Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology.

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

Hypotonic challenge reduces human sperm motility through coiling and folding of the tail.

Human ejaculates collected for in vitro procedures show variably rapid increases in osmolality, depending on enzymatic degradation of compounds. Changes in osmolality can affect cell functions due to the energy consuming processes needed to control cell volume. The aim was to examine the effects of a hypotonic challenge for spermatozoa exposed to increased osmolality. Spermatozoa were selected by density gradient centrifugation and washed in media with different osmolalities. Osmolality was measured by freezing-point depression and sperm velocities by CASA. Swimming pattern observations and assessments of tail morphology of fixed spermatozoa were done with phase contrast microscopy. Increased osmolality did not change the curvilinear velocity (VCL), while decreased osmolality reduced or abolished VCL nonreversibly. For spermatozoa first exposed to 400 mOsm/kg, reversal of osmolality to 290 mOsm/kg reduced the VCL and the average path velocity (VAP) permanently. Hypotonic challenges increased sperm tail coiling and folding in a dose-response pattern. Spermatozoa once adjusted to high osmolality in the liquefied ejaculate are likely to suffer if exposed to a medium with a lower osmolality. For improved success of Assisted Reproductive Technologies (ART), it appears to be important to minimise the duration of sperm exposure to the ejaculate, by early dilution or sperm preparation.

Possible factors influencing post-ejaculatory changes of the osmolality of human semen in vitro.

The human ejaculate is made up of secretions from the different accessory sex glands that empty in sequence at ejaculation. However, the different secretions only mix completely in vitro when the entire ejaculate is collected in a container and handled in the laboratory. At ejaculation, proteins from the seminal vesicles form a gel in the ejaculate and semen cannot be properly analysed and processed until the gel is liquefied. During and after liquefaction, there is continuous enzymatic activities and an ongoing increase in osmolality. The aim of this study was to investigate possible factors that influence the increase in semen osmolality in vitro. Osmolality was measured by freezing-point depression. Prostatic secretion was measured as zinc concentration. The presence of spermatozoa neither influenced the actual measurement of semen osmolality, nor the increase in osmolality. Enzymatic inhibitors reduced the increase in osmolality, and semen dilution prevented any increase in semen osmolality. However, the increase in osmolality covaried with the seminal zinc concentration, indicating that the observed increase was related to factors of prostatic origin. A simple and convenient procedure to reduce the risk for osmotic challenges for spermatozoa during handling for assisted reproductive technologies might be early dilution of semen.

Post-ejaculatory increase in human semen osmolality in vitro.

Spermatozoa prepared in vitro for assisted reproductive technology (ART) encounter other challenges than in vivo. One could be to survive and function despite varying extracellular osmolality. There is a lack of consistent knowledge of human semen osmolality and changes after liquefaction. The aim of this study was to investigate changes in semen osmolality that may occur in vitro after ejaculation and during sperm handling for ART. Osmolality was measured in 86 semen samples by freezing-point depression during storage in vitro at various times and temperatures. The freezing-point depression method was robust and reliable for measuring the osmolality of whole semen as shown by a low variability between repeats. At ejaculation, the osmolality was isotonic to cervical mucus and body fluids. There was a marked increase in osmolality after liquefaction, and the degree of increase varied greatly between samples. Osmolality rose with increasing temperature, and the progressive increase was blocked by denaturising temperature. Spermatozoa in each individual semen sample experienced a highly variable environment in vitro with respect to osmolality. This may be of importance regarding the handling of semen samples for the outcome of ART.

Evolving minimum standards in responsible international sperm donor offspring quota.

An international working group was established with the aim of making recommendations on the number of offspring for a sperm donor that should be allowable in cases of international use of his sperm. Considerations from genetic, psychosocial, operational and ethical points of view were debated. For these considerations, it was assumed that current developments in genetic testing and Internet possibilities mean that, now, all donors are potentially identifiable by their offspring, so no distinction was made between anonymous and non-anonymous donation. Genetic considerations did not lead to restrictive limits (indicating that up to 200 offspring or more per donor may be acceptable except in isolated social-minority situations). Psychosocial considerations on the other hand led to proposals of rather restrictive limits (10 families per donor or less). Operational and ethical considerations did not lead to more or less concrete limits per donor, but seemed to lie in-between those resulting from the aforementioned ways of viewing the issue. In the end, no unifying agreed figure could be reached; however the consensus was that the number should never exceed 100 families. The conclusions of the group are summarized in three recommendations.

The optimal number of offspring per gamete donor.

Our aim was to create a mathematical basis to calculate the risks for unintended matings of consanguineous half-siblings from a donor in a society with approximately 10 million inhabitants. The Curie-Cohen model for calculation of the risk for consanguineous mating was used. When the number of offspring per donor is limited to 10, then the model gives a yearly risk for consanguineous matings below 1%. Thus 10 offspring gives a risk for consanguineous matings of 0.9% per year, or approximately once in every 100 years. The risk increases exponentially: with 15 offspring it exceeds 2% and with 25 it reaches up above 5%.

Structure of chromatin in spermatozoa.

The specialized structure of the sperm chromatin has a dual function - first to protect the DNA from damage during storage and transport to the oocyte, and then to enable a rapid and complete unpacking of the undamaged paternal genome in the ooplasm. It is evident that zinc has a pivotal role in maintaining the structural stability and in enabling a rapid decondensation at the appropriate time. It is important for the sperm chromatin structure that the spermatozoa are ejaculated together with the zinc-rich prostatic secretion. Early exposure to zinc-binding seminal vesicular fluid can deplete the sperm chromatin of zinc and most likely induce surplus formation of disulfide bridges, likely to cause incomplete and delayed decondensation of the sperm chromatin in the oocyte. A premature decrease in sperm chromatin structure stability is likely to increase the risk for damage to the DNA due to increased access to the genome for DNA damaging compounds. The status of the sperm chromatin structure can vary in vitro depending on the exposure to zinc-depleting conditions when spermatozoa are stored in semen after ejaculation. When sperm DNA damage tests are evaluated and validated, it is therefore essential to also take into account the dynamics of zinc-dependent and zinc-independent sperm chromatin stability.

Increased androgen-related comorbidity in adolescents and adults born with hypospadias: A population-based study.

BACKGROUND: Hypospadias is a common congenital malformation often related to the effect of androgens in utero. While hypogonadism is associated with many potential health risks including metabolic and cardiovascular disease, the risk of clinical hypogonadism and comorbidities in men with hypospadias later in life has not been studied. OBJECTIVES: Investigate the risk of hypogonadism and somatic comorbidities in adolescents and men born with hypospadias. MATERIALS AND METHODS: We conducted a population-based cohort study using Swedish registers. Associations between hypospadias and hypogonadism, delayed puberty, metabolic, and cardiovascular disease respectively were estimated using Cox proportional hazards regression. Body measurements from military conscription were analysed in a subpopulation as indicators of growth and cardiometabolic risk. We used sibling comparison analyses to control for familial confounding. RESULTS: Using register data, a total of 2,165,255 men including 9,714 men born with hypospadias were followed from the age of 10 to a maximum of 60 years. We found an association between hypospadias and hypogonadism (Hazard ratio (HR) 3.27, 95% confidence interval (CI) 2.33-4.59) which was more pronounced in proximal hypospadias. Men with hypospadias had shorter average height than their brothers and the general population. We further found an increased risk of delayed puberty (HR 1.49, 95% CI 1.08-2.07), diabetes mellitus type 2 (HR 1.57, 95% CI 1.18-2.09) and cardiovascular disease (HR 1.47, 95% CI 1.27-1.71). DISCUSSION: We found an increased risk of hypogonadism, metabolic and cardiovascular disease in men born with hypospadias, increasing with severity of phenotype, as well as impacted growth. These results indicate discruptions in androgen function past childhood, although some of the associations may be due to other underlying aetiologies. CONCLUSION: Hypospadias is associated with an increased risk of androgen-related comorbidity in adolescence and adulthood. We suggest that this can be considered clinically, while further research is needed, especially in older populations.

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.

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 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.

Sexual function in men treated for testicular cancer.

INTRODUCTION: Testicular germ cell cancer (TGCC) patients may be at risk of developing sexual dysfunction after treatment. AIM: The aim of this study was to assess the prevalence of sexual dysfunctions in TGCC patients 3 to 5 years after treatment, and relate findings to biochemical hypogonadism, treatment intensity, and the expected prevalence in the Swedish male population. METHODS: A questionnaire study on 129 consecutive TGCC patients 3 to 5 years post-treatment was performed. Comparators were an age-matched nationally representative group of men (N = 916) included in a study on sexual life in Sweden. MAIN OUTCOME MEASURES: Sexual functions (including erectile dysfunctional distress), time since last intercourse, sexual satisfaction, and experience of sexological treatment seeking were assessed using the same questions used in the epidemiological study on sexual life in Sweden. The findings in TGCC patients were correlated to biochemical signs of hypogonadism and type of oncological treatment: Surveillance, adjuvant chemotherapy, adjuvant radiotherapy, or standard doses of chemotherapy. RESULTS: A higher proportion of TGCC patients than comparators were likely to report low sexual desire (odds ratio [OR] 6.7 [95% confidence interval {CI} 2.1-21]) as well as erectile dysfunction (OR 3.8 [95% CI 1.4-10]). No significant differences were observed regarding erectile dysfunctional distress, change of desire over time, interest in sex, premature or delayed ejaculation, time since last intercourse, need for or receiving sexual advice, or sexual satisfaction. Hypogonadism did not predict erectile dysfunction (OR 1.1 [95% CI 0.26-4.5]) or low sexual desire (OR 1.2 [95% CI 0.11-14]). Treatment modality had no obvious impact on sexual function. CONCLUSION: Men treated for testicular cancer had higher risk of having low sexual desire and erectile dysfunction 3 to 5 years after completion of therapy than comparators. These sexual dysfunctions were not significantly associated with treatment intensity or hypogonadism.

Cohesin protein SMC1 is a centrosomal protein.

Structural maintenance of chromosome protein 1 (SMC1) is well known for its roles in sister chromatid cohesion and DNA repair. In this study, we report a novel centrosomal localization of SMC1 within the cytoplasm in a variety of mammalian cell lines. We showed that SMC1 localized to centrosomes throughout the cell cycle in a microtubule-independent manner. Biochemically, SMC1 was cofractionated with the centrosomal protein gamma-tubulin in centrosomal preparation. Immunohistochemistry and immunoelectron microscopy performed on mouse tissue sections revealed that SMC1 antibody strongly labeled the base of cilia in ciliated epithelia, where basal bodies were located. Furthermore, we showed that SMC1 was associated with both centrioles of a centrosome at G0/G1 stage of the cell cycle. These results demonstrate that SMC1 is a centrosomal protein, suggesting possible involvement of SMC1 in centrosome/basal body-related functions, such as organization of dynamic arrays of microtubules and ciliary formation.

Normal sexual development and fertility in testatin knockout mice.

The testatin gene was previously isolated in a screen focused on finding novel signaling molecules involved in sex determination and differentiation. testatin is specifically upregulated in pre-Sertoli cells in early fetal development, immediately after the onset of Sry expression, and was therefore considered a strong candidate for involvement in early testis development. testatin expression is maintained in the adult Sertoli cell, and it can also be found in a small population of germ cells. Testatin shows homology to family 2 cystatins, a group of broadly expressed small secretory proteins that are inhibitors of cysteine proteases in vitro but whose in vivo functions are unclear. testatin belongs to a novel subfamily among the cystatins, comprising genes that all show expression patterns that are strikingly restricted to reproductive tissue. To investigate a possible role of testatin in testis development and male reproduction, we have generated a mouse with targeted disruption of the testatin gene. We found no abnormalities in the testatin knockout mice with regard to fetal and adult testis morphology, cellular ultrastructure, body and testis weight, number of offspring, spermatogenesis, or hormonal parameters (testosterone, luteinizing hormone, and follicle-stimulating hormone).

Male reproductive health statement (XIIIth international symposium on Spermatology, may 9th-12th 2018, Stockholm, Sweden.

On the occasion of the XIIIth International Symposium on Spermatology held from 9 to 13 May 2018 in Stockholm (Sweden), participants (guest speakers and audience) collectively felt the need to make a public statement on the general issue of male reproductive health. Our intention is to raise awareness of what we believe is a neglected area of research despite alarming situations around the world. The disclosure strategy desired by the co-authors is to bring it to the attention of the greatest number partly by considering co-publication in the various periodicals dealing with Reproductive Biology and Andrology. BaCA's editorial office accepted this mission and found it natural that our periodical, the official journal of the French Andrology Society (SALF), should carry this message.

A l'occasion du XIII eme Symposium international sur la Spermatologie qui s'est. tenu du 9 au 13 Mai 2018 à Stockholm (Suède), les participants (orateurs invités et l'auditoire) ont ressenti collectivement le besoin de faire une déclaration publique sur la question générale de la santé reproductive masculine. Notre intention est. de mieux faire connaître ce que nous pensons être un domaine de recherche négligé malgré des situations alarmantes dans le monde entier. La stratégie de divulgation souhaitée par les co-auteurs est. de le porter à l'attention du plus grand nombre en envisageant pour partie une co-publication dans les différents périodiques traitant de Reproduction et d'Andrologie. Le bureau éditorial de BaCA, a accepté cette mission et a trouvé naturel que notre périodique, journal officiel de la Société d'Andrologie en Langue Française (SALF) porte ce message.

Disomy 21 in spermatozoa and the paternal origin of trisomy 21 Down syndrome.

BACKGROUND: Trisomy 21 Down syndrome is the most common genetic cause for congenital malformations and intellectual disability. It is well known that in the outstanding majority of cases the extra chromosome 21 originates from the mother but only in less than 10 % from the father. The mechanism underlying this striking difference in parental origin of Trisomy 21 Down syndrome is still unknown. However, it seems likely that the main reason is a much higher stringency in the elimination of any trisomy 21 cells during fetal testicular than ovarian development. We have here focussed attention on the paternal gametic output, i.e. the incidence of disomy 21 in spermatozoa. RESULTS: We have used fluorescence in situ hybridisation (FISH) to determine the copy number of chromosome 21 in spermatozoa from 11 men with normal spermiograms. Due to the well-known risk of false positive and false negative signals using a single FISH probe, we have applied two chromosome 21q probes, and we have added a chromosome 18-specific probe to allow differentiation between disomy 21 and diploidy. Analysing a total number of 2000 spermatozoa per case, we documented an average incidence of disomy 21 at 0.13 %, with a range of 0.00-0.25 % and a SD of 0.08. There was no indication of diploidy in this cohort of 22,000 sperm. CONCLUSION: Numerous previous studies on the incidence of disomy 21 in sperm have been published, using FISH. As far as we are aware, none of these have applied more than a single chromosome 21-specific probe. Accepting our mean of 0.13 % of disomy 21, and providing there is no selective fertilisation capability of disomy 21 sperm in relation to the normal, we conclude that around 1 in 800 conceptions is expected to be trisomic for chromosome 21 of paternal origin. Bearing in mind that the maternal origin likely is at least 10 times more common, we tentatively propose that around 1 in 80 oocytes in the maternal ovarian reserve may be disomy 21. One reason for this discrepancy may be a more stringent selection against aberrant chromosome numbers during spermatogenesis than oogenesis. Further work is required to determine the relevant stages of spermatogenesis at which such a selection may take place.

THE INTRINSIC MECHANISM OF CHROMATIN DECONDENSATION AND ITS ACTIVATION IN HUMAN SPERMATOZOA.

In vitro decondensation of human sperm chromatin induced by the activation of an intrinsic mechanism was studied by light microscopy, scanning and transmission electron microscopy. Morphological evidence was provided to support the concept that this mechanism is essential for the chromatin decondensation occurring in vivo. Prostatic zinc is hypothesized to preserve this potential decondensation ability from oxidative destruction, by reversibly binding to free thiol-groups. The unique occurrence of disulphide-stabilized structures in eutherian spermatozoa may serve to protect the spermatozoon from structural degradation by its own proteolytic activity during the relatively slow passage through the eutherian egg investments.

Left-sided grade 3 varicocele may affect the biological function of the epididymis.

OBJECTIVE: Varicocele is present in approximately 30-40% of men evaluated for infertility and in 10-20% of the general male population, but the association between varicocele, compromised semen quality and male infertility remains unclear. This indicates that varicocele may impair spermatogenesis or in some other way mitigate sperm quality. Better understanding of criteria for the selection of men who will benefit from varicocele repair would improve the clinical management of men with impaired semen quality and varicocele. MATERIAL AND METHODS: To further understand the effect of varicocele repair by embolization in men with grade 3 varicocele, 50 men referred for infertility with varicocele were evaluated before and after embolization. RESULTS: There was a significant improvement in total sperm count (25.5 ± 4.7 million to 78 ± 11 million, p < 0.001) and sperm motility (slowly and rapidly progressive) (31.5 ± 2.9% to 45 ± 2.5%, p < 0.001) after spermatic vein embolization, comparing baseline to 3 month post-treatment follow-up data. Furthermore, alpha-glucosidase (specific epididymis-derived protein) increased from 61.7 ± 5.7 U to 84.7 ± 7.0 U (p < 0.05) and sperm droplets decreased from 14.2 ± 1.5% to 11.0 ± 1.0% (p < 0.05). CONCLUSION: The results indicate improved epididymal function and suggest that left-sided grade 3 varicocele may affect the epididymis in addition to effects on the testis.

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 Zn(2+). It is suggested that the chromatin stability of the ejaculated human spermatozoon is rapidly reversible due to the dual function of Zn(2+) 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 Zn(2+) 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.

Male infertility after mesh hernia repair: A prospective study.

BACKGROUND: Several animal studies have raised concern about the risk for obstructive azoospermia owing to vasal fibrosis caused by the use of alloplastic mesh prosthesis in inguinal hernia repair. The aim of this study was to determine the prevalence of male infertility after bilateral mesh repair. METHODS: In a prospective study, a questionnaire inquiring about involuntary childlessness, investigation for infertility and number of children was sent by mail to a group of 376 men aged 18-55 years, who had undergone bilateral mesh repair, identified in the Swedish Hernia Register (SHR). Questionnaires were also sent to 2 control groups, 1 consisting of 186 men from the SHR who had undergone bilateral repair without mesh, and 1 consisting of 383 men identified in the general population. The control group from the SHR was matched 2:1 for age and years elapsed since operation. The control group from the general population was matched 1:1 for age and marital status. RESULTS: The overall response rate was 525 of 945 (56%). Method of approach (anterior or posterior), type of mesh, and testicular status at the time of the repair had no significant impact on the answers to the questions. Nor did subgroup analysis of the men ≤40 years old reveal any significant differences. CONCLUSION: The results of this prospective study in men do not support the hypothesis that bilateral inguinal hernia repair with alloplastic mesh prosthesis causes male infertility at a significantly greater rate than those operated without mesh.