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LABORATORY DIAGNOSTICS OF MALE INFERTILITY. BIOMARKERS AND METHODS. Part II
Abstract
The new paradigm have been arise concerning to samples, markers and methods for assessment of male infertility. Nowadays, a sample “semen” and “sperm sediment” take a key role in examination and management of the subfertile couple. The advantages of sample “semen” such as non-invasive painless procedure as well as the wider range of diagnostic findings by number of methods (cytology, molecular and cultural) highlight the increasly importance of this diagnostic approach in comparison with conventional ones – prostate secretion and urethral scraping which obtained by invasive procedures – by massage and damaging a mucus membrane, respectively. According to the Russian register this review also presents the current situation with the work list of kits and devices for assessment of male infertility and inflammatory of male accessory glands.
Currently, the diagnosis ‘normozoospermia’ independently and in combination with ‘viscosipathy’ does not exclude male infertility and stopping further examination of patient. Moreover, these semiological diagnoses seem to be the most important diagnostic criteria for determining the cause of a significant number of idiopathic infertility in couples who had unsuccessful attempt to conceive from 12 months or more. These clinical cases demonstrate the importance of combine approach to basic semen examination made it much more extended by ancillary technique e.g. sperm sediment cytology (SSC), fragmentation of sperm DNA (DFS) and of level of reactive oxygen species (ROS) estimation.
Over the past two decades scientific and practical research and developments by Russian laboratory medicine specialists have been shown evolutionary and revolutionary achievements in the field of male infertility (MI) diagnostic technologies. Todays the current national guidelines contain information on laboratory research methods which should be reviewed and updated considering a key opinion leader of laboratory medicine. Review contains a detailed analysis of the clinical recommendations of the Ministry of Health of Russian Federation, the standard of medical care, GOST R ISO 23162–2023 and other normative legal documents which based on the algorithm of laboratory evaluation a cause of MI in ejaculate samples.
According to WHO laboratory manual for the examination and processing of human semen, sixth edition (2021) [1], there are a number of techniques available to measure ROS (Reactive Oxygen Species) and DFI levels in semen and each of them has its inherent limitations. According to WHO (2021), two methods for measuring ROS and DFI seems to be attractive due to reliability and simplifying of performance not only in laboratory conditions but also in point-of-care (POC) one. Russian technique utilizes Tetrazolium Nitroblue for measuring ROS which kit named ‘OxySperm (NBT-assay)’ and SCD-assay named ‘HaloSperm’ have been replicated and upgrade methods compared to WHO (2021) and another fundamental scientific papers. Both kits showed not only reproducible results compared with WHO (2021) but also have technological advantages over the previous described methods. This publication presents step-by-step operational procedures of novel Russian kits.
Aim. To analyze the informativeness of combined human semen analysis using a basic semen examination & SSC on the way to clarify the causes of reproductive dysfunction. Materials and methods. We examined 529 samples of semen sediment from men aged 18 – 59 years with infertility and male accessory gland infection (MAGI) between 2019 and 2022. Study was conducted in combination of two laboratory methods like ROSE (rapid onesite examination) which one of them is 1 – 'basic semen examination' and second one is 2 – 'SSC' (‘Basic semen examination+SSC’-ROSE). Results. 15.13 % of all of patients presented normozoospermia, 1.25 % of them have SSC-markers like bacteriospermia (SSC-mB «+»). Outcomes of SSC-reports clarify that 84.69 % of all men have the diagnostic significant SSC-findingsand different combination of it; 69.75 % of them were SSC-mB «+». ‘Normozoospermia’ and ‘SSC-mB’ are associated, thus, the frequency of SSC-mB in the presence of normozoospermia is recorded as follows. Conclusion. The combined approach ‘Basic semen examination+SSC’-ROSE, allows the doctors to exclude a missing in the laboratory algorithm for check-up in male with impaired reproductive function and identifying the causes of idiopathic infertility, the causes of which were mainly positioned as unspecified.
Most sexually transmitted infections (STIs) do not cause significant problems in the carrier, as a result the infection is transmitted easily to a sexual partner and may remain unrecognized with serious consequences for male reproductive health. Currently, the rapidest and most accurate method of STIs diagnosis is a molecular genetic analyses (MGA) for detection of DNA pathogens. The results of MGA do not depend on the cultural properties and morphological characteristics of the microorganism. Non-cultural methods based on a multiplex polymerase chain reaction in real time (PCR-RT) are becoming as a trend in modern laboratory medicine for identifying genes associated with antibiotic resistance. his approach can reduce turnaround time (ТАТ) for the male health benefit in time.
The study objective is to compare the results of semen analysis using the standard cell culture method and real time polymerase chain reaction (RT-PCR).Materials and methods. Between 2016 and 2018, a prospective study including 143 males who applied to the Urology Clinic of the Ivanovo State Medical Academy due to infertility and for periconceptional preparation was performed. Semen was examined using cell culture method and RT-PCR. The group 1 included 54patients with leukospermia and bacteriospermia; the group 2 included 58patients without leukospermia but with bacteriospermia; the control group consisted of 31 males without leukospermia and bacteriospermia based on RT-PCR.Results. Per RT-PCR, the most frequent microorganisms in the groups 1 and 2 were Corynebacterium spp. (51.9 and 60.3 %) and Enterobacteriaceae spp./Enterococcus spp. (51.9 and 62.1 %), but there weren’t any significant differences between the groups. The range of detected microorganisms per cell culture method and RT-PCR was the same in most cases, but in 22 (15.4 %) patients RT-PCR detected Enterobacteriaceae/Enterococcus spp., which weren’t observed in cell culture. These males were prescribed bovhyaluronidase azoximer (Longidase®) 2 times a week for 1 month for anti-inflammatory therapy and to decrease leukospermia. Post-therapy control semen cultures detected microorganisms that were previously detected by RT-PCR.Conclusion. Our study demonstrates the advantages of RT-PCR compared to the standard cell culture semen analysis. RT-PCR can be recommended as a screening method. To increase the accuracy of microorganism detection, Longidase® is recommended because it promotes prostatic acini drainage.
Infections of the reproductive tract are known to contribute to testicular inflammatory impairment, leading to an increase of pro-inflammatory cytokines such as IL-1β, and a decline in sperm quality. Prokineticin 2 (PK2), a secretory protein, is closely associated with the secretion of pro-inflammatory cytokines in inflamed tissue. It was reported that increased PK2 is related to the upregulation of IL-1β, but the underlying mechanism remains elusive. Here, we illustrated that PK2 was upregulated in testicular macrophages (TM) in a rat model of uropathogenic Escherichia coli (UPEC) infection, which induced the activation of the NLRP3 inflammasome pathway to boost IL-1β secretion. Administration of PK2 inhibitor alleviated the inflammatory damage and suppressed IL-1β secretion. Moreover, PK2 promoted NLRP3 expression and the release of cleaved IL-1β from TM to the supernatants after the challenge with UPEC in vitro. IL-1β in the supernatants affected Leydig cells by suppressing the expression of genes encoding for the enzymes P450scc and P450c17, which are involved in testosterone production. Overall, we revealed that increased PK2 levels in TM in UPEC-induced orchitis may impair testosterone synthesis via the activation of the NLRP3 pathway. Our study provides a new insight into the mechanisms underlying inflammation-associated male infertility and suggests an anti-inflammatory therapeutic target for male infertility.
Background:
Infection and inflammation of the reproductive tract are significant causes of male factor infertility. Ascending infections caused by sexually transmitted bacteria or urinary tract pathogens represent the most frequent aetiology of epididymo-orchitis, but viral, haematogenous dissemination is also a contributory factor. Limitations in adequate diagnosis and therapy reflect an obvious need for further understanding of human epididymal and testicular immunopathologies and their contribution to infertility. A major obstacle for advancing our knowledge is the limited access to suitable tissue samples. Similarly, the key events in the inflammatory or autoimmune pathologies affecting human male fertility are poorly amenable to close examination. Moreover, the disease processes generally have occurred long before the patient attends the clinic for fertility assessment. In this regard, data obtained from experimental animal models and respective comparative analyses have shown promise to overcome these restrictions in humans.
Objective and rationale:
This narrative review will focus on male fertility disturbances caused by infection and inflammation, and the usefulness of the most frequently applied animal models to study these conditions.
Search methods:
An extensive search in Medline database was performed without restrictions until January 2018 using the following search terms: 'infection' and/or 'inflammation' and 'testis' and/or 'epididymis', 'infection' and/or 'inflammation' and 'male genital tract', 'male infertility', 'orchitis', 'epididymitis', 'experimental autoimmune' and 'orchitis' or 'epididymitis' or 'epididymo-orchitis', antisperm antibodies', 'vasectomy'. In addition to that, reference lists of primary and review articles were reviewed for additional publications independently by each author. Selected articles were verified by each two separate authors and discrepancies discussed within the team.
Outcomes:
There is clear evidence that models mimicking testicular and/or epididymal inflammation and infection have been instructive in a better understanding of the mechanisms of disease initiation and progression. In this regard, rodent models of acute bacterial epididymitis best reflect the clinical situation in terms of mimicking the infection pathway, pathogens selected and the damage, such as fibrotic transformation, observed. Similarly, animal models of acute testicular and epididymal inflammation using lipopolysaccharides show impairment of reproduction, endocrine function and histological tissue architecture, also seen in men. Autoimmune responses can be studied in models of experimental autoimmune orchitis (EAO) and vasectomy. In particular, the early stages of EAO development showing inflammatory responses in the form of peritubular lymphocytic infiltrates, thickening of the lamina propria of affected tubules, production of autoantibodies against testicular antigens or secretion of pro-inflammatory mediators, replicate observations in testicular sperm extraction samples of patients with 'mixed atrophy' of spermatogenesis. Vasectomy, in the form of sperm antibodies and chronic inflammation, can also be studied in animal models, providing valuable insights into the human response.
Wider implications:
This is the first comprehensive review of rodent models of both infectious and autoimmune disease of testis/epididymis, and their clinical implications, i.e. their importance in understanding male infertility related to infectious and non-infectious/autoimmune disease of the reproductive organs.
Germline development in vivo is dependent on the environment formed by somatic cells and the differentiation cues they provide; hence, the impact of local factors is highly relevant to the production of sperm. Knowledge of how somatic and germline cells interact is central to achieving biomedical goals relating to restoring, preserving or restricting fertility in humans. This review discusses the growing understanding of how cytokines contribute to testicular function and maintenance of male reproductive health, and to the pathologies associated with their abnormal activity in this organ. Here we consider both cytokines that signal through JAKs and are regulated by SOCS, and those utilizing other pathways, such as the MAP kinases and SMADs. The importance of cytokines in the establishment and maintenance of the testis as an immune-privilege site are described. Current research relating to the involvement of immune cells in testis development and disease is highlighted. This includes new data relating to testicular cancer which reinforce the understanding that tumorigenic cells shape their microenvironment through cytokine actions. Clinical implications in pathologies relating to local inflammation and to immunotherapies are discussed.
Problem:
Antisperm antibodies (ASA) are associated with male subfertility. However, results on sperm surface autoantibodies are controversial, the relationship between ASA and semen parameters (WHO, 2010) is unknown, and data on ASA and sperm kinematics are scarce.
Method of study:
A retrospective study carried out in men undergoing routine semen analysis (WHO 2010), ASA evaluation (direct SpermMAR(™) (IgG) test), and computer-assisted sperm analysis (CASA).
Results:
A 2.6% and a 5.9% incidence of ASA-positive cases were found (cut-off 50% and 10%, respectively; n = 7492). ASA-positive samples had lower (P < 0.0001) sperm concentration, count, motility, and hypo-osmotic swelling (HOS) test score. HOS results did not correlate with sperm vitality in normozoospermic samples with high ASA levels. In unselected samples, ASA-positive samples (cut-off 50%) showed decreased sperm kinematics (VSL, VAP, LIN, ALH, STR, BCF, WOB), but in normozoospermic samples, ASA-positive and ASA-negative subgroups had similar CASA results.
Conclusions:
ASA evaluation is highly relevant in full semen assessment.
Spermatogenic cells express cell-specific molecules with the potential to be seen as "foreign" by the immune system. Owing to the time difference between their appearance in puberty and the editing of the lymphocyte repertoire around birth, local adaptations of the immune system coined immune privilege are required to confer protection from autoattack. Testicular macrophages (TM) play an important role in maintaining testicular immune privilege and display reduced proinflammatory capacity compared with other macrophages. However, the molecular mechanism underlying this macrophage phenotype remained elusive. We demonstrate that TM have a lower constitutive expression of TLR pathway-specific genes compared with peritoneal macrophages. Moreover, in TM stimulated with LPS, the NF-κB signaling pathway is blocked due to lack of IκBα ubiquitination and, hence, degradation. Instead, challenge of TM with LPS or polyinosinic-polycytidylic acid induces MAPK, AP-1, and CREB signaling pathways, which leads to production of proinflammatory cytokines such as TNF-α, although at much lower levels than in peritoneal macrophages. Pretreatment of TM with inhibitors for MAPKs p38 and ERK1/2 suppresses activation of AP-1 and CREB signaling pathways and attenuates LPS-induced TNF-α and IL-10 secretion. High levels of IL-10 production and activation of STAT3 by LPS stimulation in TM indicate a regulatory macrophage phenotype. Our results suggest that TM maintain testicular immune privilege by inhibiting NF-κB signaling through impairment of IκBα ubiquitination and a general reduction of TLR cascade gene expression. However, TM do maintain some capacity for innate immune responses through AP-1 and CREB signaling pathways.
Copyright © 2015 by The American Association of Immunologists, Inc.
Development of Myeloid Immune Cells
As leukocytes develop to maturity, they proceed through an array of phenotypically distinct intermediates. For T and B lymphocyte populations, the different developmental stages, anatomical locations, and cell signals required for progression are well established. However, until recently, much less has been known about how development proceeds in the myeloid lineage, which includes monocytes, macrophages, and dendritic cells. Geissmann et al. (p. 656 ) review our current understanding of myeloid lineage development and describe the developmental pathways and cues that drive differentiation.
Type I diabetes mellitus is caused by autoimmune destruction of pancreatic beta cells, and effective treatment of the disease might require rescuing beta cell function in a context of reinstalled immune tolerance. Sertoli cells (SCs) are found in the testes, where their main task is to provide local immunological protection and nourishment to developing germ cells. SCs engraft, self-protect, and coprotect allogeneic and xenogeneic grafts from immune destruction in different experimental settings. SCs have also been successfully implanted into the central nervous system to create a regulatory environment to the surrounding tissue which is trophic and counter-inflammatory. We report that isolated neonatal porcine SC, administered alone in highly biocompatible microcapsules, led to diabetes prevention and reversion in the respective 88 and 81% of overtly diabetic (nonobese diabetic [NOD]) mice, with no need for additional beta cell or insulin therapy. The effect was associated with restoration of systemic immune tolerance and detection of functional pancreatic islets that consisted of glucose-responsive and insulin-secreting cells. Curative effects by SC were strictly dependent on efficient tryptophan metabolism in the xenografts, leading to TGF-beta-dependent emergence of autoantigen-specific regulatory T cells and recovery of beta cell function in the diabetic recipients.
Experimental autoimmune orchitis (EAO) in the rat is the primary chronic animal model for the investigation of one of the main causes of male infertility, viz., testicular inflammation. Dendritic cells (DC) are potent antigen-presenting cells that play a fundamental role in autoimmune disease. We investigated the number of DC in normal testis and examined whether DC infiltrated the testis during the development of EAO. EAO was induced by active immunization with testis homogenate and adjuvants in two strains of rat (Wistar and Sprague Dawley). The presence of DC in testis was determined, 50 and 80 days after the first immunization, by immunohistochemical staining with specific antibodies (OX-62 and CD11c), and then the total number of DC was measured by stereological analysis. Labeled cells were found only in the interstitial compartment and within granulomas of EAO animals. The number of DC in EAO testes increased compared with control rats in both strains, whereas the number of OX-62+ and CD11c+ cells in adjuvant controls remained unchanged compared with untreated rats. Interspecies variations in the quantity of DC were found, with the total number of DC per testis in untreated and adjuvant control Sprague-Dawley rats being about three times higher than that seen in Wistar rats. Moreover, the increase in DC numbers at 80 days was less prominent in EAO testes of Sprague-Dawley rats than in the Wistar strain in which EAO was more severe and showed a higher number of granulomae. Thus, we have identified the DC population in normal and chronically inflamed testis. The increase in DC observed in EAO suggests that, under inflammatory conditions, the modified action(s) of these cells is a factor in the induction of the autoimmune response in testis.
Sperm have been known to be antigenic for more than a century. There is a strong body of evidence that in humans and in other species at least some antibodies that bind to sperm antigens can cause infertility. Therefore, these antibodies are of interest today for two practical reasons. Firstly, the association of the antibodies with infertility means that they must be detected and then the couples treated appropriately. Secondly, because these antibodies can induce infertility they have the potential to be developed for contraceptive purposes in humans and also for the control of feral animal populations.
Currently, the WHO laboratory manual for the examination and processing of human semen (5th edition, 2010) provides updated, standardized, evidence-based procedures and recommendations for laboratory managers, scientists and technicians to follow in examining human semen in a clinical or research setting. Despite the fact, there are several gaps and limitations in the interpretation of this compendium. Mostly, the WHO-protocol of estimation of peroxidase-positive cells and spermatozoa, as well as evaluation of their viability and morphology are not so affordable and applicable in Russia due to peculiarities of laboratory market. Furthermore, most of Russian manuscripts do not reflect a unified approach to the analytical stage of semen analyses. In order to standardize the protocol for human semen examination which adopted to Russian lab it was developed packing of reagents (GEMSTANDART-SEMEN ANALYSES, LLC 'GEMSTANDART', Saint Petersburg, Russia) allowing to obtain an accuracy and completeness of the examination. In summary, this approach is a necessary step in male fertility evaluation. Together with a clinical information, it is indispensable for planning the appropriate clinical management and tacking care in male health.
Background:
Infertility is an ever-increasing problem in today's world. It can be due to male or female causes. Azoospermia seen in 5-10% of infertile men is due to obstructive or non-obstructive causes. Traditionally, testicular biopsy is the gold standard for evaluation. Fine-needle aspiration (FNA), however, is minimally invasive, provides qualitative and quantitative information about spermatogenesis, and can aid in assisted reproductive techniques making it a novel technique for the evaluation of male infertility.
Objective:
We aimed to classify different causes of azoospermia into different patterns based upon FNA, and assess the utility of cell indices in classifying cases into different patterns.
Method:
We conducted a prospective and a retrospective study of 42 azoospermic males, confirmed on semen analysis, over a period of 5 years. Patients were subjected to FNA of the testes. Smears were prepared, air-dried, wet-fixed, and then stained with May-Grünwald Giemsa and Papanicolaou stains, respectively. Cells were identified using predetermined morphologic criteria, and various indices were calculated followed by statistical analysis of the observations.
Results:
The mean age of 40 patients who satisfied the adequacy criteria was 32.75 years (range 22-48 years). Thirty-four patients had primary infertility and 6 had secondary infertility. Of these, 12 had normal spermatogenesis, 8 had hypo-spermatogenesis, 3 had early and 7 had late maturation arrest, 6 had Sertoli cell-only syndrome (SCOS), and there were different results in each testicle in 4 cases. The Sperm Index (SI) was significantly higher in all cases of normal spermatogenesis than in any of the hypo-spermatogenesis cases (p = 0.009). The Sertoli Index (SEI) in cases of hypo-spermatogenesis and maturation arrest was significantly higher than in cases of normal spermatogenesis (p < 0.001). The Sperm-Sertoli Index (SSI) also showed significant differences between cases of hypo-spermatogenesis and normal spermatogenesis (p < 0.001). These indices were useful in categorising patients with azoospermia.
Conclusion:
FNA helps to easily and accurately identify all types of testicular cells without biopsy. SI, SEI, and SSI are powerful cell indices for assessing the extent of spermatogenesis and classifying various causes of azoospermia. Bilateral sampling and multiple aspirations give a better mapping of spermatogenesis within the testes. Testicular FNA can thus play a very important role in the evaluation of male infertility.
Background:
Ill-defined chronic symptoms such as itching, burning, urethral discharge, and pelvic pain are not uncommon in male urologic patients. Often, microscopic urinalysis, bacterial cultures, and laboratory testing are non-contributory. We have developed a technique for sperm sedimentation and used the cytologic examination of the sediment routinely in more than 4,000 patients with urologic complaints over the last 5 years.
Case:
We present 3 exemplary cases, documenting the diagnostic power of sperm sediment cytology (SSC). In all 3 cases, conventional laboratory testing failed to reveal a causative agent. Case 1 is that of a 28-year-old male patient with a history of occasional swelling of the lymph nodes in the left inguinal region for 1 year. Case 2 is that of a 51-year-old male patient with a history of itching and burning of the urethra of 2 months' duration. Case 3 is that of a 22-year-old female patient with copious vaginal discharge after intercourse for 18 months, non-responsive to treatment.
Conclusion:
We were able to identify causative pathologic organisms in the sediment of all patients or their partner. Subsequent specific treatment did clear and alleviate the symptoms documenting the clinical relevance of this new technique. In our experience, SSC has proven to be a valuable technique for diagnosing occult urologic infections.
The onset of spermatogenesis at puberty represents unique challenges to the immune system as neoantigens of meiotic and haploid germ cells appear long after formation of systemic self-tolerance. The protection of germ cells from autoimmune attack, the “immune privilege” of the testis, was originally attributed both to the existence of the blood-testis barrier and to a failure of the testicular immune system to respond to antigens. Recent research has now shown that the testis is by no means ignorant, but can mount well-balanced immune deviant responses that can protect the gonad from damaging inflammatory responses to pathogens. Moreover, an excessive immune response can lead to inflammatory-based male factor infertility. The mechanisms controlling immune privilege seem to involve factors that also control spermatogenesis and steroidogenesis. They appear to include androgens, a delicate balance of immunomodulatory molecules such as cytokines and chemokines and a polarizing capacity of the testicular interstitial fluids towards a tolerogenic M2 phenotype.
In the present work, the expression and secretion of transforming growth factor-beta 1 (TGF beta 1) by immature pig Leydig and Sertoli cells were investigated. Both cell types express two TGF beta 1 mRNA transcripts of 2.5 and 3.5 kilobases, and the levels were 2.6-fold higher in Leydig than in Sertoli cells. In the latter cells, mRNA levels were enhanced when cultured cells were stimulated by epidermal growth factor and phorbol ester (4-beta-phorbol 12-myristate 13-acetate) and significantly decreased by FSH and testosterone. Using a polyclonal antibody raised against a synthetic peptide that corresponded to the carboxyl-terminal region of TGF beta 1 and recognized this peptide, but not TGF beta 2 or TGF beta 3, specific immunostaining of both Leydig and Sertoli cells was demonstrated in situ, after cell isolation, and during culture. The immunostaining was more marked in Leydig cells than in Sertoli cells. Western blot analysis of Leydig or Sertoli cell-conditioned medium demonstrated a band of 25 kilod...
There is a close functional relationship between the male reproductive and immune systems. Immunological responses against sperm antigens or other elements of the reproductive tract can lead to androgen insufficiency, infertility, or chronic inflammation. Systemic or local immune activation and inflammation directly inhibit the hypothalamic-pituitary-Leydig cell axis, interfere with spermatogenic cell development, and may provoke sperm antibody formation. Traditional explanations for protection of spermatogenic cells, based on physical barriers or immune cell exclusion, are not consistent with either the organization of the reproductive tract or modern concepts of immunoregulation. Regulation of immune responses in the male reproductive tract involves immunoregulatory macrophages and lymphocytes, as well as active somatic cell suppression of antigen-specific immunity by anti-inflammatory and immunosuppressive factors, including cytokines and androgenic steroids. These restraints on antigen-specific immune mechanisms appear to be counterbalanced by enhanced innate immunity. Finally, inflammatory signaling pathways appear to play an important role in normal male reproductive function, and there is evidence that establishment of male reproduction during development is intimately linked to the maturation of the local immune environment.
Description of macrophage activation is currently contentious and confusing. Like the biblical Tower of Babel, macrophage activation encompasses a panoply of descriptors used in different ways. The lack of consensus on how to define macrophage activation in experiments in vitro and in vivo impedes progress in multiple ways, including the fact that many researchers still consider there to be only two types of activated macrophages, often termed M1 and M2. Here, we describe a set of standards encompassing three principles-the source of macrophages, definition of the activators, and a consensus collection of markers to describe macrophage activation-with the goal of unifying experimental standards for diverse experimental scenarios. Collectively, we propose a common framework for macrophage-activation nomenclature.
The mammalian testis possesses a special immunological environment because of its properties of remarkable immune privilege and effective local innate immunity. Testicular immune privilege protects immunogenic germ cells from systemic immune attack, and local innate immunity is important in preventing testicular microbial infections. The breakdown of local testicular immune homeostasis may lead to orchitis, an etiological factor of male infertility. The mechanisms underlying testicular immune privilege have been investigated for a long time. Increasing evidence shows that both a local immunosuppressive milieu and systemic immune tolerance are involved in maintaining testicular immune privilege status. The mechanisms underlying testicular innate immunity are emerging based on the investigation of the pattern recognition receptor-mediated innate immune response in testicular cells. This review summarizes our current understanding of testicular defense mechanisms and identifies topics that merit further investigation.Cellular & Molecular Immunology advance online publication, 23 June 2014; doi:10.1038/cmi.2014.38.
The rodent testis is well established as a site of immune privilege where both innate and acquired immune responses are suppressed. Immune cells and responses within human or non-human primate testes, by contrast, are poorly characterised. This study used multi-colour flow cytometry to characterise the leukocytes in testicular cells isolated from 12 young adult pigtail macaques (Macaca nemestrina) by collagenase dispersal, and to measure the cytokine responses of macaque testicular T-lymphocytes to mitogens. B-lymphocytes and granulocytes were present in very low numbers (0.24% and 3.3% of leukocytes respectively), indicating minimal blood contamination. A median of 30.8% of the recovered testicular leukocytes were CD3+ lymphocytes, with CD4 and CD8 T-lymphocyte proportions similar to those in the blood. The proportion of naïve T-lymphocytes in the testis was low, with significantly higher frequencies of central memory cells, compared with the blood. A median of 42.7% of the testicular leukocytes were CD163+ macrophages, while 4.5% were CD14(+)CD163(-) monocyte-like macrophages. Small populations of myeloid and plasmacytoid dendritic cells, NK cells and NKT cells were also detected. Following mitogen stimulation, 19.7% of blood T-lymphocytes produced IFNγ and/or TNF, whereas significantly fewer (4.4%) of the testicular T-lymphocytes responded to stimulation. Our results characterise the immune cells within the adult macaque testis and identify a suppression of T-lymphocyte responses. This study provides a baseline to examine the immunology of the primate testis and suggests that testicular immune privilege could also be present in primates.
Innate immunity has an adaptive component, which has been referred to as "memory," "trained," "imprinted" or "adaptive." Plasticity is a hallmark of cells of the monocyte-macrophage lineage. Microbial recognition and cytokines profoundly affect macrophage function causing a range of adaptive responses including activation, priming, or tolerance. These adaptive responses of macrophages include production of humoral fluid-phase pattern recognition molecules such as the prototypic long pentraxin PTX3. These components of humoral innate immunity in turn cooperate with and regulate phagocyte function. Progress has been made in defining the molecular basis underlying the polarized activation of macrophages, including signaling mediators, transcription factors, epigenetic modifications, and the microRNA network. The definition of molecules and mechanisms associated with plasticity and polarized activation of macrophages may provide a basis for innovative diagnostic and therapeutic approaches.
Urogenital infections and inflammation may contribute significantly to ejaculate parameters essential for male infertility.
For this review, data were acquired by a systematic search of the medical literature of the last 5 years.
We address the andrological relevance of male urogenital infections and inflammation on ejaculate parameters. The different classification systems of the WHO and NIH are illustrated. In most cases, a separation of the different areas of the urogenital tract, for example, of the prostate, epididymis and testicles, is not possible. The significance of bacteriospermia with common bacteria is discussed. Furthermore, HIV, ascending chlamydial, mycoplasmal and gonococcal infections are relevant. Especially, the relevance of sexually transmitted microorganisms seems to be underestimated. Leukocytospermia is not well defined in its biological significance. Seminal plasma elastase and the cytokine expression reveal better insights into the inflammatory response of the seminal pathways. Sperm antibodies and reactive oxygen species are not usable as indicators for infection and inflammation. Different aspects for an impairment of ejaculate quality have been demonstrated although a direct ascension of microorganisms to the prostate has not been confirmed. Probably, lesions of the epididymis may sustain an ongoing disturbance of sperm parameters. A potential negative influence of urogenital infections and inflammation on sperm function is under discussion. However, the severity of impairment differs according to the underlying infections and the involved compartments.
Signs of infections and inflammation in the ejaculate of infertile men are common, and the relevance is often doubtful in spite of microbiological, spermatological and immunological facilities.
We have demonstrated a role for activin A, follistatin, and FSH in male germ cell differentiation at the time when spermatogonial stem cells and committed spermatogonia first appear in the developing testis. Testis fragments from 3-day-old rats were cultured for 1 or 3 days with various combinations of these factors, incubated with bromodeoxyuridine (BrdU) to label proliferating cells, and then processed for stereological analysis and detection of BrdU incorporation. Gonocyte numbers were significantly elevated in cultures treated with activin, while the combination of FSH and the activin antagonist, follistatin, increased the proportion of spermatogonia in the germ cell population after 3 days. All fragment groups treated with FSH contained a significantly higher proportion of proliferating Sertoli cells, while activin and follistatin each reduced Sertoli cell division. In situ hybridization and immunohistochemistry on normal rat testes demonstrated that gonocytes, but not spermatogonia, contain the activin βA subunit mRNA and protein. In contrast, gonocytes first expressed follistatin mRNA and protein at 3 days after birth, concordant with the transition of gonocytes to spermatogonia. Collectively, these data demonstrate that germ cells have the potential to regulate their own maturation through production of endogenous activin A and follistatin. Sertoli cells were observed to produce the activin/inhibin βA subunit, the inhibin α subunit, and follistatin, demonstrating that these cells have the potential to regulate germ cell maturation as well as their own development. These findings indicate that local regulation of activin bioactivity may underpin the coordinated development of germ cells and somatic cells at the onset of spermatogenesis.
The ability of spermatogenic cells to evade the host immune system and the ability of systemic inflammation to inhibit male reproductive function represent two of the most intriguing conundrums of male reproduction. Clearly, an understanding of the underlying immunology of the male reproductive tract is crucial to resolving these superficially incompatible observations. One important consideration must be the very different immunological environments of the testis, where sperm develop, and the epididymis, where sperm mature and are stored. Compared with the elaborate blood-testis barrier, the tight junctions of the epididymis are much less effective. Unlike the seminiferous epithelium, immune cells are commonly observed within the epithelium, and can even be found within the lumen, of the epididymis. Crucially, there is little evidence for extended allograft survival (immune privilege) in the epididymis, as it exists in the testis, and the epididymis is much more susceptible to loss of immune tolerance. Moreover, the incidence of epididymitis is considerably greater than that of orchitis in humans, and susceptibility to sperm antibody formation after damage to the epididymis or vas deferens increases with increasing distance of the damage from the testis. Although we still know relatively little about testicular immunity, we know less about the interactions between the epididymis and the immune system. Given that the epididymis appears to be more susceptible to inflammation and immune reactions than the testis, and thereby represents the weaker link in protecting developing sperm from the immune system, it is probably time this imbalance in knowledge was addressed.
Plasticity is a hallmark of cells of the myelomonocytic lineage. In response to innate recognition or signals from lymphocyte subsets, mononuclear phagocytes undergo adaptive responses. Shaping of monocyte-macrophage function is an essential component of resistance to pathogens, tissue damage and repair. The orchestration of myelomonocytic cell function is a key element that links inflammation and cancer and provides a paradigm for macrophage plasticity and function. A better understanding of the molecular basis of myelomonocytic cell plasticity will open new vistas in immunopathology and therapeutic intervention.
Experimental autoimmune orchitis (EAO) is a useful model to study organ-specific autoimmunity and chronic testicular inflammation. EAO is characterized by an interstitial lymphomononuclear cell infiltration and damage of the seminiferous tubules showing germ cell sloughing and apoptosis. Using flow cytometry, we analysed the phenotype and number of T lymphocytes present in the testicular interstitium of rats during EAO development. A large increase in the number of testicular CD3+ T lymphocytes was detected. The number of CD4+ and CD8+ effector T lymphocytes (T(effector) cells) dramatically increased in the testis at EAO onset, with the CD4+ cell subset predominating. As the severity of the disease progressed, CD4+ T(effector) cells declined in number while the CD8+ T(effector) cell subset remained unchanged, suggesting their involvement in maintenance of the chronic phase of EAO. As a novel finding, we detected by immunohistochemistry and flow cytometry Foxp3 expressing CD4+ and CD8+ regulatory T lymphocytes (T(regs)) in chronically inflamed testis of EAO rats. The numbers of both T(reg) cell subsets increased in the testis of rats with orchitis, mainly at the onset of EAO; CD4+Foxp3+ T(reg) cells were more abundant than CD8+Foxp3+ T(reg) cells. Unexpectedly, CD25- T lymphocytes were more abundant than CD25+ cells within CD4+Foxp3+ and CD8+Foxp3+ T(reg) cell populations. Although T(reg) subsets are actively accumulated into the testis in EAO rats, these cells are outnumbered by an even more vigorously expanding T(effector) subset. Further, it is possible that factors present in the inflamed testis might limit the ability of T(regs) to abrogate tissue damage.
To investigate whether testicular cytology by fine needle aspiration (FNA) may be considered a diagnostic parameter in the evaluation of the azoospermic subject.
Cytologic smears were obtained using a 23-G needle, stained with May-Grünwald-Giemsa stain and examined under a light Orthoplan microscope (Wild, Leitz, Germany) for qualitative and quantitative analysis.
Fifty-four azoospermic patients were analyzed, and the findings were compared with those obtained from 40 normozoospermic infertile subjects used as controls.
Two hundred spermatogenic cells were counted and classified at the various steps of spermatogenesis. Spermatic index and Sertoli index provided further elucidations and more comprehensible results.
No sign of traumatization was observed. Cytologic analysis was proved to have high statistical reproducibility (P less than 0.01 for spermatogonia and secondary spermatocytes and P less than 0.001 for the other cell types, when compared between differential counts) and permitted identification of different situations associated with azoospermia: Sertoli cell-only syndrome, germ depopulation (hypospermatogenesis), spermatogonial arrest, spermatidic arrest, and obstructive azoospermia. These findings agreed with clinical and hormonal parameters and with the results of bilateral surgical biopsies, when performed.
The results support use of FNA of the testis as a noninvasive diagnostic parameter for the assessment of azoospermic subjects.
In testicular imprint smears from 100 infertile men (both testicles), stained using the Pappenheim and Papanicolaou methods, the cell forms (light and dark spermatogonia, primary and secondary spermatocytes, spermatids, spermatozoa and Sertoli cells) were identified and quantified by the counting of 500 consecutive cells. Identification of the cell types, which are described and illustrated, was consistent and reproducible; the advantages of the different stainings in their analysis is documented. The cell counts were tested for reproducibility and compared to the histologic diagnoses and sperm counts. Statistical analysis showed the highest reproducibility for cells frequently encountered in smears (0.99 for Sertoli cells and 0.98 for spermatozoa) and the lowest but still satisfactory reproducibility for rare or arbitrarily defined cell forms (0.71 for dark spermatogonia and 0.76 for secondary spermatocytes). The high reproducibility of the smear quantification permits the introduction of a number of indices, defining clinically useful relations between cell types that are indicative of various types of infertility. The data obtained by cytologic quantification showed reasonably good correlation with the histologic diagnoses of desquamation and focal fibrosis and excellent correlation with Sertoli cells only, arrested spermatogenesis and complete fibrosis. The cytologic quantification of testicular smears adds considerable information to the diagnosis of impaired fertility and should be instituted in properly equipped centers.
The appearances in cytologic specimens of the principal cell types in the normal human seminiferous epithelium are described and illustrated. Sertoli cells, which are larger than spermatogenic cells, are characterized by a slightly basophilic, ill-defined cytoplasm of triangular, elongated or columnar shape; the cytoplasm may be vacuolated and may contain spermatozoa. The nuclei of Sertoli cells are round, with a uniformly finely granulated chromatin and a single nucleolus. Spermatogenic cells are round or oval and show scanty cytoplasm with deeper basophilia and well-defined cytoplasmic borders. Multinucleation is common in spermatogenic cells. The Sertoli cells constitute a very homogeneous cell population as compared to the spermatogenic cells, which show several distinct cell types (spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa) whose nuclear structures depend on the stage of meiosis. Both cell types may occur as naked nuclei. Some problems of cell classification are discussed.
Enriched fractions of spermatogenic cells were isolated by unit gravity sedimentation and analyzed both for the presence of secreted tumor necrosis factor-alpha (TNF alpha) in vitro by bioassay and for the presence of TNF alpha mRNA by Northern blot analysis. Small quantities of bioactive TNF alpha were consistently detected in medium conditioned by round spermatid fractions. Both pachytene spermatocyte and round spermatid fractions contained RNA that hybridized with murine cDNA probes for TNF alpha, with pachytene spermatocytes containing a normal 1.9-kilobase (kb) transcript, while round spermatids contained principally an approximately 2.8-kb transcript. Both the normal size transcript and the larger haploid-specific transcript were enriched when total RNA from pachytene spermatocyte and round spermatid fractions was passed through an oligo(dT) column. The normal 1.9-kb transcript within pachytene spermatocytes could be induced by exposing the spermatogenic cells to lipopolysaccharides in vitro, yet the approximately 2.8-kb transcript within round spermatids appeared uninduced by LPS treatment. In situ hybridization for the TNF alpha message by using digoxigenin label antisense TNF alpha riboprobe labeled pachytene spermatocytes, round spermatids, and presumptive interstitial macrophages. Spermatogonia and elongating spermatids as well as other interstitial cells were unlabeled or very lightly labeled. Hybridization of 16-day-old prepuberal testis resulted in the labeling of spermatocytes and presumptive interstitial macrophages. RNA from Sertoli cells, but not pachytene spermatocytes or round spermatids, hybridized with human TNF alpha receptor p60 probe in Northern blot analysis. These results are consistent with the working hypothesis that spermatids release TNF alpha, which is detected by Sertoli cells and may serve as a paracrine factor, regulating an as yet unidentified process in spermatogenesis.
The testis is an immunologically privileged site, and transplantation data suggest that this privilege may be enhanced in cryptorchidism. Although alphabetaT cells, which mediate and promote the immune response, have access to the normal testis, relatively little is known about these cells in the abdominally located testis. An increase in testicular lymphocyte-inhibiting cytokines has also been implicated in enhanced graft survival following the experimental induction of cryptorchidism. Consequently, T cell traffic and lymphocyte-inhibiting activity in testes of cryptorchid adult rats were examined in the following study. Numbers of alphabetaT cells and the cytotoxic CD8(+) T cell subset in the testis were unaffected following 1 month of cryptorchidism. In contrast, subcutaneous testosterone implants, which inhibit Leydig cell function through suppression of gonadotrophin secretion, reduced these parameters in both scrotal and abdominal testes. Testicular T cell numbers were positively correlated with the number of testicular resident macrophages, which also were reduced by subcutaneous testosterone implants. The concentration of lymphocyte-inhibiting activity in the testicular interstitial fluid was reduced by 80% in short-term (1 month) and longer-term (3 months) cryptorchidism. These data indicate that the T cell population, and in particular the CD8(+) T cell subset, in the rat testis is functionally related to the resident macrophages or Leydig cells. On the other hand, testicular lymphocyte-inhibiting activity does not appear to be a determinant of the number of testicular T cells, and may not be a major factor in the prolonged survival of certain grafts in the abdominal testis.
The majority of macrophages in the rat testis can be identified by the tissue-resident macrophage marker ED2. A smaller population of intratesticular macrophages do not express the ED2 antigen but are positive for the monocyte/macrophage marker ED1. Treatment of adult rats with the inflammatory stimulus lipopolysaccharide (LPS) had no effect on the number of testicular resident (ED2(+)) macrophages but caused a transient increase in ED1(+)ED2(-) monocyte-like macrophages (an average three-fold increase 12 h later). In both control and LPS-treated rat testes, a majority of macrophages that expressed ED1 and all Leydig cells were immuno-positive for the inducible isoform of nitric oxide synthase (iNOS). However, less than 6% of ED2(+) macrophages showed any iNOS expression, even after LPS treatment. This deficiency was confirmed by the finding that isolated ED2(+) testicular macrophages (>98% pure) stimulated with LPS did not produce NO in vitro. In contrast, resident macrophages from the peritoneum showed the expected NO response, and purified Leydig cells produced significant NO regardless of the presence or absence of LPS. Collectively, these data indicate the presence of at least two macrophage subsets in the adult rat testis: (1) the ED2(+) resident macrophages, which do not alter following LPS-treatment and mostly do not express iNOS or produce NO in response to an inflammatory stimulus, and (2) the ED1(+)ED2(-) monocyte-like macrophages, which increase in number after LPS-treatment and express iNOS even in the absence of exogenous inflammatory stimulation. It is highly probable that these different subsets have different functional roles within the testis.
Controversy exists over levels of DNA integrity in the sperm of fertile and infertile men. In addition, the effect of leukocytospermia on sperm DNA in these 2 groups is unclear. We decided to address these questions by collecting semen samples from men known or presumed to be fertile and men from infertile couples. Samples were analyzed and assessed for sperm concentration, motility, and morphology. Samples failing to meet World Health Organization (WHO) standards in one or more of these parameters were judged abnormal. Samples were then arbitrarily assigned normalized scores in each of the above parameters, and scores were summed to give a normalized value for overall sperm quality. DNA abnormality was determined by an in situ DNA denaturation test with acridine orange and expressed as a percentage of cells with abnormal DNA integrity (ADI). Assessment of 187 samples revealed a moderate inverse correlation between ADI and sperm quality (r =.58), although a large degree of ADI dispersion was observed in abnormal semen samples. The average ADI for normal and abnormal semen samples was 18% +/- 2.8% and 36% +/- 5.8%, respectively, with the threshold of 95% probability set at 30%. When sorted for leukocytospermia, the difference in ADI between normal and abnormal semen groups without leukocytospermia was much smaller (17% +/- 2.2% and 22% +/- 4.6%; P =.023). Leukocytospermia had no significant effect on ADI in the normal semen group (P = .46); however, ADI was more than double the ADI in the abnormal semen group (18% +/- 2.4% and 50% +/- 11%; P < .001). The results of our analysis show that at least 3 factors affect net DNA integrity in leukocytospermic samples that fail to meet WHO standards: 1) primary DNA damage, which is moderately inverse to sperm quality, in particular to sperm concentration; 2) effect of leukocytes increasing primary or provoking potential DNA damage in a cascade-like manner, particularly in sperm with poor morphology and motility; and 3) a decreasing proportion of cells with damaged DNA in semen with the worst quality.
Plasticity and functional polarization are hallmarks of the mononuclear phagocyte system. Here we review emerging key properties of different forms of macrophage activation and polarization (M1, M2a, M2b, M2c), which represent extremes of a continuum. In particular, recent evidence suggests that differential modulation of the chemokine system integrates polarized macrophages in pathways of resistance to, or promotion of, microbial pathogens and tumors, or immunoregulation, tissue repair and remodeling.
Most laboratory methods used to evaluate semen quality have not correlated highly with fertilizing capacity. The discovery of a variety of fluorochromes and compounds conjugated to fluorescent probes has enabled a more widespread analysis of sperm attributes, and in conjunction with the flow cytometer, permit the evaluation of a large number of spermatozoa. A number of characteristics of sperm integrity, viability and function can be assessed by flow cytometry. The DNA status of spermatozoa has been determined using the metachromatic properties of acridine orange (AO). AO staining, when used in the sperm chromatin structure assay (SCSA), correlates with fertility in a number of species. DNA fragmentation can also be assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, which identifies DNA strand breaks by labeling free 3'-OH termini with modified nucleotides. The status of the sperm acrosome can be determined using fluorescently labeled lectins and LysoTracker Green DND-26, a fluorescent acidotropic probe. Capacitation status has been observed through calcium-mediated changes using chlortetracycline (CTC) or by changes in membrane fluidity monitored by the binding of the fluorescent amphiphilic probe, Merocyanine 540. Fluorescently labeled annexin-V, C6NBD and Ro-09-0198 can also be used to detect changes in membrane phospholipid distribution. Cell viability can be determined using the propensity of propidium iodide (PI), ethidium homodimer-1 (EthD-1) or Yo-Pro-1 to permeate damaged membranes. These are generally more adaptable to clinical flow cytometry than the bisbenzimide membrane impermeable stain, Hoechst 33258, which excites in the ultraviolet range and requires UV laser equipment. Mitochondrial function can be determined using rhodamine 123 (R123) and MitoTracker Green FM (MITO) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Flow cytometry is a tool that may be used in the future to monitor many new potential markers of sperm function.
To investigate whether there is an impact of different sources of reactive oxygen species (ROS) on sperm functions.
Prospective study.
Patients at the Center for Dermatology and Andrology, Giessen, Germany.
Semen collected from 63 randomly collected patients attending the IVF unit of the University of Giessen, Germany.
Only patients with nonleukocytospermia were included in this study.
Sperm count and motility before and after sperm separation by swim-up, morphology, DNA fragmentation, and extrinsic (by leukocytes) and intrinsic ROS production (by spermatozoa) were evaluated.
Leukocytes correlated significantly with extrinsic ROS production (r = 0.576), but markedly less with intrinsic ROS production (r = 0.296). Sperm count, morphology, and motility in the ejaculate were markedly more affected by extrinsic than by intrinsic ROS. The DNA fragmentation was strongly positively correlated with intrinsic ROS production, whereas this correlation was weaker for extrinsic ROS production. No correlation was found between DNA fragmentation and the number of leukocytes, whereas the correlations with motility in the ejaculate and the motile sperm count after swim-up were highly significant. Moreover, significant differences were observed for extrinsic and intrinsic ROS production between groups of patients having a high (> or = 1 x 10(6)/mL) and a low number (<1 x 10(6)/mL) of leukocytes in the ejaculate.
The origin of ROS seems to have an influence on the site of the damage. Because leukocyte counts <1 x 10(6)/mL caused a significant decrease of motility and DNA integrity, the threshold given by the World Health Organization (WHO) should be re-evaluated.
To report clinical findings associated with an increased incidence of mumps orchitis in Liverpool, UK, as in the last 2 years there has been a four-fold increase in the number of notified viral mumps cases in the UK.
In the 8 months before April 2005, patients presenting to the Accident and Emergency/Urology Departments of the Royal Liverpool University Hospital with a diagnosis of epididymo-orchitis were identified from the emergency urological admission database.
Of 195 males presenting with an acute history of testicular pain and swelling, 25 gave a history of mumps parotitis 4-11 days earlier. Three had bilateral orchitis and two needed scrotal exploration to exclude torsion. Scrotal ultrasonography findings varied from increased vascularity to abnormal testicular echo texture. Treatment included analgesia, scrotal support, re-hydration and broad-spectrum antibiotics. Of the 10 patients followed-up to date, four had testicular abnormalities; one had persistent testicular pain, one a change in testicular consistency, one a noticeably reduced size of one testis, and one significant testicular atrophy.
As postpubertal mumps in males is associated with a 40% incidence of orchitis, parents should be made aware that failing to immunize their children threatens the future fertility of their sons. Young men not immunized as children should be counselled and offered urgent vaccination. Clinicians should be aware that epididymo-orchitis may be secondary to mumps infection even with no history or clinical evidence of parotitis.
There is a consensus on the diagnostic management of bacterial prostatitis (acute and chronic). In chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) the diagnostic approach remains unclear, because inflammatory and noninflammatory CP/CPPS might be one entity with varying findings over time. The WHO definition of male accessory gland infection does not differentiate between prostatitis, epididymitis, and other inflammatory alterations of the urethral compartment. The definition therefore cannot be further accepted as a rational tool for the diagnosis of prostatitis and related diseases in urological andrology. Therapy in infectious prostatitis is standardised and antibiotics are the primary agents. Andrological implications are well defined, side-effects are minimal. CP/CPPS therapy has the goal to reduce pelvic pain. However, treatment regimens are not as standardised. Andrological side-effects are well defined and mainly due to the functional background of these agents.
Antisperm antibodies: etiology, pathogenesis, diagnosis, and treatment
- S Mazumdar
- A S Levine
Unification of procedures of cytochemical staining of human ejaculate to determine the true fertility of men. Laboratornaya diagnostika. Vostochnaya Evropa
- Sapozhkova Zh
- K Eremin
- V Dolgov
Urogenital Infection as a Risk Factor for Male Infertility
- H C Schuppe
- A Pilatz
- H Hossain
Issledovanie osadka jejakuljata v diagnostike infekcij, peredavaemyh polovym putem
- Sapozhkova Zhju
- I P Shabalova
- K T Kasojan
VisionCyto® SpermSediment: New diagnostic possibilities of cytological examination of ejaculate sediment
- Sapozhkova Zhyu
Patent on Invention. Method for combined measurement of concentration of peroxidase-positive cells (neutrophilic granulocytes) and sperm cells in human ejaculate using variations based on cytochemical staining
- Sapozhkova Zhyu
- K I Eremin
Method of Microscopic Diagnostics of Sperm Quality after Ejaculate Sedimentation
- Sapozhkova Zhyu