Citation Rodríguez‐Martínez H, Kvist U, Ernerudh J, Sanz L, Calvete JJ. Seminal Plasma Proteins: What Role Do They Play? Am J Reprod Immunol 2011; 66 (Suppl. 1): 11–22Problem  Semen is a heterogenous and complex cell suspension in a protein‐rich fluid with different functions, some of them well known, others still obscure.Method of study  This paper reviews, comparatively, our current knowledge on the growing field of proteomics of the SP and its relevance in relation to the in vivo situation, for the sake of reproductive biology, diagnostics and treatment.Results  Ejaculated spermatozoa, primarily bathing in cauda epididymal fluid, are (in vitro) bulky, exposed to most, if not all, secretions from the accessory sexual glands. In vivo, however, not all spermatozoa are necessarily exposed to all secretions from these glands, because sperm cohorts are delivered in differential order and bathe in seminal plasma (SP) with different concentrations of constituents, including peptides and proteins. Proteins are relevant for sperm function and relate to sperm interactions with the various environments along the female genital tract towards the oocyte vestments. Specific peptides and proteins act as signals for the female immune system to modulate sperm rejection or tolerance, perhaps even influencing the relative intrinsic fertility of the male and/or couple by attaining a status of maternal tolerance towards embryo and placental development.Conclusions  Proteins of the seminal plasma have an ample panorama of action, and some appear responsible for establishing fertility.

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.

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 group's 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.

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.

This practical, extensively illustrated handbook covers the procedures that are undertaken in andrology and ART laboratories to analyse and assess male-factor infertility, and to prepare spermatozoa for use in assisted conception therapy. The content is presented as brief, authoritative overviews of the relevant biological background for each area, plus detailed, step-by-step descriptions of the relevant analytical procedures. Each technical section includes pertinent quality control considerations, as well as the optimum presentation of results. In addition to the comprehensive 'basic' semen analysis, incorporating careful analysis of sperm morphology, the handbook provides established techniques for the use of computer-aided sperm analysis and sperm functional assessment. Throughout the handbook the interpretation of laboratory results in the clinical context is highlighted, and safe laboratory practice is emphasized. It is an invaluable resource to all scientists and technicians who perform diagnostic testing for male-factor infertility.

Open testicular biopsy is a classic method of investigation in men with azoospermia. Recently, percutaneous needle biopsy of the testis has been used in attempts to obtain material for histopathological diagnosis in such cases and to retrieve spermatozoa for intracytoplasmic sperm injection (ICSI). To determine whether a 19 gauge (G) and a 21G butterfly needle could be used for percutaneous aspiration of testicular tissue to determine the presence of mature spermatids and assess spermatogenesis, 10 patients (16 testes) and 12 patients (17 testes) underwent 19G or 21G needle biopsy respectively, immediately followed by open testicular biopsy, with both procedures under local anaesthesia. Biopsy with each needle size was compared with open biopsy. With the 19G needle, in the 14 cases where material was obtained there was full agreement with open biopsy regarding the presence or absence of mature spermatozoa, whereas with the 21G needle only nine of the 13 biopsies yielding material were predictive in this respect. Each needle size correlated poorly with open biopsy regarding evaluation of spermatogenesis. We conclude that percutaneous biopsy with a 19G butterfly needle is a quick and reliable method for demonstrating spermatozoa for ICSI. But for a detailed histopathological diagnosis, however, the needle biopsies gave poor results, whereas the material from the open testicular biopsies was assessable.

Nuclear chromatin decondensation (NCD) of human ejaculated spermatozoa exposed to sodium dodecyl sulphate (SDS) has been studied. A high proportion of NCD reacting spermatozoa was only found in semen samples with a relatively low activity of some prostatic factor(s) (i.e. zinc/fructose ratio below 0.18) in the seminal plasma. Exposure to SDS for one h was found sufficient to reveal the main proportion of spermatozoa undergoing NCD in such a solution. Addition of seminal plasma with an apparently normal composition to a sperm population with a high NCD reactivity restored the sperm SDS resistance to normal, i.e. blocked the NCD-response. Other results indicated that NCD reactivity was decreased or abolished upon prolonged storage of the spermatozoa in the seminal plasma. The various results indicated that some factor(s) in the seminal plasma can preserve the nuclear chromatin stability of human spermatozoa and that this factor most likely is of prostatic origin.

Nuclear chromatin decondensation (NCD) of ejaculated human spermatozoa was studied in vitro. Spermatozoa subjected to membrane disintegration induced by the detergent sodium dodecyl sulphate (SDS) were found to undergo NCD if previously or afterwards treated with substances known to deplete the spermatozoa of zinc (albumin and EDTA). Zn2+, but not other, "prostatic" cations (Ca2+, Mg2+), inhibited the experimentally induced NCD and the NCD of spermatozoa from men with impaired prostatic function. It is suggested that the human spermatozoon has an intrinsic mechanism for NCD, that is preserved by temporary zinc inhibition and might be reactivated by zinc removal within the female genital tract.