Leukocytes In Semen Research Articles

Hydrogels containing monocaprin have potent microbicidal activities against sexually transmitted viruses and bacteria in vitro.

OBJECTIVE: To investigate the in vitro microbicidal and cytocidal potency of monocaprin dissolved in pharmaceutical hydrogel formulations and to evaluate their potential use as vaginal microbicides against sexually transmitted pathogens...

Chlamydia trachomatis detected by ligase chain reaction in the semen of asymptomatic patients without pyospermia or pyuria.

To determine whether an absence of leukocytes in semen and urine predicts an absence of Chlamydia trachomatis in asymptomatic infertile men, chlamydial DNA was detected in subjects' semen and urine by ligase chain reaction (LCR). Ninety-eight infertile men were studied, including 39 cases of oligozoospermia, 19 of azoospermia, 16 of asthenozoospermia, and 24 of normozoospermia. None of the subjects had pyospermia or pyuria. C. trachomatis was detected by LCR. Antichlamydial and antisperm antibody were also measured. C. trachomatis was detected by LCR in the semen of only 1 of 98 patients (1.02%), but not in the urine samples. In C. trachomatis-positive patients LCR, IgG, and IgA levels were higher than normal. No antisperm antibody was detected. Even if leukocytes are not observed in the semen and urine of asymptomatic infertile men, the presence of C. trachomatis in semen specimens is rarely observed. Therefore, it should be noted that the presence of C. trachomatis in such cases is addressed in the context of artificial insemination and other assisted reproductive technologies.

Chlamydia trachomatis infection in male infertility--the clinical usefulness of the detection of antibodies against Chlamydia trachomatis

The objective of this study is to investigate the clinical usefulness of the detection of antibodies against Chlamydia trachomatis (C. trachomatis) in potential male infertility patients. Analysis of serum samples for the presence of antibodies against C. trachomatis by enzyme immunoassay was performed in 83 male infertility patients. Immunoglobulin A (IgA) and G (IgG)-antibodies against C. trachomatis were found in 16 and 10 of 83 serum samples (19.3 and 12.0%) respectively. IgA and/or IgG-antibodies against C. trachomatis were found in 20 of 83 serum samples (24.1%). Serum IgA, IgG, or IgA and/or IgG positive patients showed increased semen and urine leukocytes numbers and reduced semen volume and sperm numbers, compared with negative patients, respectively, although these effects are not statistically significant. By Spearman's correlation analysis, however, significant positive correlations with serum IgA or IgG antibody index were found in semen and urine leukocytes numbers. Significant negative correlations with serum IgA antibody index was found in semen volume. In serum IgA positive patients, only 1 of 8 first-voided urine samples was positive for C. trachomatis-DNA by PCR. Moreover, the positive rates of surum IgA antibody and cervical antigen in the 8 female partners of IgA positive patients were only 25% (2 of 8 serum samples) and 12.5% (1 of 8 cervical specimens) respectively. There was a high prevalence (24.1%) of serum IgA and/or IgG-antibodies against C. trachomatis from infertility patients. The existance of antibodies against C. trachomatis in serum samples possibly influence semen quality, suggesting that C. trachomatis infection may play an important role in male infertility.

Immature germ cells in semen and their correlations with other semen parameters.

The significance of the presence of leukocytes and immature germ cells in semen and other parameters of semen is currently a subject of controversy. Semen from 572 subfertile patients was analyzed according to WHO criteria and leukocytes as well as immature germ cells were assessed by identifying the round cells in semen by peroxidase staining. Microbiological investigation was carried out in cases with leukocyte counts of >1,000,000/ml. It was found that as the concentration of spermatozoa decreased the rate of immature germ cells increased and this increase was accompanied by a decrease in motility and in the number of spermatozoa with normal morphology. As the sperm count increased, motility, number of spermatozoa with normal morphology and of immature germ cells also increased whereas an increase in sperm motility was accompanied by an increase in the percentage of spermatozoa with normal morphology. Microbiological investigations were negative in patients with semen leukocyte counts of >l,000,000/ml. Although it is possible to establish that the leukocyte and immature germ cell counts correlate with other parameters of semen, these correlations are not statistically significant. The most significant finding is that as the number of sperms decreases, the ratio of immature germ cells to total germ cells increases. While assessing immature germ cells instead of giving special attention to the number of immature germ cells in semen, the ratio of immature germ cells to total germ cells should be considered. The increase of leukocyte count in the semen of oligospermic patients may not always mean leukospermia.

Analysis of human immunodeficiency virus in semen: indications of a genetically distinct virus reservoir

It is well established that HIV is found in semen, either as cell-free or cell associated virus, yet many questions remain about the source of the virus. A number of factors, including anatomic features of the male reproductive tract, the restricted access of the immune system to the germ cell compartment, and the results from sexually transmitted virus studies, suggest that the source of HIV in semen may be different from that in the peripheral blood. In this study, we examine the HIV in the infected cells of semen as indicators of the virus producing reservoir. The frequency of HIV positive leukocytes in semen is compared to that of concurrent blood samples from eight donors and these values are found to be highly variable and frequently discordant. The protease gene sequences of HIV strains isolated from semen cells and blood cells were determined and phylogenetic analyses were performed which indicate the virus populations in the two sources are genetically distinct. In one patient receiving anti-HIV protease inhibitor therapy, gene sequences indicative of protease inhibitor resistance were found in the blood, but not the semen cell compartment. These results suggest that HIV in the semen and blood compartments are distinct, and further, may respond differently to antiviral therapy.

VASECTOMY REVERSAL ASSOCIATED WITH INCREASED REACTIVE OXYGEN SPECIES PRODUCTION BY SEMINAL FLUID LEUKOCYTES AND SPERM

Purpose Reactive oxygen species, which are primarily produced by leukocytes, are generally detrimental to sperm. High reactive oxygen species levels are found in men with abnormal sperm function. Since men often have poor sperm characteristics and infertility after vasectomy reversal, fertile men to determine if reactive oxygen species were elevated in the former group. Materials and Methods We studied semen samples of men with proved fertility (39) and those with previously proved fertility who had undergone vasectomy reversal (45). The presence of leukocytes was determined by Bryan-Leishman staining. Reactive oxygen species endogenous activity was monitored by luminol dependent chemiluminescence in washed cells, including all cells in the semen, and Percoll density gradient purified sperm. Results After vasovasostomy men had significantly lower sperm concentration, motility and computerized motility measurements than fertile men. Mean reactive oxygen species in washed seminal cells after vasovasostomy was 684 relative light units per second compared to 49 for fertile controls (p <0.0001). Density gradient purified sperm had 53 and 0.64 relative light units per second, respectively (p <0.0001). When men with leukocytospermia were excluded from analysis, differences between the groups remained, although 9 times more reactive oxygen species were detected in men after vasectomy reversal with than those without leukocytes in semen. Conclusions Higher levels of reactive oxygen species are found in washed seminal cells and purified sperm after vasectomy reversal than in those of fertile men. Although leukocytes are probably a significant source of reactive oxygen species in these groups, they may not account for all of the increased reactive oxygen species after vasovasostomy. Low motility after vasectomy reversal may be related to the detrimental effects of reactive oxygen species produced by leukocytes or sperm, even in men without clinical leukocytospermia.

T lymphocytes and macrophages, but not motile spermatozoa, are a significant source of human immunodeficiency virus in semen.

The cellular fraction of semen contains spermatozoa, immature germ cells, leukocytes, and epithelial cells. Recent evidence implicates seminal cells as a major source of sexually transmitted human immunodeficiency virus (HIV) in semen, but the identity and infectious potential of infected cells remains poorly understood. HIV provirus was found in 75% of viable semen cell samples by polymerase chain reaction and in 88% of paired blood cell samples from HIV-seropositive men. When semen cell subpopulations were isolated by an immunomagnetic bead technique, T cells were found to be most commonly HIV-infected (75% of samples), followed by macrophages (38% of samples). Viral DNA was never detected in motile spermatozoa or immature germ cell populations. Semen leukocytes proliferated in response to mitogenic and antigenic challenge and produced p24 following stimulation with irradiated allogeneic cells. These data provide evidence that both T cells and macrophages, but not germ cells, are cellular vectors of HIV transmission in semen.

Leucocytes in semen from spinal injury patients

The objectives of the present study were to test the effect of coconut water solution (CWS), TES–TRIS and ACP-118® on the seminal cooling and cryopreservation of semen from capuchin monkeys (Sapajus apella). Semen was collected from six males by electro-ejaculation, diluted in TES–TRIS, CWS or ACP-118®, and maintained at 4 °C for 28 h. Semen was subsequently evaluated (Experiment I) or cryopreserved in the presence of different glycerol concentrations (3%, 5% or 7%) (Experiment II). ACP-118® was the preferred extender to preserve sperm motility and viability after 28 h incubation at 4 °C. Cooled sperm were successfully frozen–thawed in a medium containing 3% glycerol. After thawing, sperm retained the capacity to fertilize oocytes and zygotes were obtained. In conclusion, ACP-118® can be effectively and efficiently used as extender for the cooling of S. apella semen. Furthermore, cryopreservation using ACP-118® by adding 3% glycerol is suitable to maintain sperm morphology and the capacity of these cells to fertilize in vitro.

Interactions between leukocytes and the male reproductive system. The unanswered questions.

It is well established that leukocytes are often present in seminal fluid and consequently there exists an overwhelming potential for interactions between different types of leukocytes and spermatozoa. However, the precise role of leukocytes in the semen and the fate of spermatozoa are not fully understood. For comprehensive descriptions of the biological significance of leukocytes in semen we recommend reviews by Wolff (1995) and Aitken and Baker (1995) and for a discussion of the immunosuppressive mechanisms in semen we suggest a recent review by Kelly (1995). The purpose of this mini-review is to highlight some of the pertinent, largely unanswered, questions regarding the role of interactions between inflammatory cells and the male reproductive system. In addition, we discuss the ill-defined fate of senescent spermatozoa in comparison to the relatively well understood fate of apoptotic somatic cells.

Glass wool filtration reduces reactive oxygen species by elimination of leukocytes in oligozoospermic patients with leukocytospermia.

Two types of glass wool were used to remove leukocytes in semen for evaluation of reactive oxygen species production by spermatozoa in oligozoospermic patients with leukocytospermia. Semen samples were prepared using fine-structure glass wool (SpermFertil) and coarse-structure glass wool. In each treatment group, native semen was evaluated for sperm concentration, percentage motility, viability, leukocyte concentration, and production of reactive oxygen species. Electron microscopically, SpermFertil showed a higher number of leukocytes attached to the fibers compared to coarse-structure glass wool. Leukocytes in native semen and after glass wool filtration as determined by peroxidase cytochemistry confirmed this observation. Reactive oxygen species decreased from 45.303 counts/10(7) viable cells in native semen to 15.806 counts/10(7) cells in coarse structure wool and 7.465 counts/10(7) cells in Spermfertil, respectively. Removal of leukocytes from semen of oligozoospermic patients by means of glass wool filtration is a useful method to distinguish production of reactive oxygen species by leukocytes versus sperm cells.

Evaluation of the Infertile Couple-Reply

In Reply. —Dr Seaman and colleagues appeal for a more complete investigation of the male partner as a prerequisite to the diagnosis of unexplained infertility. I agree with their contention that the male partner should see a physician. The male partner's physician might be a family physician, a urologist, or an andrologist. Alternatively, as in the Clinical Crossroads article, both partners might consult an infertility specialist. Whatever the choice, the physician should have an interest in the broad range of issues that arise in the management of infertility and should be prepared to participate in a coordinated treatment plan that meets the wishes of the couple. In the discussion section of the article, the definitions for a normal semen analysis were based on the World Health Organization laboratory manual. 1 This manual cites normal values for leukocytes in semen, an analysis that should be done routinely, and it includes methods

Enumeration of semen leucocytes by fluorescence in situ hybridisation technique

Aim-To determine whether the fluorescent in situ hybridisation technique (FISH) using a total human DNA genomic probe can be used to enumerate semen leucocytes.Methods-Semen samples from five donors were subjected to a mild KC1 solution. These samples were then biotin labelled under FISH conditions using a total human DNA genomic probe and the leucocyte counts were determined. To check the accuracy of the technique a monoclonal antibody against the common leucocyte antigen CD45 [KC56(T-200)] served as a control. An isotypic control for [KC56(T-200)], the immunoglobulin [MsIgG1], served as a secondary control.Results-Semen leucocytes stained by the FISH technique were easily detected because of their distinct bright yellow colour, while the sperm cells were red. The leucocyte count ranged from 0.5 to 4.9 x 10(6) per ml of semen. KC56(T-200) and its isotypic control MsIgG1, which served as control for the FISH technique, accurately identified 94% and 97% of the semen leucocytes of a control donor, respectively.Conclusions-The FISH technique using a total human DNA probe can accurately and effectively enumerate the overall leucocyte population in semen.

INFECTION AND PYOSPERMIA IN MALE INFERTILITY: Is It Really a Problem?

Controversy exists concerning the exact role of genitourinary tract infections in male infertility. This ambiguity is due to the currently conflicting literature coupled with present limitations in obtaining and evaluating the significance of diagnostic tests such as semen cultures and leukocyte counts. In the evaluation of the infertile man for a possible infectious cause, a complete history, physical examination, and urinalysis identify most infectious disorders. In the majority of infertile men who are asymptomatic, evaluation for a subclinical genital tract infection focuses on the accurate determination of seminal leukocytes. The determination of pyospermia may identify those men with a subclinical genital tract infection who require further investigation. Prospective studies using clearly defined criteria, new diagnostic tools, and effective control groups are needed to evaluate better the impact of infection and pyospermia on male infertility.

Prospective study of leukocytes and leukocyte subpopulations in semen suggests they are not a cause of male infertility

To determine the effects of leukocytes in semen on sperm quality and the ability to achieve conception. A prospective analysis of 512 couples attending a regional infertility clinic. Leukocyte subsets were quantified using a monoclonal antibody-based staining procedure. In addition to basic seminal parameters (density, motility, morphology, and antisperm antibodies), reactive oxygen species and immature germ cells were also quantified in the semen of each patient. The presence or absence of a treatment-independent conception was determined 22 months after the start of the study. Semen parameters were then related to the ability to conceive. University-based center for reproductive medicine. Success or failure to conceive was recorded from 512 couples. Couples were then selected to minimize the influence of any pathology of the female on outcome. A final study group of 229 couples, in which the women had regular menstrual cycles, ovulatory midluteal serum P levels of > 18 nmol/L, and patent fallopian tubes was finally selected for analysis. Pregnancy. Leukocyte concentration (total or individual subsets) was not associated with either reduced semen quality or conception rates. Similarly, neither reactive oxygen species or antisperm antibody (immunobead) concentration had any bearing on the outcome. Of all semen parameters measured, only the level of immature germ cells was found to be negatively associated with the rate of conception. Measurement of seminal leukocytes in routine semen analysis appears to be of little prognostic value with regard to male fertilizing potential. As reactive oxygen species and antisperm measurement were of similar predictive value, the term "immunologic male infertility" should be redefined.

Subpopulation of leucocytes in the semen from fertile and infertile men--immunohistochemical and flow-cytometric analysis

It is hard to differentiate leucocytes from immature germ cells among many round cells in ejaculated semen. Recently monoclonal antibodies (MoAbs) which recognize surface antigen of various subtypes of leucocytes are available. In this study we applied a panel of MoAbs for leucocyte subpopulations (granulocytes, T-lymphocytes, B-lymphocytes, Monocytes/Macrophages and pan-leucocytes). Semen smears from 64 mild oligozoospermic patients and 5 fertile volunteers were stained by immunohistological technique (avidin-biotin-immunoperoxidase method). We found highly varying total leucocyte numbers ranging from 1200 to 2.2 x 10(8) per ejaculate. When comparing semen from infertile patients with those from fertile donors, statistically significant higher medians of total leucocytes, total granulocytes and monocytes/macrophages were seen in infertile group. We compared a total number of leucocytes obtained from conventional Papanicolaou staining with that from immunohistochemical staining. Both were almost identical but the former gave a little bit higher value, because of the difficulty in differentiating leucocytes from immature germ cell by Papanicolaou staining. Moreover we performed an experiment to evaluate the applicability of flow-cytometry (FCM) to count leucocytes in semen. Cells in the semen smears were stained with MoAbs recognizing the surface antigen shared by every leucocyte, and percentage of the leucocytes which were thus stained with MoAbs was compared with that determined by FCM. Results from FCM constantly gave lower value than these from immunostaining. This is probably because FCM could not differentiate leucocytes from contaminated non cell substances (e.g. cell debris). From the present study it is not adequate to use FCM for the detection of leucocytes in semen as applied in haematology. Further techniques to remove non-cellular substances should be established for its application in semen analysis.

Differentiation of round cells in semen by means of monoclonal antibodies and relationship with male fertility

To differentiate round cells in semen samples of subfertile men and evaluate the clinical significance during infertility investigation. One hundred and eight randomly chosen couples with a median duration of infertility of 4 (range, 1 to 20) years presenting at the outpatient infertility clinic of the University of Heidelberg, Germany. Differentiation of round cells in semen by means of monoclonal antibodies (mABs) and a streptavidin-biotin system for staining. Correlation of results with medical history, outcome of clinical examination, sperm analysis, microbial screening of both partners, evaluation of sperm functional capacity in vivo by means of the postcoital test (PCT) and in vitro with the standardized crossed sperm-cervical mucus penetration test (SCMPT) and the subsequent fertility in a prospective study. The method used for differentiation of round cells proved to be practical and suitable for routine use. The percentage of leukocytes ranged from 0% to 58% with a median of 3%. Number of round cells and percentage of leukocytes did not differ markedly with regard to andrologic history, clinical findings, for example, varicocele, results of standard sperm analysis, and microbial colonization of semen samples. However, high rates of leukocytes of the round cells correlated with reduced sperm count and morphology and results of PCT. Leukocyte-positive (> 15% leukocytes) specimens were also significantly more frequent in case of inadequate SCMPT and reduced sperm penetration ability in vitro. In asymptomatic patients (in terms of genital tract infection), the majority of round cells consist of immature germ cells and < 5% are white blood cells. The streptavidin-biotin system and the mABs used in this study proved to be useful to identify patients with elevated rates of leukocytes in semen possibly reflecting subclinical genital tract infection with influence on sperm functional capacity and subsequent fertility. Thus the procedure can be recommended to be included in a comprehensive evaluation of male fertility.

Prognostic factors in assessment and management of male infertility.

Evaluation of 304 infertile couples with at least one abnormal semen analysis (sperm density < 20 x 10(6)/ml and/or motility < 50%) and no apparent female factors was performed in a multicentre prospective cohort study. In 73 cases therapeutic donor insemination was performed (TDI group) with a resulting pregnancy rate of 48%. The remaining 231 couples (non-TDI group) had an overall pregnancy rate of 25%. The TDI group had a shorter duration of infertility. The ages of both partners were comparable in TDI and non-TDI groups. In the non-TDI group, univariate analysis resulted in identification of six clinical variables associated with a change in pregnancy rates. The strongest association was noted for length of infertility. There was a weaker association for semen volume, concentration of leukocytes in semen, history of pregnancy in the female partner and laparoscopy. Multiple variable analysis of data from the non-TDI group revealed that independent predictors of pregnancy were 'duration of infertility' and 'history of pregnancy in the female partner'. The multiple variable modelling suggested that (i) an increase in the length of infertility by 1 month prolongs the time to pregnancy by an additional 1.6% (95% confidence interval: 1.5-1.7%); and (ii) a history of past pregnancy in the female partner reduces the time of pregnancy by 51% (95% confidence interval: 47-56%).

Fertility Parameters in Men Infected with Human Immunodeficiency Virus

The effect of human immunodeficiency virus type 1 (HIV) infection on semen parameters that assess fertility was investigated in 50 semen specimens from 21 asymptomatic or minimally symptomatic HIV-seropositive men and 3 specimens from 3 men with AIDS. HIV was isolated from 15 (30%) of 50 specimens from asymptomatic or minimally symptomatic persons and from 1 of 3 specimens from patients with AIDS. The men with AIDS all had pyosemia and grossly abnormal sperm. In contrast, semen specimens from other seropositive men did not differ significantly from semen specimens from healthy seronegative semen donors. No abnormality in sperm count, morphology, numbers or types of leukocytes in semen, or other seminal parameters was associated with HIV shedding in semen. Zidovudine therapy did not affect sperm morphology or seminal characteristics. Thus, although patients with AIDS had abnormal semen, the laboratory parameters that assess fertility were not affected by shedding of HIV in semen or concomitant therapy with zidovudine.

An automated chemiluminescence test for diagnosis of leukocytospermia

A new rapid and sensitive chemiluminescence (CL) test was developed for the analysis of leucocytes in semen. Opsonized zymosan induced luminol-dependent CL of diluted (1:500) semen was measured in samples from 64 fertile and infertile men with or without leukocytospermia, using an automated luminometer set-up. The white-blood-cell (WBC) count in semen was determined using a conventional leucocyte-peroxidase staining method. A good linear correlation (r = 0.932) was observed between the seminal white-blood-cell number and the CL response. The coefficients of inter-assay variations for the CL method were 9.6% and 1.8% for semen samples with 0.3 x 10(6) WBC ml-1 and 3.2 x 10(6) WBC ml-1, respectively. The results also suggest that the previously reported inhibitory effect of seminal plasma on CL activity of human phagocytes is due to the quenching of light-producing reactions and that this can be circumvented by using appropriate semen dilution. It is concluded that the simple and high-capacity CL test is an especially convenient method for routine diagnosis of leukocytospermia.

Differential diagnosis of immature germ cells in semen utilizing monoclonal antibody MHS-10 to the intra-acrosomal antigen SP-10

The monoclonal antibody (mAb) MHS-10 (IgG1) is a mouse antihuman sperm antibody which recognizes a polymorphic sperm protein, (SP-10), which has previously been localized within the acrosomal matrix and the acrosomal membranes. The SP-10 antigen has been shown to be sperm-specific and is not found in somatic tissues. It is stage specific, having been immunohistologically localized to Golgi phase spermatids and all subsequent phases of spermiogenesis. In the present study, acetone-dried smears from washed human semen containing significant numbers of round cells were probed with mAb MHS-10. Monoclonal antibody-labeled cells were visualized by a standard streptavidin-biotin immunoperoxidase method using a light microscope. The MHS-10 mAb immunoreacted with mature sperm and with a subset of round cells diagnosed as developing spermatids, which had been sloughed off from the testis at varying stages of acrosome formation. To rule out possible cross-reactivity of the mAb with leukocytes in semen, a leukocyte surface marker (anti-HLe-1) was used in conjunction with MHS-10. Round cell populations staining with MHS-10 did not stain with anti-HLe-1. The mAb MHS-10 is thus a promising probe for the identification and quantitation of immature germ cells in human semen.