scheme and appropriate training of laboratory personnel. However, journals have not had appropriate systems to assess if these methods have been used. STUDY DESIGN, SIZE, DURATION After discussion at a series of Associate Editor Meetings of the ESHRE Journals the authors of the present text were asked to propose a tool for authors, reviewers and editors of the ESHRE Journals to ensure a high quality assessment of submitted manuscripts which rely on semen analysis data, including a detailed verification of the relevance and the quality of the methods used. PARTICIPANTS/MATERIALS, SETTING, METHODS N/A. MAIN RESULTS AND THE ROLE OF CHANCE A basic checklist for authors, reviewers and editors is presented. The checklist contains key points which should be considered by authors when designing studies and which provides essential information for when the submitted manuscript is evaluated. For published articles the answers in the checklist are suitable to be available as supplementary data, which will also reduce the space necessary for technical details in the printed article. LIMITATIONS, REASONS FOR CAUTION Guidelines such as these should not be used uncritically. It is therefore important that submitting authors, in situations where their study does not comply with the basic requirements for semen analysis, not only explain all methodological deviations but also declare the level of uncertainty in their analyses and how it complies with, or might confound, the aims of the study. WIDER IMPLICATIONS OF THE FINDINGS The fundamental importance of appropriate and robust methodology to facilitate advances in scientific understanding and patient management and treatment, is now accepted as being paramount. Use of the semen analysis checklist should be part of this process, and when completed and signed by the corresponding author at the time of submitting a manuscript should result in greater transparency, and ultimately uniformity. It is hoped that this initiative will pave the way for wider adoption of the methodology/reporting by other biomedical, epidemiological and scientific journals, and ultimately become the standard of practice for papers reporting semen analysis results obtained in laboratory and clinical andrology. Systems to assist referees, authors and editors to present high quality findings should have a significant impact on the field of reproductive medicine. STUDY FUNDING/COMPETING INTERESTS No funding was obtained for this work. The authors have no competing interests in relation to the present publication and checklist. TRIAL REGISTRATION NUMBER N/A.

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.

The usefulness and significance of assessing rapidly progressive spermatozoa.

It is possible and clinically relevant to distinguish between slow and rapid progressive spermatozoa in basic semen analysis. This is discussed in light of the different purposes of semen analysis for the subfertile couple and the male patient. The two groups of progressive spermatozoa should be distinguished to help ensure that pertinent information available in the semen sample is not neglected.

A model for the importance of zinc in the dynamics of human sperm chromatin stabilization after ejaculation in relation to sperm DNA vulnerability

The focus of this review is the dual functions of the sperm chromatin stabilization and how external factors can interfere with these functions. Zinc depletion after ejaculation allows for rapid and total sperm chromatin decondensation without addition of exogenous disulfide cleaving agents. Zinc depletion without concomitant repulsion of chromatin fibers induces another type of stability that requires exogenous disulfide cleaving agents to allow decondensation. It is essential to extend the present concept, that the sperm chromatin stability is based on disulfide bridges only, to include also the functions of Zn2+. It is suggested that the chromatin stability of the ejaculated human spermatozoon is rapidly reversible due to the dual function of Zn2+ that stabilizes the structure and prevents the formation of excess disulfide bridges by a single mechanism: the formation of zinc bridges involving protamine thiols of cysteine and potentially also imidazole groups of histidine. Extraction of zinc from the freshly ejaculated spermatozoon allows two totally different biological results: (1) immediate decondensation if chromatin fibers concomitantly are induced to repel (e.g., through phosphorylation in the ooplasm) and (2) thiols freed from Zn2+ are available to form disulfide bridges creating a superstabilized chromatin. Spermatozoa in the zinc rich prostatic fluid (in first ejaculated fraction) represent physiology. Extraction of chromatin zinc can be caused by unphysiological exposure of spermatozoa to the zinc chelating and oxidative seminal vesicular fluid, a situation common to most assisted reproductive techniques (ART) laboratories where the entire ejaculate is collected into a single container in which spermatozoa and secretions are mixed during at least 30 min. Some men in infertile couples have low content of sperm chromatin zinc due to loss of zinc during ejaculation and liquefaction. Tests for sperm DNA integrity may give false negative results due to decreased access for the assay to the DNA in superstabilized chromatin.

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.

What is normal semen quality? On the use and abuse of reference limits for the interpretation of semen analysis results

Semen analysis is the corner stone in the basic evaluation of the man in the subfertile couple. The recent WHO manual identifies and recommends robust, controllable methods. However, the distribution of results from recent fathers and men in the general population introduces significant problems. This review discusses the pitfalls with reference limits and the proper use of such limits for the interpretation of semen analysis results. It is critical to understand the statistical basis upon which the assumptions of reference ranges and cut-off limits are built and the importance of standardising methods and practical laboratory training. These are indispensable for qualitative laboratory work as well as for future prospective studies aimed at providing prognostic information for spontaneous pregnancies and successful Assisted Reproductive Techniques (ART) treatment. Proper understanding of biological and physiological variability is also essential for the correct interpretation of semen analysis results. Understanding all the factors influencing semen analyses is of great importance for the development of the entire field of reproductive medicine.