Leukocytes In Semen Research Articles

A study of quantitative and qualitative analysis of semen in smokers and non-smokers

Numerous health effects of smoking are well-known; associations with semen quality are uncertain. Cigarette smoking may be associated with sub-fertility in males and may result in decreased sperm concentration, lower sperm motility, and a reduced percentage of morphologically normal sperm respectively Semen analysis is a simple, cost effective screening test for evaluation of male in laboratory & clinics, which provides information on quantitative and qualitative aspects of testicular function. Except in cases of azoospermia, semen analysis does not separate patients in to sterile and fertile group. Objective: The present study was undertaken to perform the quantitative and qualitative Analysis of Semen and to compare the sperm parameters in smokers and non-smokers. Materials and methods: The following males were excluded from the study groups: 1. Those suffering from azoospermia or secondary infertility; 2. Ex-smokers and ex-alcoholics, or those with history of tobacco/betel nut chewing or substance abuse; 3. Those with history of prolonged medication, intake of indigenous medications/herbal preparations/tonics, occupational exposure to chemicals; or excessive heat; 4. History of injury to testes, varicocele, hydrocele, undescended testis. A total of 200 cases, retrospective and prospective from September 2018 to January 2021 were included in the study. Results: Compared with non-smokers, smokers had a significant decrease in semen volumes, rapid progressive motility and sperm viability moreover, smokers had a significant increase in the levels of immotile sperms and semen leukocytes, pH and sperm concentration were not significant. Sperm motion parameters were all lower in the smokers. Normal morphology sperm was decreased significantly in smokers. The sperm morphology was worse with increasing degree of smoking. Compared, the overall quality of sperm was significantly decreased in smokers when compared with non-smokers. Conclusion: Semen parameters like, volume, motility, count, liquifraction time, viscocity morphologically normal spermatozoa were reduced in cigarette smokers when compared to Non-smokers, but sperm count was found to have a decrease in Smokers, when compared to Non-smokers. Liquifraction time and viscocity of semen was variable .In concordance with other researchers, results of present study support that cigarette smoking have a relatively significant effect on semen.

Leukocytospermia in late adolescents: possible clinical interpretations

BackgroundNo data are currently available on the implication of amicrobial leukocytospermia in male adolescents. Therefore, the primary aim of this study was to evaluate the prevalence of amicrobial leukocytospermia among non-smoker late adolescents who were exposed to other risky lifestyles for the andrological health. The main andrological clinical features of adolescents with leukocytospermia were also reported.MethodsThis is a cross-sectional study carried out in 80 boys. Each adolescent underwent a physical examination, and to the assessment of sperm conventional parameters, seminal leukocytes concentration and immature germ cell evaluation. A possible correlation between seminal leukocytes and immature germ cells and testicular volume (TV) was tested.ResultsThe adolescents enrolled in this study had 18.0 ± 0.4 (range 18.1–18.9) years. Unprotected sexual intercourse was referred by 38% of them. Sexual dysfunctions were found in 25% and isolated hypoactive sexual desire in 12.5% of boys. Low TV and penile length in flaccidity were found in 44% and 30% of them, respectively. Only 41% had normozoospermia at the sperm analysis, whereas 19% had isolated oligozoospermia, 15% oligo-asthenozoospermia, and 25% oligo-astheno-teratozoospermia. Leukocytospermia occurred in 25% (20 out of 80) of adolescents. No seminal infection was detected in 19% (15 out of 80) of them. Adolescents with leukocytospermia had lower progressive sperm motility, percentage of normal forms, TV, and a higher percentage of immature germ cells compared to those without leukocytospermia. Semen leukocyte concentration correlated negatively with TV and positively with the percentage of immature germ cells in the ejaculate.ConclusionLeukocytospermia, increased immature germ cell number, and low TV identify a distinct phenotype suggestive of testicular tubulopathy. Primary prevention of male infertility and the counselling for andrological risky lifestyles is mandatory and should be started as early as possible.

Фрагментация ДНК сперматозоидов. Есть ли связь с основными параметрами спермы и возрастом?

Introduction. TStandard sperm examination, performed according to WHO guidelines, remains the main method of assessment of male fertility. At the same time, this research method has a number of significant limitations, including a low predictive value regarding the outcomes of assisted reproductive technologies (ART) programs and the outcome of naturally occurring pregnancies. The limitations of standard sperm examination dictate the need for additional methods for assessing male fertility. The most promising and widely used test is the assessment of the level of sperm DNA fragmentation. Aim. To evaluate the associations between the levels of sperm DNA fragmentation and the age of the patients along with the following parameters used as a part of standard sperm analysis: semen volume, total number of spermatozoa, percentages of progressively-mobile, non-progressively-mobile and immobile forms, percentages of morphologically normal forms and the forms bearing head defects within the structure of total number of morphological anomalies, as well as semen leukocyte count. Materials and methods. Study materials were the examination results from 121 males aged from 21 to 53 years old (mean age 32.7±4.5 years old), undergoing an examination within the Clinical Institution «Mother and Child – Yaroslavl» during a time period from January 2019 until April 2020. Standard sperm analysis procedures were carried out according to latest edition of WHO Guidelines (2010). The determinations of germ cell DNA fragmentation levels were performed using the TUNEL method. Results. The test results have revealed a weak negative relation between the sperm DNA fragmentation (%) and the percentage of progressively-mobile forms (%) – r = -0.26 (p <0.01). The correlation of sperm DNA fragmentation with age and other parameters was considered statistically insignificant. The second stage of the analysis have demonstrated that the increased degree sperm DNA fragmentation is 1.8-fold more often found in the patients having signs of astenozoospermia (23.6%) comparing to the patients showing normal degree of spermatozoa mobility (13.1%), (p <0.05). Conclusions. The level of sperm DNA fragmentation correlates with the percentage of progressively-mobile forms of spermatozoa (negative relation) and no correlations were found with other semen parameters and with the age. The rates of increased levels of sperm DNA fragmentation are 1.8-fold more often found in astenozoospermia patients comparing to the patients showing normal degrees of spermatozoa mobility.

Impaired spermatogenesis in COVID-19 patients.

BackgroundThe current study aimed to determine the impact of SARS-CoV-2 infection on male fertility.MethodsThis is a single-center, hospital-based observational study that included autopsied testicular and epididymal specimens of deceased COVID-19 male patients (n=6) and recruited recovering COVID-19 inpatients (n=23) with an equal number of age-matched controls, respectively. We performed histopathological examinations on testicular and epididymal specimens, and also performed TUNEL assay and immunohistochemistry. Whereas, we investigated the semen specimen for sperm parameters and immune factors.FindingsAutopsied testicular and epididymal specimens of COVID-19 showed the presence of interstitial edema, congestion, red blood cell exudation in testes, and epididymides. Thinning of seminiferous tubules was observed. The number of apoptotic cells within seminiferous tubules was significantly higher in COVID-19 compared to control cases. It also showed an increased concentration of CD3+ and CD68+ in the interstitial cells of testicular tissue and the presence of IgG within seminiferous tubules. Semen from COVID-19 inpatients showed that 39.1% (n=9) of them have oligozoospermia, and 60.9% (n=14) showed a significant increase in leucocytes in semen. Decreased sperm concentration, and increased seminal levels of IL-6, TNF-α, and MCP-1 compared to control males were observed.InterpretationImpairment of spermatogenesis was observed in COVID-19 patients, which could be partially explained as a result of an elevated immune response in testis. Additionally, autoimmune orchitis occurred in some COVID-19 patients. Further research on the reversibility of impairment and developing treatment are warranted.FundingThis study was supported by Ministry of Science and Technology of China Plan, Hubei Science and Technology Plan, National Key Research and Development Program of China, HUST COVID-19 Rapid Response Call, China and National Natural Science Foundation of China; these funding bodies are public institutions, and they had no role in study conception, design, interpretation of results, and manuscript preparation.

Reactive Oxygen Species Secreted by Leukocytes in Semen Induce Self-Expression of Interleukin-6 and Affect Sperm Quality.

Reproductive tract inflammation is considered an important cause of male infertility. Increased leukocytes in semen can produce many reactive oxygen species (ROS), which affect sperm function. The aim of this study is to identify the main source of ROS in seminal plasma and to assess the effect of ROS on leukocytes. Semen samples (n = 20) with leukocyte concentration >1 × 106 were collected from a male infertility clinic. This study mainly compares the sperm function parameters of the normal group and the semen white blood cell group >1 × 106. The results identified that ROS in semen was closely related to sperm function parameters, and CD45+ leucocytes were the main source of ROS. Compared with the control group, the concentration of IL-2, IL-4, IL-6, IFN-γ, and TNF-α was higher in the experimental group. Leukocytes in semen may regulate the secretion of ROS through the mammalian target of rapamycin (mTOR) pathway. A considerable amount of ROS can upregulate the expression of IL-6 in leukocytes via the nuclear factor kappa-B (NF-kB) pathway.

The importance of semen leukocytes in HIV-1 transmission and the development of prevention strategies

ABSTRACT HIV-1 sexual transmission occurs mostly through contaminated semen, which is a complex mixture of soluble factors with immunoregulatory functions and cells. It is well established that semen cells from HIV-1-infected men are able to produce the virus and that are harnessed to efficiently interact with mucosal barriers exposed during sexual intercourse. Several cofactors contribute to semen infectivity and may enhance the risk of HIV-1 transmission to a partner by increasing local HIV-1 replication in the male genital tract, thereby increasing the number of HIV-1-infected cells and the local HIV-1 shedding in semen. The introduction of combination antiretroviral therapy has improved the life expectancy of HIV-1 infected individuals; however, there is evidence that systemic viral suppression does not always reflect full viral suppression in the seminal compartment. This review focus on the role semen leukocytes play in HIV-1 transmission and discusses implications of the increased resistance of cell-mediated transmission to immune-based prevention strategies.

Broadly neutralizing antibodies potently inhibit cell-to-cell transmission of semen leukocyte-derived SHIV162P3.

BackgroundHIV-1 sexual transmission occurs mostly through infected semen, which contains both free virions and infected leukocytes. Transmission initiated by infected cells has been shown by several in vitro and in vivo studies and a reduced capacity of broadly neutralizing antibodies (bNAbs) to inhibit cell-to-cell transmission has also been reported. However, due to limitations of available experimental models, there is yet no clarity to which extend bNAbs can prevent transmission mediated by semen leukocytes.MethodsWe developed a novel in vitro assay to measure cell-cell transmission that makes use of splenocytes or CD45+ semen leukocytes collected from acutely SHIV162P3-infected cynomolgus macaques. A panel of 11 bNAbs was used either alone or in combination to assess their inhibitory potential against both cell-free and cell-cell infection.FindingsSplenocytes and semen leucocytes displayed a similar proportion of CD4+T-cell subsets. Either cell type transferred infection in vitro to target TZM-bl cells and PBMCs. Moreover, infection of macaques was achieved following intravaginal challenge with splenocytes. The anti-N-glycans/V3 loop bNAb 10–1074 was highly efficient against cell-associated transmission mediated by infected spleen cells and its potency was maintained when transmission was mediated by CD45+ semen leukocytes.InterpretationThese results support the use of bNAbs in preventative or therapeutic studies aiming to block transmission events mediated not only by free viral particles but also by infected cells. Our experimental system could be used to predict in vivo efficacy of bNAbs.FundingThis work was funded by the 10.13039/501100003323ANRS and the European Commission.

Follicle-Stimulating Hormone Treatment and Male Idiopathic Infertility: Effects on Sperm Parameters and Oxidative Stress Indices according to FSHR c. 2039 A/G and c. -29 G/A Genotypes.

Scientific evidence shows that the administration of follicle-stimulating hormone (FSH) to infertile patients with normal serum FSH concentrations improves sperm parameters in oligozoospermic men. The aim of this study was to evaluate the effects of highly purified urofollitropin (hpFSH) on conventional and bio-functional sperm parameters and on oxidative stress indices in patients with idiopathic infertility. We also evaluated the response to hpFSH on these parameters in relationship to FSHR c. 2039 A/G and FSHR c. -29 G/A genotypes. A prospective longitudinal study was conducted on 42 patients with idiopathic male infertility, 23 of whom underwent to FSHR c. 2039 A/G and FSHR c. -29 G/A genotyping. Each patient was asked to collect two semen samples before and after administration of 150 IU hpFSH three times a week for 16 weeks. Patients were divided into responders or non-responders based on whether their total sperm count had at least doubled or was less than double at the end of treatment, respectively. Responders showed a significantly higher semen volume, sperm concentration, spermatids, and leukocytes. Non-responders had a significant decrease of the percentage of spermatozoa in early apoptosis after hpFSH administration. Oxidative stress indexes did not differ significantly after FSH administration in both groups. Conventional and bio-functional sperm parameters did not differ in patients with FSHR c. 2039 GG and AA genotypes, and FSHR c. -29 GG genotype both before and after FSH administration. The FSHR c. 2039 and FSHR -29 G/A genotypes and allelic distribution did not differ between responders and non-responders. FSH showed to be capable of ameliorating sperm parameters in about half patients treated, therefore it may be helpful in patients with idiopathic infertility.

MP58-17 PREVALENCE OF POSITIVE SEMEN CULTURES OF SPERM BANKED SPECIMENS

INTRODUCTION AND OBJECTIVE: Semen culture is routinely obtained prior to in-vitro fertilization (IVF), and efforts to treat positive cultures are important to optimize success. Leukocytospermia and bacteriospermia′s influence on semen and fertility is widely debated. Currently no guidelines on the role of semen culture before sperm cryopreservation exists. Survival of bacteria after cryopreservation has been reported in the literature. The goal of this study was to assess the prevalence of positive semen cultures and define the role of culture prior to cryopreservation. METHODS: Retrospective review was conducted on men with cryopreserved specimens over a 10 month period. Genital Benzalkonium chloride wipe was added to standard hand washing protocol after 8 months. An incubating solution containing Gentamicin for cryopreservation was used. Demographics, semen parameters, and semen culture results with organisms and antibiotic sensitivity were recorded. RESULTS: 63 men provided ejaculated semen for cryopreservation. Mean age was 30.5±10.4 years. 33 men cryopreserved because of malignancy, 14 for sub-fertility, 7 were transgender, and 9 were for other reasons. 36 men (57.1%) had positive semen cultures and 27 men (42.9%) had negative cultures. Potential pathogenic bacteria were found in 25 men (69.4%) with positive culture; enterococcus (8) and β-hemolytic streptococcus (7) were most common. 16 men (44%) had non-pathogenic bacteria, coagulase negative staph (13) was most common. 17 men had bacteria not sensitive or not tested against Gentamicin, 25 men had Gentamicin sensitive bacteria. After institution of genital wipes, 9 out of 20 men (45%) had positive culture. 43 men provided a sample prior to addition of the genital wipe, and 27 men (62.8%) had a positive culture. No differences were found with culture positivity before or after use of genital wipes, p>0.05. No differences were found when comparing semen parameters and leukocytes between positive and negative semen cultures. CONCLUSIONS: Greater than 50% of men cryopreserving semen had positive cultures with over 69% harboring a potentially pathogenic organism, many of which are not sensitive to Gentamicin. Semen parameters and seminal leukocyte presence were not indicative of culture positivity. The institution of a hand washing protocol did not change the frequency of positive cultures. Work is needed to assess the survivability of bacteria after thawing and evaluation of the effect of culture positivity on IVF. Source of Funding: None

Leukocytospermia is not an informative predictor of positive semen culture in infertile men: results from a validation study of available guidelines.

STUDY QUESTIONAre the European Association of Urology (EAU) guidelines for performing semen culture accurate enough for detecting a positive semen culture in Caucasian-European infertile men?SUMMARY ANSWERThe majority (80%) of asymptomatic infertile men with a positive sperm culture may miss a proper diagnostic assessment when relying on EAU guidelines; no single parameter can assist in medical decision-making.WHAT IS KNOWN ALREADYThe EAU guidelines suggest performing semen culture in case of increased leukocytes in semen (>106 peroxidase positive white blood cells/ml, i.e. leukocytospermia).STUDY DESIGN, SIZE, DURATIONA cross-sectional validation study including 523 infertile men was carried out during 2010–2018.PARTICIPANTS/MATERIALS, SETTING, METHODSInfertile men who were asymptomatic for genital infections were enrolled at a single academic center, and a semen culture was obtained in every case. A concentration of >103 cfu/ml urinary tract pathogens in the ejaculate was considered indicative of significant bacteriospermia. Semen analysis values were assessed on the basis of 2010 World Health Organization reference criteria. EAU guidelines for semen culture were used to predict positive semen culture in our cohort and thus validated. Moreover, we tested the predictive performance and accuracy of several clinical parameters and compared them to EAU guidelines.MAIN RESULTS AND THE ROLE OF CHANCEA positive semen culture was found in 54 men (10%). The application of EAU guidelines would have missed 43 out of 54 (80%) positive semen cultures with 120/131 (92%) useless examinations. EAU guidelines specificity, sensitivity and discrimination were 74%, 20% and 47%, respectively. When trying to improve positive semen culture prediction, we were unable to find any informative baseline parameter except for serum neutrophil-to-lymphocyte ratio (odds ratio 1.70 (95% CI 1.04–2.77)), although without any improvement in terms of discrimination (P = 0.10).LIMITATIONS, REASONS FOR CAUTIONThe study was limited by the lack of a control group of fertile men its retrospective nature. Moreover, monoclonal antibodies were not used for leukocyte assessment.WIDER IMPLICATIONS OF THE FINDINGSSince it is not possible to identify infertile men at risk of semen infection, further studies are needed to tailor the execution of semen culture.STUDY FUNDING/COMPETING INTEREST(S)No funding was received for this study. There are no competing interests.

SC301 - Prevalence of positive semen cultures in infertile men without leukocytospermia: A cross sectional study

INTRODUCTION AND OBJECTIVES:The EAU guidelines suggest performing semen culture in case of increased leukocytes in semen. However, the clinical significance of this parameter is controversial. Furt...

Strategies to Minimize Various Stress-Related Freeze-Thaw Damages During Conventional Cryopreservation of Mammalian Spermatozoa.

The aim of the article is to report a review on different sperm cryopreservation techniques, various stress-related freeze-thaw damages altering sperm structure and function during conventional cryopreservation, and strategies to minimize these stresses. Sperm cryopreservation has allowed indefinite storage and successful transportation of valuable germplasm from proven sites at distant locations, for genetic upgradation through implementation of reproductive techniques, such as artificial insemination. Different techniques for sperm cryopreservation have been proposed such as conventional freezing techniques, directional freezing, and sperm vitrification. Drawbacks related to conventional freezing methods, such as heterogeneous ice nucleation and repeated freeze-thaw cycles at the ice front that disrupts and kill sperm cells, led to the emergence of the directional freezing technique. Sperm vitrification is advantageous as there is no ice crystal-induced physical damages to sperm. However, sperm vitrification has less applicability as encouraging results are only reported in human, dog, and cat. In spite of several drawbacks, conventional freezing techniques are still most widely used for sperm cryopreservation. Spermatozoa experience stresses in the form of cold shock, osmotic stress, and mainly oxidative stress during conventional cryopreservation ultimately reduces the sperm viability and fertility. Several attempts have been made in the past to minimize all these stresses individually or in combination. Membrane fluidity was increased to prevent the cold shock and cryocapacitation-like changes by the addition of cholesterol to the membrane. Antifreeze proteins were added in semen extender to minimize freeze-thaw damages due to heterogeneous ice nucleation and ice recrystallization. Oxidative stress was reduced either by neutralizing reactive oxygen species (ROS) through enzymatic, nonenzymatic, plant-based antioxidants or reductants; or by minimizing the level of sources like the semen radiation exposure, leucocytes, and dead and defective spermatozoa, which lead to ROS production during the semen cryopreservation process. A novel approach of minimizing oxidative stress was to reduce the oxygen tension in sperm microenvironment that is, extender by partial deoxygenation process, as a number of literatures pointed out direct link of O2 with ROS production. When compared with other strategies, partial deoxygenation of semen extender with N2 gassing is found as a cost-effective, comparatively easy and a potential approach to large-scale frozen semen production.

MP75-20 HIGHER SPERM LEUKOCYTE COUNT IS ASSOCIATED WITH INCREASED SPERM DNA FRAGMENTATION INDEX IN MEN WITH NEGATIVE SEMEN CULTURES: A PATHOPHYSIOLOGICAL PATHWAY

INTRODUCTION AND OBJECTIVES:Semen leukocytes are thought to be involved in the reactive oxygen species (ROS)-mediated sperm damage. However, conflicting findings have been reported so far. Aim of t...

Mother-fetus immunogenetic dialogue as a factor of progeny immune system development

Despite the advances in medicine, about 4 million children under the age of 6 months die annually around the world due to infection, which is 450 deaths per hour (UNISEF, 2009). The degree of development of the immune system of children born in time is determined by many factors, including the immunogenetic similarity or difference of mother and fetus organisms, which, in turn, is due to the genotypes of mating pairs, as well as the selection of surrogate mothers duringin vitrofertilization. From our review of the literature, it follows that immunogenetic interactions of mother and fetus organisms, which occur at all stages of pre- and postnatal development, have a signifcant effect on the resistance of offspring to infections and allergens. Before implantation, the mother’s immune responses are formed under the influence of semen fluid antigens, leukocytes and cytokines, as well as under the influence of the genes of the major histocompatibility complex, which are expressed in embryos at the stage of two cells. After implantation, transplacental transfer of immunoglobulins and immunocompetent cells becomes of immunomodulating importance. It is important to emphasize that, although substances with a high molecular weight usually do not pass through the placenta, this rule does not apply to immunoglobulin G (IgG), which, with a molecular weight of about 160 kDa, overcomes the transplacental barrier due to binding to the fetal Fc receptor. The level of IgG in newborns usually correlates with the level of maternal antibodies. During the period of natural feeding, the immune protection of newborns is provided by the mechanisms of innate immunity and the factors of humoral immunity of mothers. It has been shown that immunoglobulins from the milk of many animal species are transferred through the neonatal intestinal epithelium to the blood. Since breast milk contains large amounts of various immunoactive components, including proteins, cytokines, hormones, immunoglobulins, exosomes containing micro-RNA, and viable immune cells, the immunomodulating effects of breast milk persist even after elimination of maternal immunoglobulins from the blood of the offspring, up to maturation. Analysis of a large body of experimental data shows that the study of mechanisms of “motherfetus” and “mother-newborn” interactions are the basis of a knowledge base needed to fnd means of life-long directed modulation of the descendants’ immune status.

Pregnancy Rate after Intrauterine Insemination with the Presence or Absence of Leukocytospermia in Sperms Prepared using Density Gradient Method

Objective: To examine the association between different concentrations of leukocyte and sperm recovery rate after sperms are prepared using density gradient method and pregnancy rate after intrauterine insemination (IUI). Increased leukocytes in semen have been associated with increased reactive oxygen species (ROS) that reduces sperm quality. Methods: Semen samples that were collected from 31 male partners of couples undergoing infertility investigation were analyzed for sperm concentration, motility, and leucocytes concentration. Semen samples were then divided in two groups based on their leucocytes concentrations (category A: >0 to 1 x 106/mL. Semen samples were processed using density-gradient centrifugation technique. Results: There was a significant difference in the number of sperms harvested and sperm motility after preparation. Interestingly, pregnancy rate after IUI was higher (p<0.05) in non-leukocytospermia semen (39%) when compared to leukocytospermia semen (30%). Conclusions: Seminal leukocytes (PMNL) concentration affects pregnancy rate after intrauterine insemination. Keywords: Density gradient method, sperm recovery rate, intrauterine insemination, pregnancy rate DOI: 10.15850/ijihs.v6n2.1318

Microbiological profiles of semen culture in male infertility

Background: Primary infertility and secondary infertility of men in the reproductive age represent 1.9 and 10.5%, respectively. Many etiological factors are involved, among which urogenital bacterial infections play an important role.Materials and Methods:Semen analysis, bacteriological culture, and sensitivity analyses were carried out to investigate the effect of genitourinary infections on semen parameters of infertile men.Results: Staphylococcus aureus was the most common isolated pathogen (46.2%) followed by urogenic gram-negative pathogens (24.1%). The isolated microorganisms are highly sensitive to piperacillin/tazobactam, imipenem, meropenem, gentamicin, doxycycline, amikacin, and nitrofurantoin. These antibiotics could be used empirically while awaiting the results of semen culture.Conclusion: Semen culture is an important diagnostic tool in all patients undergoing fertility investigations to detect genitourinary infections, pyospermia, and bacteriospermia. Moreover, early treatment should be considered according to the results of culture whenever possible. Wide range of broad-spectrum antibiotics can be used as an empirical treatment for infertile patients to adjust the seminal parameters and reduce the number of leukocytes in semen ejaculates.

Significance of differentiation of round cell count in semen in infertile men: A cost-effective study

Background: The significance of the presence of immature germ cells (IGCs) and leukocytes in semen have not very frequently been highlighted in the diagnostic criteria of male infertility. Round cell count differentiation aids in better clinical correlation. Aim and Objectives: To differentiate the round cells into IGCs and leukocytes in the semen and to find their correlation with the total sperm count and sperm motility. Materials and Methods: Semen samples from patients at Bharati hospital, Sangli for evaluation of infertility from July 2014 to January 2015 were analyzed as per the WHO criteria. After the consent and collection of the semen sample, routine semen analysis was done. Smears of semen were prepared and stained by Leishman stain. The round cells were counted and differentiated into IGCs and leukocytes. Results: We studied 103 cases. Semen parameters such as total sperm count, motility were noted. The smears were studied for differential counts of round cell count into IGC count and leukocyte. The cases were divided into four groups based on total sperm count. Maximum number of cases was found in group III. Our study shows that round cell count is 5%–10% of the total sperm count. Negative correlation was noted between the total sperm count and IGCs. The correlation of total sperm count and leukocytes was negative and significant. Conclusion: The present study shows that differentiation of round cells in the semen can be effectively differentiated into spermatogenic and nonspermatogenic cells with the use of simple and cost-effective stain like Leishman stain. The correlation of sperm count with the IGCs and leukocytes and sperm motility is of immense use to find out the possible cause of infertility.

Semen leukocytes and oxidative-dependent DNA damage of spermatozoa in male partners of subfertile couples with no symptoms of genital tract infection.

The influence of seminal leukocytes on generation of oxidative damage to sperm DNA was here investigated on male partners of subfertile couples asymptomatic for a genital tract infection. The study included 111 ejaculates from men attending the Andrology Centre at University of L'Aquila. Semen leukocytes subset included round cells expressing pan-leukocyte CD45 antigen, monocyte/macrophage lineage antigen CD14, and activated macrophages HLA-DR antigen. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression identified spermatozoa with DNA oxidative adducts while terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick end labeling (TUNEL) assay detected spermatozoa with DNA fragmentation. Flow cytometry and immunocytochemistry was used for determinations. Main outcome measure was the association of semen leukocyte subpopulations with spermatozoa showing oxidative-related DNA damage and with routine semen parameters. Leukocyte subpopulations were strictly correlated (p<0.0001), but no association was found between the concentration of leukocytes, semen parameters, the percentage of TUNEL-positive and of 8-OHdG-positive spermatozoa. The percentage of 8-OHdG-positive spermatozoa was positively correlated with the percentage of TUNEL-positive spermatozoa (r=0.48; p<0.0001) and negatively correlated with sperm concentration (r=-0.44; p<0.0001). Sperm concentration and the percentage of TUNEL-positive spermatozoa independently contributed (β=-0.25, p=0.008; β=0.23, p=0.05, respectively) to the variation in percentage of 8-OHdG-positive spermatozoa after adjusting for age, abstinence time, and smoking. In conclusion, oxidative-dependent DNA damage in spermatozoa was associated to poor semen quality but not to different leukocyte subpopulations in ejaculates of men asymptomatic for a genital tract infection.

Hemospermia is not associated with the production of antisperm antibody

Objective To investigate whether hemospermia is involved in the production of antisperm antibodies (ASA). Methods A total of 40 males with hemospermia were enrolled in this study. These males were divided into 3 groups according to their fertile status: 18 cases were patients who visited the infertility clinic (group A), 8 had fertility (group B), and 14 did not know their fertile status (group C). Semen samples were collected and ASA levels were detected by the direct immunobead test (D-IBT). Leukocytes in semen were assessed by the peroxidase assay. Results None of these males with hemospermia were found to display significant levels of ASA in the 3 groups. The number of cases who had increased levels of leukocytes in the semen samples of groups A, B and C were 15 (83.3%), 6 (75.0%) and 12 (85.7%), respectively. Conclusion Hemospermia is not associated with the production of ASA. It is worth noting that the leukocyte levels are increased in semen of males with hemospermia.

Involvement of seminal leukocytes, reactive oxygen species, and sperm mitochondrial membrane potential in the DNA damage of the human spermatozoa.

Measurement of reactive oxygen species (ROS) producing leukocytes in semen has been a standard component of the semen analysis, but its true significance remains still unknown. In this study, we have correlated the number of seminal leukocytes to various semen parameters. We found a negative correlation between the leukocyte number and sperm concentration (rs =-0.22; p=0.01) and motility (rs =-0.20; p=0.02). In contrast, a positive correlation between the number of leukocytes and both seminal ROS (rs =0.70, p<0.001; n=125) and the number of spermatozoa with DNA fragmentation (rs =0.43, p=0.032; n=25) was found. However, only a trend of positive correlation between ROS and the number of spermatozoa with TUNEL-detected DNA fragmentation was observed. Moreover, this latter was not correlated with loss of sperm mitochondrial membrane potential (MMP) (10% vs 35%, rs =0.25, p=0.08; n=50). Overall these results indicate that the presence of high number of leukocytes in the ejaculate negatively affects key semen parameters, as sperm concentration and motility, associated with infertility conditions. Moreover, they suggest that leukocytes are the major source of the seminal ROS and cause of sperm DNA fragmentation. However, the absence of a clear correlation between ROS and sperm DNA fragmentation, and spermatozoa with damaged DNA and MMP loss, suggest that ROS produced by leukocytes might be not the only cause of DNA damage in spermatozoa and that intrinsic mitochondrial-dependent apoptotic pathways might not have a major impact on sperm DNA fragmentation.