- Email: [email protected]
Determination of neutrophil concentration in semen by measurement of superoxide radical formation*
FERTILITY AND STERILITY
Vol. 56, No.5, November 1991
Copyright © 1991 The American Fertility Society
Printed on acid-free paper in U.S.A.
Determination of neutrophil concentration in semen measurement of superoxide radical formation*
by
Neil Kovalski, M.D. Eve de Lamirande, Ph.D. Claude Gagnon, Ph.D.t Urology Research Laboratory, Royal Victoria Hospital and Faculty of Medicine, McGill University, Montreal, Quebec, Canada
Objective: To develop an assay that measures the concentration of functional neutrophils in human semen. Design: Human blood neutrophils were first isolated to establish linearity and proportionality for the determination of functional neutrophil concentration. Thereafter blood neutrophils in seminal plasma and neutrophils in semen of patients were measured. Setting: Blood and human samples were obtained from clinics of the Royal Victoria Hospital. Patients, Participants: Blood from normal men or from patients presenting with chest pain or trauma were used. Semen samples were also obtained from healthy fertile donors or from unselected patients attending the infertility clinic. Main Outcome Measures: Active neutrophil concentration in semen by a colorimetric assay. Results: The assay developed is based on the reduction of nitroblue tetrazolium of pale yellow color to blue formazan by the superoxide anions produced by stimulated neutrophils. The intensity of the derived blue color is proportional to the concentration of active neutrophils. This assay is simple, requires only 10 minutes of preparation time, and detects neutrophil concentrations > 0.5 X 106 neutrophils/mL of semen, independent of sperm concentration. Fertil Steril 56:946, 1991
The relevance of leukocytes in semen remains controversial. Multiple studies have presented conflicting results in their attempts to correlate leukocyte concentration to the presence of subclinical genital infection. 1- 3 One of the key problems in any such study relates to the difficulty in distinguishing leukocytes from sperm precursor cells in the ejaculate. Furthermore, the presence of multiple leukocyte subpopulations implies that any form of correlation made with total leukocyte number would likely be inadequate. Among the commonly used methods for determining leukocyte concentration in the ejaculate are myeloperoxidase stains, 4 indirect immunoperoxidase
Received January 28, 1991: revised and accepted July 16, 1991.
* Supported by a grant from the Medical Research Council of Canada to C.G. t Reprint requests: Claude Gagnon, Ph.D., Urology Research Laboratory, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1Al.
946
Kovalski et al.
Neutrophil concentration in semen
stains employing specific monoclonal antibodies for leukocyte subpopulations, 1•5 and granulocyte elastase level determinations. 2•3 These tests yield a static value for leukocyte concentration, but they do not provide any clear indication of leukocyte viability or activity. A newer approach to detecting the presence of leukocytes in any cell suspension is based on the ability of leukocytes to generate reactive oxygen species. These reactive oxygen species (such as superoxide anion ["02], hydrogen peroxide [H 2 0 2 ], hydroxyl radical ["OH], and hypochlorite radical ["OHCl]) are considered to be responsible for much of the target cell and/or tissue injury induced by leukocytes. Ryan et al. 6 used flow cytometry to measure fluorescence as an indicator of reactive oxygen species output from immune-complex stimulated human blood neutrophils and went on to study in detail the kinetics of the production of reactive oxygen species by neutrophils. The system of Ryan et al., 6 although expensive and technically demanding Fertility and Sterility
to operate, offers the opportunity to quantify and then correlate neutrophil generation of reactive oxygen species to neutrophil concentration. As such, one has. a method for comparing neutrophil populations on the basis of their cytotoxic capability rather than just according to the sheer number of cells present. The aim of the present study was to develop a quantitative and functional test of neutrophil concentration that would be applicable to semen, based on the same principle of the neutrophil's capacity to produce reactive oxygen species. However, to be suitable for an infertility clinic, such a test would necessarily be inexpensive, technically simple, fast to use, easy to interpret, and require minimal technician time per sample. It was realized that the cost and technical complexity of this new test would rest primarily on the choice of neutrophil stimulating agent and reactive oxygen species output detecting system. Because of its key characteristic of being reduced by "02 to yield blue formazan (clearly distinguishable blue color), nitroblue tetrazolium (NBT; pale yellow color) was selected as the indicator of reactive oxygen species produced by the neutrophils. This characteristic of NBT was translated into a colorimetric assay for neutrophil function. To stimulate the neutrophils to produce reactive oxygen species, phorbol myristate acetate, a protein kinase C activator, was chosen for its stability, efficacy, ready availability, and low cost. In this paper, we describe and validate a new assay (referred to henceforth as the NBT assay) of neutrophil concentration in human semen. In contrast to most previous tests, the present assay measures the functional capacity of the seminal neutrophils. The NBT assay clearly detects neutrophils at concentrations ~ 0.5 X 106 /mL. This level of sensitivity is appropriate for detecting the 1.0 X 106 neutrophil polymorphonuclear leukocytes (PMNL)/mL considered by the World Health Organization (WH0)1 to be an indicator of possible genital tract infection. MATERIALS AND METHODS Reagents
Percoll was purchased from Pharmacia Fine Chemicals (Dorval, Quebec, Canada). Phorbol 12myristate 13-acetate (PMA) was purchased from ICN Biochemicals (Cleveland, OH). Nitroblue tetrazolium, cetylpyridinium chloride (CPC), the Sigma Diagnostics Leukocyte Myeloperoxidase Stain (procedure no. 390), and bovine serum albumin (BSA) were obtained from Sigma Chemical ComVol. 56, No. 5, November 1991
pany (St. Louis, MO). Superoxide dismutase (SOD) was purchased from Calbiochem Corporation (La Jolla, CA). Diethylenetriaminepentaacetic acid (DETAPAC) was obtained from Aldrich Chemical Company (Milwaukee, WI). Blood Neutrophil Separation
Peripheral blood neutrophils were isolated from ethylenediaminetetraacetic acid-anticoagulated venous blood of normal human donors and from patients presenting to the emergency room for treatment of chest pain or trauma. Twelve milliliters of whole blood were divided into six identical aliquots. To each aliquot was added 10 mL of 0.87% NH4Cl. All six mixes were then allowed to stand for 20 minutes at room temperature (RT) to induce an osmotic lysis of the erythrocytes. The hemolyzed blood was then centrifuged at 600 X g at RT to yield six leukocyte-enriched pellets. The supernatants were removed, and each of the six pellets was resuspended in 2 mL of 0.87% NH4Cl. All six cell suspensions were pooled and then layered onto a single discontinuous Percoll gradient made up of 1 mL 55%, 1.5 mL 40%, 1 mL 30%, and 1 mL 20% Percoll. All Percoll solutions were buffered with Hepes balanced saline (130 mM NaCl, 4 mM KCl, 2.5 mM CaC12 , 14 mM fructose, and 10 mM N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid [Hepes] adjusted to pH 8) and supplemented with 10 mg/mL ofBSA. The Percoll gradient, with cell suspension on top, was then centrifuged at RT at 1,300 X g for 30 minutes. Leukocytes were recovered from the 20% to 30% interface (L1), 30% to 40% interface (L2), and the 40% to 55% interface (L3, neutrophil layer). All interfaces were devoid of contaminating erythrocytes, but only L3 was devoid of debris. Cell viability was determined by trypan blue exclusion. Semen Collection and Processing
Semen samples collected by masturbation after 3 days of sexual abstinence were obtained from healthy, fertile donors and patients attending the Infertility Clinic at the Royal Victoria Hospital. Aliquots of 200 #LL of liquefied semen were layered onto identical discontinuous Percoll gradients 1 hour after semen collection. Each of these gradients consisted of 30 #LL 80% and 500 #LL 20% Percoll in Hepes balanced saline and was prepared in a microcentrifuge tube (1.5 mL capacity). These gradients, with semen sample on top, were spun at 6,500 X g for 1 minute at RT in a Johns Scientific Microcentaur microcentrifuge (Biotech, Montreal, Quebec, CanKovalski et al.
Neutrophil concentration in semen
947
ada). The upper 530 ~tL of each centrifuged sample was then discarded to yield a 200-~tL seminal plasma-free cell isolate of the original semen. We refer to this process of separating seminal plasma from the cellular components as centrifugation-separation. The remainder of each original semen sample was then individually layered onto a separate Percoll gradient identical to those used for the neutrophil separation from whole blood. The 20% to 30%,30% to 40%, and 40% to 55% interfaces were collected and assessed to determine if this more refined separation technique had any benefits over that using the microcentrifuge. Myeloperoxidase Stain for Neutrophils
Myeloperoxidase staining for neutrophils was performed according to WHO. 7 One milligram of the Peroxidase Indicator Reagent {p-phenylenediamine diHCl [1 part] and catechol [2 parts]) and 4 ~tL of 3% H 2 0 2 were added to 500 ~tL of TRIZMAL 6.3 buffer at RT. Then, 180 ~tL of this solution were mixed with 20 ~tL of the cell suspension being tested. This mix was incubated in the dark at 37°C for 30 minutes. With the hematocytometer, the individual concentrations of the variously stained leukocytes were determined: neutrophils show brown-black intracellular granulation, monocytes stain less intensely, and lymphocytes (and immature germ cells) do not show peroxidase activity. Measurement of Reactive Oxygen Species Output From Blood Neutrophils With NBT
Various concentrations of freshly isolated (within 4 hours of blood collection) human blood neutrophils were incubated at 37°C with 8 X 10-8 M PMA and 0.1 mM NBT in a final volume of 200 JLL. Reactive oxygen species production was followed by measuring the formation of blue formazan at 630 nm with an MR 580 Dynatech Microelisa Auto Reader spectrophotometer (Chantilly, VA) at incubation times 0, 8, 15, 22, 30, 45, 60, 90, and 120 minutes. Assay of NBT for Blood Neutrophils in the Presence of Spermatozoa
Purified human blood neutrophils were incubated with 8 X 10-8 M PMA and 0.1 mM NBT in the presence and absence of different concentrations of Percoll-washed human spermatozoa from healthy fertile donors. 8 As controls for sample turbidity secondary to the spermatozoa themselves, different concentrations of spermatozoa were incubated alone 948
Kovalski et al.
Neutrophil concentration in semen
in BSA-supplemented Hepes balanced saline buffer. After an incubation period of 60 minutes, the absorbance at 630 nm was measured. In another series of experiments, purified human blood neutrophils were incubated in the absence and presence of 200 X 106 spermatozoa/mL with 8 X 10-8 M PMA and 0.1 mM NBT in a final volume of 200 JLL. Controls were 200 X 106 spermatozoa/mL alone in BSA-supplemented Hepes balanced saline buffer. After 60 minutes of incubation at 37°C, 10 ~tL of 50 mM DETAPAC {pH 8), 100 ~tL of 20% CPC, and 200 ~tL of 3 M MgC12 were added, in sequence, to and vigorously mixed with each of the incubated cell preparations to solubilize all cellular material and precipitated blue formazan. An effective iron chelating agent, DET APAC is essential to eliminate the direct reduction of NBT by intracellular components that are released into the milieu by solubilization. 9 To maximize solubilization, the mixtures (each now with a volume of 500 ~tL) were then incubated for another 15 minutes at 37°C after which they were again vigorously mixed. All mixtures were then centrifuged for 3 minutes at 11,000 X g in the microcentrifuge. A 300 ~tL aliquot was removed from each mixture (leaving the postcentrifugation pellet undisturbed), placed in an enzyme-linked immunosorbent assay plate well, and kept at 37°C. Absorbance at 630 nm was determined as described above. Assay of NBT for Human Semen Neutrophils
Duplicate seminal plasma-free cell isolates were prepared from each original semen sample and incubated in the presence and absence of PMA and NBT. After 60 minutes of incubation at 37°C, the absorbance at 630 nm was measured. The NBT assay was carried out with one cell isolate designated as the control (without PMA and NBT) and the duplicate isolate mixed with PMA and NBT, thereby designated as the stimulated sample. Statistical Analysis
Regression analysis was used to evaluate the correlation between the variables analyzed. The 0.05 level of probability was used as the criterion of significance. RESULTS Isolation of Blood Neutrophils on Discontinuous Percoll Gradients
After NH 4 Cl hemolysis of the erythrocytes in the original whole blood and centrifugation to yield the Fertility and Sterility
blood leukocyte enriched fraction, further purification was carried out by centrifugation on Percoll gradients made of 55%, 40%, 30%, and 20% Percoll layers. The L3 interface (40% to 55% Percoll) was comprised of dark brown-staining leukocytes, consistent with a neutrophil population of purity> 98%. The L1 (20% to 30% Percoll) and L2 (30% to 40% Percoll) interfaces were comprised mostly of intermediate and non-staining cells consistent with monocyte and lymphocyte populations, respectively. The ratio of neutrophil concentration between interfaces was 8:1:1 (L3:L2:L1). Preincubation ofblood neutrophils in seminal plasma followed by centrifugation-separation in the microcentrifuge allowed a >98% recovery of neutrophils and yielded no change in staining pattern. Attempted staining in the presence of seminal plasma resulted in markedly reduced coloration of the cells. All neutrophil populations, recovered from the L3 interface, were :2::98% viable. Final neutrophil recovery (total of L1, L2, and L3 interfaces) was an average of 80% of the original whole blood neutrophil content. Measured Reactive Oxygen Species Output of Human Blood Neutrophils With NBT
Phorbol 12-meristate 13-acetate-stimulated human blood neutrophils, incubated in the presence of NBT, generated a progressive increase in absorbance, indicative of superoxide radical production.
I 150 Time (minutes)
Figure 1 Time course of measured superoxide output from human blood neutrophile. Human blood neutrophile, isolated on a Percoll gradient, were incubated in the presence of NBT, with and without PMA, at 37°C. The formation of blue formazan was followed by measuring the absorbance at 630 nm at 0, 8, 15, 22, 30, 45, 60, 90, and 120 minutes of incubation. Data are presented for 2 X 106 and 4 X 106 neutrophils/mL. All values are expressed as a percentage of the maximum absorbance recorded for 4 X 106 PMA stimulated neutrophils/mL. •-•, 2 X 106 unstimulated neutrophils/mL; +- +, 2 X 106 stimulated neutrophils/mL; X- X, 4 X 106 unstimulated neutrophils/mL; A- A, 4 X 106 PMA-stimulated neutrophils/mL. Vol. 56, No. 5, November 1991
NeutrophD concentration (mlDion/ml)
Figure 2 Correlation between measured superoxide output from PMA-stimulated neutrophile and neutrophil concentration. Blood neutrophile were incubated with NBT and PMA for 60 minutes at 37°C. The formation of blue formazan was determined by measuring the absorbance at 630 nm. These samples mixes were assessed directly without the addition of DETAPAC or solubilization with CPC and MgCl2 • Values depicted are the means ± SEM for eight experiments. The correlation coefficient for 0 to 4 X 106 neutrophils/mL was 0.97.
Absorbance change, for any given sample, plateaus at its maximum by 30 to 45 minutes of incubation (Fig. 1). This time course is consistent with that previously reported for reactive oxygen species output from PMA-stimulated human neutrophils, as determined by chemiluminescence with luminol. 10 The absorbance of the formed blue formazan was linearly correlated to neutrophil concentration, within the range of 0 to 4 million neutrophils/mL (Fig. 2). Such a linear correlation over this range of neutrophil concentration was previously reported with PMA-stimulated mouse neutrophils, using chemiluminescence with luminol and polynoidon to determine reactive oxygen species output. 11 Equal concentrations of neutrophils, recovered from the L1 and L2 interfaces, generated 50% less absorbance change, upon PMA stimulation, than the equivalent concentration of neutrophils from the L3 interface. The absorbance change induced by stimulated neutrophils (L3 interface), at concentrations up to 10 X 106 /mL, was reduced by >95% in the presence of superoxide dismutase (660 U /mL) or seminal plasma (50% vol/vol). This effect of seminal plasma on absorbance change was consistent among different donors (n = 10) and different infertile patients (n = 10). This effect was unchanged by freezing and thawing of the seminal plasma. Preincubation of neutrophils with 50% (voljvol) seminal plasma followed by centrifugation -separation in the microcentrifuge had no effect on the generated absorbance change after PMA stimulation. Kovalski et al.
Neutrophil concentration in semen
949
0.4
100 :::;-
~
=
80
0.3
0
... C)
0
~ e..
4>
4>
a ~
20
-e ~
a
60 40
C)
C)
-e0
0.2
"'
~
0.1
o~~--~-.~-.~~.-~.-~
0 100
0
200
0
300
Spenn concentration (million/mil
Figure 3 Effect of sperm concentration on measured superoxide output from PMA -stimulated neutrophils. Various concentrations of washed spermatozoa were incubated with a fixed concentration of Percoll-washed blood neutrophils in the presence of NBT and PMA. The formation of blue formazan was determined by measuring the absorbance at 630 nm. Values depicted are the means ± SEM for four experiments. The value of 100% corresponds to an absorbance of 0.43 generated by 6 X 106 neutrophils/mL incubated without spermatozoa.
1
2
3
4
5
6
Neutrophil concentration (million/mil
Figure 4 Correlation between measured superoxide output generated by PMA-stimulated neutrophils and neutrophil concentration in the presence of 200 X 106 spermatozoa/mL. After the 60-minute incubation period at 37°C with PMA and NBT, all samples of human blood neutrophils, both with and without spermatozoa, were mixed with DETAPAC, CPC, and MgCl2 , and incubated for another 15 minutes at 37°C before the determination of absorbance at 630 nm. Values depicted are means ± SD for five experiments. The correlation coefficient between 0 to 3 X 106 neutrophils/mL was 0.99. D-D, neutrophils alone; +-+, neutrophils + 200 X 106 spermatozoa/mL.
Effect of Spermatozoa on the NBT Assay
In the presence of spermatozoa, blue formazan precipitated as it was being actively generated from NBT by stimulated neutrophils. As such, the absorbance change generated by samples of PMAstimulated neutrophils, incubated with spermatozoa, was markedly reduced as compared with samples of PMA-stimulated neutrophils incubated without spermatozoa (Fig. 3). However, after the addition of DETAPAC, solubilization with CPC and MgCl2 , and final microcentrifugation, the absorbance change (defined as the absorbance of the stimulated sample minus the absorbance of the control sample) for any given concentration of neutrophils was now independent of initial sperm concentration in the incubation mixture (data shown for 200 X 106 spermatozoa/mL) (Fig. 4). Furthermore, the marked turbidity observed with high concentrations of spermatozoa was reduced by >90%. For at a least 1 hour after solubilization, the blue color generated remained clearly measurable and easily distinguishable to the eye.
patients had neither positively staining cells nor a measurable absorbance change by NBT assay. Five patients had positively staining cells, judged to be neutrophils, but at concentrations < 0.5 X 106 /mL. Two patients had positively staining cells, read as 2 X 106 neutrophils/mL. With the NBT assay, the seminal neutrophil concentration of these two patients was determined to be 1.0 X 106 and 2.0 X 106 neutrophils/mL, respectively. These results were independent of Percoll centrifugation technique, that is, minigradient of 30% to 80% Percoll layers versus larger gradient of 20%, 30%, 40%, 55% Percoli layers.
Table 1 Detection of Neutrophils in Human Semen by the NBT Assay and Myeloperoxidase Staining Neutrophils
Patient group"
With myeloperoxidase staininif
WithNBT assay'
10'/mL
Myeloperoxidase Staining and NBT Testing of Semen Round Cells
An unselected population of 67 patients from the Infertility Clinic, all presenting with round cells in their ejaculate, as noted by light microscopy during routine semen analysis, was studied. Neutrophil concentration was assessed both by myeloperoxidase staining and the NBT assay (see Table 1). Sixty 950
Kovalski et al.
Neutrophil concentration in semen
1 (n = 2 (n = 3 (n = (n =
60)
0
5) 1) 1)
<0.5 2.0 2.0
0 0 1.0
2.0
a Semen samples were obtained from 67 unselected patients, consulting for infertility. b Only myeloperoxidase-stained cells that were dark brown were judged to be neutrophils. ' Each NBT assay was assessed visually and spectrophotometrically.
Fertility and Sterility
To further validate the NBT assay for neutrophils, different quantities of purified blood neutrophils were added to semen samples from five normal volunteers who had no endogenous neutrophils. The concentration of blood neutrophils in semen was determined by the NBT and myeloperoxidase assay. Values obtained were then used to calculate the original blood neutrophil concentration. Although the calculated initial concentration of blood neutrophils was referred at 100%, the NBT and myeloperoxidase assays estimated the concentration at 111% ± 8% (mean ± SEM) and 105% ± 8%. These values were not significantly different from each other. DISCUSSION
The production of reactive oxygen species by PMA -stimulated neutrophils is an index of function that can be quantitated and correlated to neutrophil concentration. The advantages of NBT as the reactive oxygen species output indicator are multiple. Nitroblue tetrazolium is inexpensive, stable, and readily available. As well, the conversion of NBT to blue formazan, in the presence of PMA-stimulated neutrophils, yields, after DETAPAC addition and CPC /MgC1 2 solubilization, a blue color that is clearly visible to the eye, measurable with the spectrophotometer, and independent of the concentration of spermatozoa present. The clarity and reproducibility of the blue color, for a given neutrophil concentration, are such that one can determine neutrophil concentration on a day-to-day basis, solely by reference to a standard color chart. As such, one can even make use of the NBT assay without a spectrophotometer. In the development of the NBT assay, there was initial concern over the ability to standardize neutrophil response to generate the standard curve for reactive oxygen species output and the reference color chart. With PMA stimulation of the neutrophils, no significant difference was noted in measured reactive oxygen species output between neutrophil populations isolated from very different sources: healthy blood donors, patients with acute myocardial ischemia, and trauma victims. The implications of this feature of PMA stimulation are as yet unclear. It may be that the 20% to 30% to 40% to 55% Percoll gradient, used for purification of the enriched leukocyte fraction, has the ability to consistently isolate a particular neutrophil subpopulation that possesses a specific set of characteristics. Alternatively, by directly stimulating protein kinase Vol. 56, No.5, November 1991
C within the neutrophil, PMA may be bypassing regulatory pathways that could alter the degree of neutrophil response. Regardless of the underlying mechanism, it is the lack of variability in neutrophil response to PMA stimulation, along with its low cost, easy storage, and ready availability that make PMA a superior choice for neutrophil stimulator. Any preparation of immune complexes, such as that used by Ryan et al. 6 could be a substitute for PMA. However, these preparations would necessarily be more expensive and less stable, notwithstanding the cost and complexity involved in operating a flow cytometer. Sample preparation has been kept to a minimum. Because of the stability of the Percoll gradients, it is a simple matter to prepare the minigradients in bulk (10 to 20) the night before or the morning of the clinic. Total sample preparation time is not > 10 minutes/sample, including the time for centrifugations in the microcentrifuge. The total volume of original semen sample required for the NBT assay is 400 JLL because of the need for a control for measurements with the spectrophotometer. The NBT assay, using the spectrophotometer, clearly detects a concentration of neutrophils ~ 0.5 X 106 /mL, even in the presence of the high concentrations of spermatozoa tested, and reliably distinguishes differences in concentration down to 0.5 X 106 neutrophils/mL. This degree of sensitivity is more than appropriate for the WHO definition of possible significant genital infection, established as the presence of ~1 X 106 neutrophil PMNL/mL of semen. 7 The coefficients of variations for intra-assays and interassays were relatively low with values of <10% and 15%, respectively. Bare eye comparison of NBT assay-generated colors to the standard color chart also detects a concentration of neutrophils ~ 0.5 X 106 /mL and reliably distinguishes differences in concentration down to 1 X 106 neutrophils/mL. Difficulties that may arise from the NBT assay are often showed by other assays. Pipetting a representative sample of semen is often a difficulty when the aliquot taken is small or when the semen is very viscous. In this respect, the sample required for the NBT assay is generally bigger than that required for the myeloperoxidase assay described in the WHO Laboratory Manual. 7 However, viscosity and density of a semen sample may cause some problem for the Percoll gradients used before the NBT assay. Occasionally, a semen sample may have a density sufficiently high to penetrate the 20% Percolllayer. This situation can be corrected by simply diluting the semen sample twofold with a physioKovalski et al.
Neutrophil concentration in semen
951
logical buffer such as Hepes balanced saline or Ham's F-10. The NBT assay has the disadvantage of most assays requiring live cells, that is, it has to be performed the same day. However, this disadvantage is compensated by the ease of performing the NBT assay, the minimal working time required to run it, and the fact that results are obtained the same day. The role of the neutrophil 12 is to seek out, ingest, and kill pathogens. It is often thought of and referred to as the body's first line of defense against bacterial infections. Monocytes also invade areas of infection and phagocytose bacteria, other foreign material, and dead cells. They follow the neutrophils into the area of infection and as such, they constitute a second line of defense against invading pathogens. Because neutrophils have a recognized t 1; 2 of 6 hours in the circulation, and less so in the tissues, their presence tends to imply a relatively acute and/or significant infectious assault. 13 As such, the presence of active neutrophils may better distinguish a genital tract infection requiring immediate and specific antibiotic treatment. In contrast, a chronic inflammatory cell infiltrate composed of macrophages, and perhaps even lymphocytes, may only reflect the clearing away of dead or immotile spermatozoa. In trying to correlate leukocyte concentration to positive bacterial cultures, one must also consider the possibility of commensal growth. The lack of a significant neutrophilic infiltrate in the face of a positive semen bacterial culture might imply the relative nonpathogenicity of the cultured organisms. With this in mind, it would be worthwhile to correlate NET-assayed neutrophil function as an indicator of fresh and active neutrophils in the semen to positive bacterial cultures. It should be emphasized that NBT does not cross the cell membrane and, as such, is an indicator of reactive oxygen species present in the extracellular milieu. Strictly speaking, one would not necessarily expect a correlation between an assay of neutrophil function and the static measurement techniques that yield a value for neutrophil concentration based on the absolute number of neutrophils present in the sample. Even with fresh human blood, it was noted that L1 and L2 interface neutrophils had suboptimal superoxide output even though they clearly stained as neutrophils. In the same light, there exists the possibility that seminal neutrophils may not respond to PMA stimulation as do the L3 interface neutrophils. Therefore, their concentration in semen would be underestimated by the NBT assay. However, by standardizing all concentration determinations to a 952
Kovalski et al.
Neutrophil concentration in semen
specified neutrophil population in blood, we may find that "functional equivalents" ofneutrophils (versus strict numerical concentration equivalents) correlate far better to the results obtained with bacterial cultures. From our data in Table 1, it is difficult to make any firm conclusions in light of the rarity of semen samples that contain neutrophils; however, these data are in accordance with the findings of Aitken and W est. 14 These authors used a monoclonal antibody-based technique for leukocyte detection to evaluate Percoll-gradient separated semen fractions of 109 consecutive infertile patients. It was found that seminal leukocytes were few or absent for most of these patients. These results reinforce the need for a quick and inexpensive technique for verifying all semen samples that are provided to an infertility clinic. Immunocytochemical techniques could be useful in the detection and identification of the various classes of leukocytes other than neutrophils but mainly only once a neutrophil-positive semen has been identified. Evidently, classification of round cells found in semen solely on the basis of gross morphology is completely inadequate, 15 at least with respect to the determination of neutrophil concentration. Our future interest is to evaluate the ability of the NBT assay to help identify those bacterial genital infections, both in symptomatic and asymptomatic patients, that definitely require antibacterial treatment (such as chlamydia and gonorrhea). The lack of an effect of sperm, even at concentrations of 200 X 106/mL, on measured reactive oxygen species output from neutrophils, yields an important message as to the biological characteristics of semen. It is apparent that the simple presence of even large quantities of cellular material, namely sperm, is incapable of scavenging reactive oxygen species to any significant degree. This apparently contradicts the findings of Alvarez et al. 16 These authors established that there is a large variation in human spermatozoan SOD concentration that can be correlated to the duration of sperm motility. However, sperm cytosolic SOD does not have access to reactive oxygen species present in the extracellular medium. Even upon sperm destruction and release of the SOD into the extracellular milieu, the released SOD would likely be diluted to an insignificant concentration. It seems clear, then, that the presence of seminal plasma is critical for the protection of sperm against neutrophil-induced injury. If so, the suppression by seminal plasma of measured superoxide radical output from stimulated neutrophils introduces the question as to whether these Fertility and Sterility
neutrophils could have any deleterious effect in vivo on sperm function after ejaculation. To address this issue, a series of experiments is presently being conducted to determine critical concentrations of stimulated neutrophils, with and without seminal plasma present, at which sperm motility is adversely affected. With such results, we aim to correlate the neutrophil concentration, determined with the NBT assay, of a given semen sample to a specified cytotoxic effect on sperm. In summary, the NBT assay provides a new tool in the study of the role of leukocytes in human semen. This assay will help to determine whether neutrophil function rather than concentration is a better indicator of genital infection. As well, this assay could even be applied to other cell suspensions, such as vaginal and cervical secretions, to assess the degree to which neutrophils are present and active within the female genital tract. One of the most important features of the NBT assay is that it is simple to incorporate into any sperm analysis protocol, even when operating conditions are technologically limited.
4.
5.
6.
7.
8.
9.
10.
11.
Acknowledgments. The authors are grateful to Togas Tulandi, M.D., and Ms. Kim Wood of the Infertility Center, Royal Victoria Hospital, Montreal, Quebec, for their help in providing semen specimens, and to the nurses of the Royal Victoria Hospital Emergency Department for their help in providing fresh blood samples. Neil Kovalski, M.D., is also grateful to his brother, Mr. Shlomo Kovalski, M.Sc., and Rebono Shel Olam for general support and inspiration.
12.
13. 14.
REFERENCES 1. El Demiry MIM, Young H, Elton RA, Hargreave TB, James K, Chisholm GD: Leukocytes in the ejaculate from fertile and infertile men. Br J Urol 58:715, 1986 2. Jochum M, Pabst W, Schill WB: Granulocyte elastase as a sensitive diagnostic parameter of silent genital tract inflammation. Andrologia 18:413, 1986 3. Cumming JA, Dawes J, Hargreave TB: Granulocyte elastase levels do not correlate with anaerobic and aerobic bacterial
Vol. 56, No.5, November 1991
15.
16.
growth in seminal plasma from infertile men. Int J Androl 13:273, 1990 Barratt CLR, Robinson A, Spencer RC, Kinghorn GR, White A, Harrison PE, Kessopoulou E, Cooke ID: Seminal peroxidase positive cells are not an adequate indicator of asymptomatic urethral genital infection. lnt J Androl 13:361, 1990 Wolff H, Anderson DJ: Immunohistologic characterization and quantitation of leukocyte subpopulations in human semen. Fertil Steril 49:497, 1988 Ryan TC, Weil GJ, Newburger PE, Haughland R, Simons ER: Measurement of superoxide release in the phagovacuoles of immune complex-stimulated human neutrophils. J Immunol Methods 130:223, 1990 World Health Organization: WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. 2nd edition. Cambridge, The Press Syndicate of the University of Cambridge, 1987, p 8 de Lamirande E, Sherins RJ, Gagnon C: The presence of a motility inhibitor within spermatozoa may explain the poor sperm motility of some infertile men. J Androl 7:215, 1986 Spitz DR, Oberley LW: An assay for superoxide dismutase activity in mammalian tissue homogenates. Anal Biochem 179:8, 1989 Allen RC: Phagocytic leukocyte oxygenation activities and chemiluminescence: a kinetic approach to analysis. In Bioluminescence and Chemiluminescence: Methods in Enzymology, Vol. 133; Edited by MA DeLuca, WD McElroy. Orlando, Academic Press, 1986, p 481 Colepicolo P, Camarero VCPC, Nicolas MT, Bassot JM, Karnovsky ML, Hastings JW: A sensitive and specific assay for superoxide anion released by neutrophils or macrophages based on bioluminescence of polynoidon. Anal Biochem 184: 369, 1990 Fantone JC, Ward PA: Role of oxygen-derived free radicals and metabolites in leukocyte dependent inflammatory reactions. Am J Pathol107:397, 1982 Ganong WF: Review of Medical Physiology. East Norwalk, Lange Medical Publications, 1981, p 407 Aitken RJ, West KM: Analysis of the relationship between reactive oxygen species production and leukocyte infiltration in fractions of human semen separated on Percoll gradients. lnt J Androl 13:433, 1990 Barratt CLR, Bolton AE, Cooke ID: Functional significance of white blood cells in the male and female reproductive tract. Hum Reprod 5:639, 1990 Alvarez JG, Touchstone JC, Blasco L, Storey TS: Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major major enzyme protectant against oxygen toxicity. J Androl 8:338, 1987
Kovalski et al.
Neutrophil concentration in semen
953
Vol. 56, No.5, November 1991
Copyright © 1991 The American Fertility Society
Printed on acid-free paper in U.S.A.
Determination of neutrophil concentration in semen measurement of superoxide radical formation*
by
Neil Kovalski, M.D. Eve de Lamirande, Ph.D. Claude Gagnon, Ph.D.t Urology Research Laboratory, Royal Victoria Hospital and Faculty of Medicine, McGill University, Montreal, Quebec, Canada
Objective: To develop an assay that measures the concentration of functional neutrophils in human semen. Design: Human blood neutrophils were first isolated to establish linearity and proportionality for the determination of functional neutrophil concentration. Thereafter blood neutrophils in seminal plasma and neutrophils in semen of patients were measured. Setting: Blood and human samples were obtained from clinics of the Royal Victoria Hospital. Patients, Participants: Blood from normal men or from patients presenting with chest pain or trauma were used. Semen samples were also obtained from healthy fertile donors or from unselected patients attending the infertility clinic. Main Outcome Measures: Active neutrophil concentration in semen by a colorimetric assay. Results: The assay developed is based on the reduction of nitroblue tetrazolium of pale yellow color to blue formazan by the superoxide anions produced by stimulated neutrophils. The intensity of the derived blue color is proportional to the concentration of active neutrophils. This assay is simple, requires only 10 minutes of preparation time, and detects neutrophil concentrations > 0.5 X 106 neutrophils/mL of semen, independent of sperm concentration. Fertil Steril 56:946, 1991
The relevance of leukocytes in semen remains controversial. Multiple studies have presented conflicting results in their attempts to correlate leukocyte concentration to the presence of subclinical genital infection. 1- 3 One of the key problems in any such study relates to the difficulty in distinguishing leukocytes from sperm precursor cells in the ejaculate. Furthermore, the presence of multiple leukocyte subpopulations implies that any form of correlation made with total leukocyte number would likely be inadequate. Among the commonly used methods for determining leukocyte concentration in the ejaculate are myeloperoxidase stains, 4 indirect immunoperoxidase
Received January 28, 1991: revised and accepted July 16, 1991.
* Supported by a grant from the Medical Research Council of Canada to C.G. t Reprint requests: Claude Gagnon, Ph.D., Urology Research Laboratory, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1Al.
946
Kovalski et al.
Neutrophil concentration in semen
stains employing specific monoclonal antibodies for leukocyte subpopulations, 1•5 and granulocyte elastase level determinations. 2•3 These tests yield a static value for leukocyte concentration, but they do not provide any clear indication of leukocyte viability or activity. A newer approach to detecting the presence of leukocytes in any cell suspension is based on the ability of leukocytes to generate reactive oxygen species. These reactive oxygen species (such as superoxide anion ["02], hydrogen peroxide [H 2 0 2 ], hydroxyl radical ["OH], and hypochlorite radical ["OHCl]) are considered to be responsible for much of the target cell and/or tissue injury induced by leukocytes. Ryan et al. 6 used flow cytometry to measure fluorescence as an indicator of reactive oxygen species output from immune-complex stimulated human blood neutrophils and went on to study in detail the kinetics of the production of reactive oxygen species by neutrophils. The system of Ryan et al., 6 although expensive and technically demanding Fertility and Sterility
to operate, offers the opportunity to quantify and then correlate neutrophil generation of reactive oxygen species to neutrophil concentration. As such, one has. a method for comparing neutrophil populations on the basis of their cytotoxic capability rather than just according to the sheer number of cells present. The aim of the present study was to develop a quantitative and functional test of neutrophil concentration that would be applicable to semen, based on the same principle of the neutrophil's capacity to produce reactive oxygen species. However, to be suitable for an infertility clinic, such a test would necessarily be inexpensive, technically simple, fast to use, easy to interpret, and require minimal technician time per sample. It was realized that the cost and technical complexity of this new test would rest primarily on the choice of neutrophil stimulating agent and reactive oxygen species output detecting system. Because of its key characteristic of being reduced by "02 to yield blue formazan (clearly distinguishable blue color), nitroblue tetrazolium (NBT; pale yellow color) was selected as the indicator of reactive oxygen species produced by the neutrophils. This characteristic of NBT was translated into a colorimetric assay for neutrophil function. To stimulate the neutrophils to produce reactive oxygen species, phorbol myristate acetate, a protein kinase C activator, was chosen for its stability, efficacy, ready availability, and low cost. In this paper, we describe and validate a new assay (referred to henceforth as the NBT assay) of neutrophil concentration in human semen. In contrast to most previous tests, the present assay measures the functional capacity of the seminal neutrophils. The NBT assay clearly detects neutrophils at concentrations ~ 0.5 X 106 /mL. This level of sensitivity is appropriate for detecting the 1.0 X 106 neutrophil polymorphonuclear leukocytes (PMNL)/mL considered by the World Health Organization (WH0)1 to be an indicator of possible genital tract infection. MATERIALS AND METHODS Reagents
Percoll was purchased from Pharmacia Fine Chemicals (Dorval, Quebec, Canada). Phorbol 12myristate 13-acetate (PMA) was purchased from ICN Biochemicals (Cleveland, OH). Nitroblue tetrazolium, cetylpyridinium chloride (CPC), the Sigma Diagnostics Leukocyte Myeloperoxidase Stain (procedure no. 390), and bovine serum albumin (BSA) were obtained from Sigma Chemical ComVol. 56, No. 5, November 1991
pany (St. Louis, MO). Superoxide dismutase (SOD) was purchased from Calbiochem Corporation (La Jolla, CA). Diethylenetriaminepentaacetic acid (DETAPAC) was obtained from Aldrich Chemical Company (Milwaukee, WI). Blood Neutrophil Separation
Peripheral blood neutrophils were isolated from ethylenediaminetetraacetic acid-anticoagulated venous blood of normal human donors and from patients presenting to the emergency room for treatment of chest pain or trauma. Twelve milliliters of whole blood were divided into six identical aliquots. To each aliquot was added 10 mL of 0.87% NH4Cl. All six mixes were then allowed to stand for 20 minutes at room temperature (RT) to induce an osmotic lysis of the erythrocytes. The hemolyzed blood was then centrifuged at 600 X g at RT to yield six leukocyte-enriched pellets. The supernatants were removed, and each of the six pellets was resuspended in 2 mL of 0.87% NH4Cl. All six cell suspensions were pooled and then layered onto a single discontinuous Percoll gradient made up of 1 mL 55%, 1.5 mL 40%, 1 mL 30%, and 1 mL 20% Percoll. All Percoll solutions were buffered with Hepes balanced saline (130 mM NaCl, 4 mM KCl, 2.5 mM CaC12 , 14 mM fructose, and 10 mM N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid [Hepes] adjusted to pH 8) and supplemented with 10 mg/mL ofBSA. The Percoll gradient, with cell suspension on top, was then centrifuged at RT at 1,300 X g for 30 minutes. Leukocytes were recovered from the 20% to 30% interface (L1), 30% to 40% interface (L2), and the 40% to 55% interface (L3, neutrophil layer). All interfaces were devoid of contaminating erythrocytes, but only L3 was devoid of debris. Cell viability was determined by trypan blue exclusion. Semen Collection and Processing
Semen samples collected by masturbation after 3 days of sexual abstinence were obtained from healthy, fertile donors and patients attending the Infertility Clinic at the Royal Victoria Hospital. Aliquots of 200 #LL of liquefied semen were layered onto identical discontinuous Percoll gradients 1 hour after semen collection. Each of these gradients consisted of 30 #LL 80% and 500 #LL 20% Percoll in Hepes balanced saline and was prepared in a microcentrifuge tube (1.5 mL capacity). These gradients, with semen sample on top, were spun at 6,500 X g for 1 minute at RT in a Johns Scientific Microcentaur microcentrifuge (Biotech, Montreal, Quebec, CanKovalski et al.
Neutrophil concentration in semen
947
ada). The upper 530 ~tL of each centrifuged sample was then discarded to yield a 200-~tL seminal plasma-free cell isolate of the original semen. We refer to this process of separating seminal plasma from the cellular components as centrifugation-separation. The remainder of each original semen sample was then individually layered onto a separate Percoll gradient identical to those used for the neutrophil separation from whole blood. The 20% to 30%,30% to 40%, and 40% to 55% interfaces were collected and assessed to determine if this more refined separation technique had any benefits over that using the microcentrifuge. Myeloperoxidase Stain for Neutrophils
Myeloperoxidase staining for neutrophils was performed according to WHO. 7 One milligram of the Peroxidase Indicator Reagent {p-phenylenediamine diHCl [1 part] and catechol [2 parts]) and 4 ~tL of 3% H 2 0 2 were added to 500 ~tL of TRIZMAL 6.3 buffer at RT. Then, 180 ~tL of this solution were mixed with 20 ~tL of the cell suspension being tested. This mix was incubated in the dark at 37°C for 30 minutes. With the hematocytometer, the individual concentrations of the variously stained leukocytes were determined: neutrophils show brown-black intracellular granulation, monocytes stain less intensely, and lymphocytes (and immature germ cells) do not show peroxidase activity. Measurement of Reactive Oxygen Species Output From Blood Neutrophils With NBT
Various concentrations of freshly isolated (within 4 hours of blood collection) human blood neutrophils were incubated at 37°C with 8 X 10-8 M PMA and 0.1 mM NBT in a final volume of 200 JLL. Reactive oxygen species production was followed by measuring the formation of blue formazan at 630 nm with an MR 580 Dynatech Microelisa Auto Reader spectrophotometer (Chantilly, VA) at incubation times 0, 8, 15, 22, 30, 45, 60, 90, and 120 minutes. Assay of NBT for Blood Neutrophils in the Presence of Spermatozoa
Purified human blood neutrophils were incubated with 8 X 10-8 M PMA and 0.1 mM NBT in the presence and absence of different concentrations of Percoll-washed human spermatozoa from healthy fertile donors. 8 As controls for sample turbidity secondary to the spermatozoa themselves, different concentrations of spermatozoa were incubated alone 948
Kovalski et al.
Neutrophil concentration in semen
in BSA-supplemented Hepes balanced saline buffer. After an incubation period of 60 minutes, the absorbance at 630 nm was measured. In another series of experiments, purified human blood neutrophils were incubated in the absence and presence of 200 X 106 spermatozoa/mL with 8 X 10-8 M PMA and 0.1 mM NBT in a final volume of 200 JLL. Controls were 200 X 106 spermatozoa/mL alone in BSA-supplemented Hepes balanced saline buffer. After 60 minutes of incubation at 37°C, 10 ~tL of 50 mM DETAPAC {pH 8), 100 ~tL of 20% CPC, and 200 ~tL of 3 M MgC12 were added, in sequence, to and vigorously mixed with each of the incubated cell preparations to solubilize all cellular material and precipitated blue formazan. An effective iron chelating agent, DET APAC is essential to eliminate the direct reduction of NBT by intracellular components that are released into the milieu by solubilization. 9 To maximize solubilization, the mixtures (each now with a volume of 500 ~tL) were then incubated for another 15 minutes at 37°C after which they were again vigorously mixed. All mixtures were then centrifuged for 3 minutes at 11,000 X g in the microcentrifuge. A 300 ~tL aliquot was removed from each mixture (leaving the postcentrifugation pellet undisturbed), placed in an enzyme-linked immunosorbent assay plate well, and kept at 37°C. Absorbance at 630 nm was determined as described above. Assay of NBT for Human Semen Neutrophils
Duplicate seminal plasma-free cell isolates were prepared from each original semen sample and incubated in the presence and absence of PMA and NBT. After 60 minutes of incubation at 37°C, the absorbance at 630 nm was measured. The NBT assay was carried out with one cell isolate designated as the control (without PMA and NBT) and the duplicate isolate mixed with PMA and NBT, thereby designated as the stimulated sample. Statistical Analysis
Regression analysis was used to evaluate the correlation between the variables analyzed. The 0.05 level of probability was used as the criterion of significance. RESULTS Isolation of Blood Neutrophils on Discontinuous Percoll Gradients
After NH 4 Cl hemolysis of the erythrocytes in the original whole blood and centrifugation to yield the Fertility and Sterility
blood leukocyte enriched fraction, further purification was carried out by centrifugation on Percoll gradients made of 55%, 40%, 30%, and 20% Percoll layers. The L3 interface (40% to 55% Percoll) was comprised of dark brown-staining leukocytes, consistent with a neutrophil population of purity> 98%. The L1 (20% to 30% Percoll) and L2 (30% to 40% Percoll) interfaces were comprised mostly of intermediate and non-staining cells consistent with monocyte and lymphocyte populations, respectively. The ratio of neutrophil concentration between interfaces was 8:1:1 (L3:L2:L1). Preincubation ofblood neutrophils in seminal plasma followed by centrifugation-separation in the microcentrifuge allowed a >98% recovery of neutrophils and yielded no change in staining pattern. Attempted staining in the presence of seminal plasma resulted in markedly reduced coloration of the cells. All neutrophil populations, recovered from the L3 interface, were :2::98% viable. Final neutrophil recovery (total of L1, L2, and L3 interfaces) was an average of 80% of the original whole blood neutrophil content. Measured Reactive Oxygen Species Output of Human Blood Neutrophils With NBT
Phorbol 12-meristate 13-acetate-stimulated human blood neutrophils, incubated in the presence of NBT, generated a progressive increase in absorbance, indicative of superoxide radical production.
I 150 Time (minutes)
Figure 1 Time course of measured superoxide output from human blood neutrophile. Human blood neutrophile, isolated on a Percoll gradient, were incubated in the presence of NBT, with and without PMA, at 37°C. The formation of blue formazan was followed by measuring the absorbance at 630 nm at 0, 8, 15, 22, 30, 45, 60, 90, and 120 minutes of incubation. Data are presented for 2 X 106 and 4 X 106 neutrophils/mL. All values are expressed as a percentage of the maximum absorbance recorded for 4 X 106 PMA stimulated neutrophils/mL. •-•, 2 X 106 unstimulated neutrophils/mL; +- +, 2 X 106 stimulated neutrophils/mL; X- X, 4 X 106 unstimulated neutrophils/mL; A- A, 4 X 106 PMA-stimulated neutrophils/mL. Vol. 56, No. 5, November 1991
NeutrophD concentration (mlDion/ml)
Figure 2 Correlation between measured superoxide output from PMA-stimulated neutrophile and neutrophil concentration. Blood neutrophile were incubated with NBT and PMA for 60 minutes at 37°C. The formation of blue formazan was determined by measuring the absorbance at 630 nm. These samples mixes were assessed directly without the addition of DETAPAC or solubilization with CPC and MgCl2 • Values depicted are the means ± SEM for eight experiments. The correlation coefficient for 0 to 4 X 106 neutrophils/mL was 0.97.
Absorbance change, for any given sample, plateaus at its maximum by 30 to 45 minutes of incubation (Fig. 1). This time course is consistent with that previously reported for reactive oxygen species output from PMA-stimulated human neutrophils, as determined by chemiluminescence with luminol. 10 The absorbance of the formed blue formazan was linearly correlated to neutrophil concentration, within the range of 0 to 4 million neutrophils/mL (Fig. 2). Such a linear correlation over this range of neutrophil concentration was previously reported with PMA-stimulated mouse neutrophils, using chemiluminescence with luminol and polynoidon to determine reactive oxygen species output. 11 Equal concentrations of neutrophils, recovered from the L1 and L2 interfaces, generated 50% less absorbance change, upon PMA stimulation, than the equivalent concentration of neutrophils from the L3 interface. The absorbance change induced by stimulated neutrophils (L3 interface), at concentrations up to 10 X 106 /mL, was reduced by >95% in the presence of superoxide dismutase (660 U /mL) or seminal plasma (50% vol/vol). This effect of seminal plasma on absorbance change was consistent among different donors (n = 10) and different infertile patients (n = 10). This effect was unchanged by freezing and thawing of the seminal plasma. Preincubation of neutrophils with 50% (voljvol) seminal plasma followed by centrifugation -separation in the microcentrifuge had no effect on the generated absorbance change after PMA stimulation. Kovalski et al.
Neutrophil concentration in semen
949
0.4
100 :::;-
~
=
80
0.3
0
... C)
0
~ e..
4>
4>
a ~
20
-e ~
a
60 40
C)
C)
-e0
0.2
"'
~
0.1
o~~--~-.~-.~~.-~.-~
0 100
0
200
0
300
Spenn concentration (million/mil
Figure 3 Effect of sperm concentration on measured superoxide output from PMA -stimulated neutrophils. Various concentrations of washed spermatozoa were incubated with a fixed concentration of Percoll-washed blood neutrophils in the presence of NBT and PMA. The formation of blue formazan was determined by measuring the absorbance at 630 nm. Values depicted are the means ± SEM for four experiments. The value of 100% corresponds to an absorbance of 0.43 generated by 6 X 106 neutrophils/mL incubated without spermatozoa.
1
2
3
4
5
6
Neutrophil concentration (million/mil
Figure 4 Correlation between measured superoxide output generated by PMA-stimulated neutrophils and neutrophil concentration in the presence of 200 X 106 spermatozoa/mL. After the 60-minute incubation period at 37°C with PMA and NBT, all samples of human blood neutrophils, both with and without spermatozoa, were mixed with DETAPAC, CPC, and MgCl2 , and incubated for another 15 minutes at 37°C before the determination of absorbance at 630 nm. Values depicted are means ± SD for five experiments. The correlation coefficient between 0 to 3 X 106 neutrophils/mL was 0.99. D-D, neutrophils alone; +-+, neutrophils + 200 X 106 spermatozoa/mL.
Effect of Spermatozoa on the NBT Assay
In the presence of spermatozoa, blue formazan precipitated as it was being actively generated from NBT by stimulated neutrophils. As such, the absorbance change generated by samples of PMAstimulated neutrophils, incubated with spermatozoa, was markedly reduced as compared with samples of PMA-stimulated neutrophils incubated without spermatozoa (Fig. 3). However, after the addition of DETAPAC, solubilization with CPC and MgCl2 , and final microcentrifugation, the absorbance change (defined as the absorbance of the stimulated sample minus the absorbance of the control sample) for any given concentration of neutrophils was now independent of initial sperm concentration in the incubation mixture (data shown for 200 X 106 spermatozoa/mL) (Fig. 4). Furthermore, the marked turbidity observed with high concentrations of spermatozoa was reduced by >90%. For at a least 1 hour after solubilization, the blue color generated remained clearly measurable and easily distinguishable to the eye.
patients had neither positively staining cells nor a measurable absorbance change by NBT assay. Five patients had positively staining cells, judged to be neutrophils, but at concentrations < 0.5 X 106 /mL. Two patients had positively staining cells, read as 2 X 106 neutrophils/mL. With the NBT assay, the seminal neutrophil concentration of these two patients was determined to be 1.0 X 106 and 2.0 X 106 neutrophils/mL, respectively. These results were independent of Percoll centrifugation technique, that is, minigradient of 30% to 80% Percoll layers versus larger gradient of 20%, 30%, 40%, 55% Percoli layers.
Table 1 Detection of Neutrophils in Human Semen by the NBT Assay and Myeloperoxidase Staining Neutrophils
Patient group"
With myeloperoxidase staininif
WithNBT assay'
10'/mL
Myeloperoxidase Staining and NBT Testing of Semen Round Cells
An unselected population of 67 patients from the Infertility Clinic, all presenting with round cells in their ejaculate, as noted by light microscopy during routine semen analysis, was studied. Neutrophil concentration was assessed both by myeloperoxidase staining and the NBT assay (see Table 1). Sixty 950
Kovalski et al.
Neutrophil concentration in semen
1 (n = 2 (n = 3 (n = (n =
60)
0
5) 1) 1)
<0.5 2.0 2.0
0 0 1.0
2.0
a Semen samples were obtained from 67 unselected patients, consulting for infertility. b Only myeloperoxidase-stained cells that were dark brown were judged to be neutrophils. ' Each NBT assay was assessed visually and spectrophotometrically.
Fertility and Sterility
To further validate the NBT assay for neutrophils, different quantities of purified blood neutrophils were added to semen samples from five normal volunteers who had no endogenous neutrophils. The concentration of blood neutrophils in semen was determined by the NBT and myeloperoxidase assay. Values obtained were then used to calculate the original blood neutrophil concentration. Although the calculated initial concentration of blood neutrophils was referred at 100%, the NBT and myeloperoxidase assays estimated the concentration at 111% ± 8% (mean ± SEM) and 105% ± 8%. These values were not significantly different from each other. DISCUSSION
The production of reactive oxygen species by PMA -stimulated neutrophils is an index of function that can be quantitated and correlated to neutrophil concentration. The advantages of NBT as the reactive oxygen species output indicator are multiple. Nitroblue tetrazolium is inexpensive, stable, and readily available. As well, the conversion of NBT to blue formazan, in the presence of PMA-stimulated neutrophils, yields, after DETAPAC addition and CPC /MgC1 2 solubilization, a blue color that is clearly visible to the eye, measurable with the spectrophotometer, and independent of the concentration of spermatozoa present. The clarity and reproducibility of the blue color, for a given neutrophil concentration, are such that one can determine neutrophil concentration on a day-to-day basis, solely by reference to a standard color chart. As such, one can even make use of the NBT assay without a spectrophotometer. In the development of the NBT assay, there was initial concern over the ability to standardize neutrophil response to generate the standard curve for reactive oxygen species output and the reference color chart. With PMA stimulation of the neutrophils, no significant difference was noted in measured reactive oxygen species output between neutrophil populations isolated from very different sources: healthy blood donors, patients with acute myocardial ischemia, and trauma victims. The implications of this feature of PMA stimulation are as yet unclear. It may be that the 20% to 30% to 40% to 55% Percoll gradient, used for purification of the enriched leukocyte fraction, has the ability to consistently isolate a particular neutrophil subpopulation that possesses a specific set of characteristics. Alternatively, by directly stimulating protein kinase Vol. 56, No.5, November 1991
C within the neutrophil, PMA may be bypassing regulatory pathways that could alter the degree of neutrophil response. Regardless of the underlying mechanism, it is the lack of variability in neutrophil response to PMA stimulation, along with its low cost, easy storage, and ready availability that make PMA a superior choice for neutrophil stimulator. Any preparation of immune complexes, such as that used by Ryan et al. 6 could be a substitute for PMA. However, these preparations would necessarily be more expensive and less stable, notwithstanding the cost and complexity involved in operating a flow cytometer. Sample preparation has been kept to a minimum. Because of the stability of the Percoll gradients, it is a simple matter to prepare the minigradients in bulk (10 to 20) the night before or the morning of the clinic. Total sample preparation time is not > 10 minutes/sample, including the time for centrifugations in the microcentrifuge. The total volume of original semen sample required for the NBT assay is 400 JLL because of the need for a control for measurements with the spectrophotometer. The NBT assay, using the spectrophotometer, clearly detects a concentration of neutrophils ~ 0.5 X 106 /mL, even in the presence of the high concentrations of spermatozoa tested, and reliably distinguishes differences in concentration down to 0.5 X 106 neutrophils/mL. This degree of sensitivity is more than appropriate for the WHO definition of possible significant genital infection, established as the presence of ~1 X 106 neutrophil PMNL/mL of semen. 7 The coefficients of variations for intra-assays and interassays were relatively low with values of <10% and 15%, respectively. Bare eye comparison of NBT assay-generated colors to the standard color chart also detects a concentration of neutrophils ~ 0.5 X 106 /mL and reliably distinguishes differences in concentration down to 1 X 106 neutrophils/mL. Difficulties that may arise from the NBT assay are often showed by other assays. Pipetting a representative sample of semen is often a difficulty when the aliquot taken is small or when the semen is very viscous. In this respect, the sample required for the NBT assay is generally bigger than that required for the myeloperoxidase assay described in the WHO Laboratory Manual. 7 However, viscosity and density of a semen sample may cause some problem for the Percoll gradients used before the NBT assay. Occasionally, a semen sample may have a density sufficiently high to penetrate the 20% Percolllayer. This situation can be corrected by simply diluting the semen sample twofold with a physioKovalski et al.
Neutrophil concentration in semen
951
logical buffer such as Hepes balanced saline or Ham's F-10. The NBT assay has the disadvantage of most assays requiring live cells, that is, it has to be performed the same day. However, this disadvantage is compensated by the ease of performing the NBT assay, the minimal working time required to run it, and the fact that results are obtained the same day. The role of the neutrophil 12 is to seek out, ingest, and kill pathogens. It is often thought of and referred to as the body's first line of defense against bacterial infections. Monocytes also invade areas of infection and phagocytose bacteria, other foreign material, and dead cells. They follow the neutrophils into the area of infection and as such, they constitute a second line of defense against invading pathogens. Because neutrophils have a recognized t 1; 2 of 6 hours in the circulation, and less so in the tissues, their presence tends to imply a relatively acute and/or significant infectious assault. 13 As such, the presence of active neutrophils may better distinguish a genital tract infection requiring immediate and specific antibiotic treatment. In contrast, a chronic inflammatory cell infiltrate composed of macrophages, and perhaps even lymphocytes, may only reflect the clearing away of dead or immotile spermatozoa. In trying to correlate leukocyte concentration to positive bacterial cultures, one must also consider the possibility of commensal growth. The lack of a significant neutrophilic infiltrate in the face of a positive semen bacterial culture might imply the relative nonpathogenicity of the cultured organisms. With this in mind, it would be worthwhile to correlate NET-assayed neutrophil function as an indicator of fresh and active neutrophils in the semen to positive bacterial cultures. It should be emphasized that NBT does not cross the cell membrane and, as such, is an indicator of reactive oxygen species present in the extracellular milieu. Strictly speaking, one would not necessarily expect a correlation between an assay of neutrophil function and the static measurement techniques that yield a value for neutrophil concentration based on the absolute number of neutrophils present in the sample. Even with fresh human blood, it was noted that L1 and L2 interface neutrophils had suboptimal superoxide output even though they clearly stained as neutrophils. In the same light, there exists the possibility that seminal neutrophils may not respond to PMA stimulation as do the L3 interface neutrophils. Therefore, their concentration in semen would be underestimated by the NBT assay. However, by standardizing all concentration determinations to a 952
Kovalski et al.
Neutrophil concentration in semen
specified neutrophil population in blood, we may find that "functional equivalents" ofneutrophils (versus strict numerical concentration equivalents) correlate far better to the results obtained with bacterial cultures. From our data in Table 1, it is difficult to make any firm conclusions in light of the rarity of semen samples that contain neutrophils; however, these data are in accordance with the findings of Aitken and W est. 14 These authors used a monoclonal antibody-based technique for leukocyte detection to evaluate Percoll-gradient separated semen fractions of 109 consecutive infertile patients. It was found that seminal leukocytes were few or absent for most of these patients. These results reinforce the need for a quick and inexpensive technique for verifying all semen samples that are provided to an infertility clinic. Immunocytochemical techniques could be useful in the detection and identification of the various classes of leukocytes other than neutrophils but mainly only once a neutrophil-positive semen has been identified. Evidently, classification of round cells found in semen solely on the basis of gross morphology is completely inadequate, 15 at least with respect to the determination of neutrophil concentration. Our future interest is to evaluate the ability of the NBT assay to help identify those bacterial genital infections, both in symptomatic and asymptomatic patients, that definitely require antibacterial treatment (such as chlamydia and gonorrhea). The lack of an effect of sperm, even at concentrations of 200 X 106/mL, on measured reactive oxygen species output from neutrophils, yields an important message as to the biological characteristics of semen. It is apparent that the simple presence of even large quantities of cellular material, namely sperm, is incapable of scavenging reactive oxygen species to any significant degree. This apparently contradicts the findings of Alvarez et al. 16 These authors established that there is a large variation in human spermatozoan SOD concentration that can be correlated to the duration of sperm motility. However, sperm cytosolic SOD does not have access to reactive oxygen species present in the extracellular medium. Even upon sperm destruction and release of the SOD into the extracellular milieu, the released SOD would likely be diluted to an insignificant concentration. It seems clear, then, that the presence of seminal plasma is critical for the protection of sperm against neutrophil-induced injury. If so, the suppression by seminal plasma of measured superoxide radical output from stimulated neutrophils introduces the question as to whether these Fertility and Sterility
neutrophils could have any deleterious effect in vivo on sperm function after ejaculation. To address this issue, a series of experiments is presently being conducted to determine critical concentrations of stimulated neutrophils, with and without seminal plasma present, at which sperm motility is adversely affected. With such results, we aim to correlate the neutrophil concentration, determined with the NBT assay, of a given semen sample to a specified cytotoxic effect on sperm. In summary, the NBT assay provides a new tool in the study of the role of leukocytes in human semen. This assay will help to determine whether neutrophil function rather than concentration is a better indicator of genital infection. As well, this assay could even be applied to other cell suspensions, such as vaginal and cervical secretions, to assess the degree to which neutrophils are present and active within the female genital tract. One of the most important features of the NBT assay is that it is simple to incorporate into any sperm analysis protocol, even when operating conditions are technologically limited.
4.
5.
6.
7.
8.
9.
10.
11.
Acknowledgments. The authors are grateful to Togas Tulandi, M.D., and Ms. Kim Wood of the Infertility Center, Royal Victoria Hospital, Montreal, Quebec, for their help in providing semen specimens, and to the nurses of the Royal Victoria Hospital Emergency Department for their help in providing fresh blood samples. Neil Kovalski, M.D., is also grateful to his brother, Mr. Shlomo Kovalski, M.Sc., and Rebono Shel Olam for general support and inspiration.
12.
13. 14.
REFERENCES 1. El Demiry MIM, Young H, Elton RA, Hargreave TB, James K, Chisholm GD: Leukocytes in the ejaculate from fertile and infertile men. Br J Urol 58:715, 1986 2. Jochum M, Pabst W, Schill WB: Granulocyte elastase as a sensitive diagnostic parameter of silent genital tract inflammation. Andrologia 18:413, 1986 3. Cumming JA, Dawes J, Hargreave TB: Granulocyte elastase levels do not correlate with anaerobic and aerobic bacterial
Vol. 56, No.5, November 1991
15.
16.
growth in seminal plasma from infertile men. Int J Androl 13:273, 1990 Barratt CLR, Robinson A, Spencer RC, Kinghorn GR, White A, Harrison PE, Kessopoulou E, Cooke ID: Seminal peroxidase positive cells are not an adequate indicator of asymptomatic urethral genital infection. lnt J Androl 13:361, 1990 Wolff H, Anderson DJ: Immunohistologic characterization and quantitation of leukocyte subpopulations in human semen. Fertil Steril 49:497, 1988 Ryan TC, Weil GJ, Newburger PE, Haughland R, Simons ER: Measurement of superoxide release in the phagovacuoles of immune complex-stimulated human neutrophils. J Immunol Methods 130:223, 1990 World Health Organization: WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. 2nd edition. Cambridge, The Press Syndicate of the University of Cambridge, 1987, p 8 de Lamirande E, Sherins RJ, Gagnon C: The presence of a motility inhibitor within spermatozoa may explain the poor sperm motility of some infertile men. J Androl 7:215, 1986 Spitz DR, Oberley LW: An assay for superoxide dismutase activity in mammalian tissue homogenates. Anal Biochem 179:8, 1989 Allen RC: Phagocytic leukocyte oxygenation activities and chemiluminescence: a kinetic approach to analysis. In Bioluminescence and Chemiluminescence: Methods in Enzymology, Vol. 133; Edited by MA DeLuca, WD McElroy. Orlando, Academic Press, 1986, p 481 Colepicolo P, Camarero VCPC, Nicolas MT, Bassot JM, Karnovsky ML, Hastings JW: A sensitive and specific assay for superoxide anion released by neutrophils or macrophages based on bioluminescence of polynoidon. Anal Biochem 184: 369, 1990 Fantone JC, Ward PA: Role of oxygen-derived free radicals and metabolites in leukocyte dependent inflammatory reactions. Am J Pathol107:397, 1982 Ganong WF: Review of Medical Physiology. East Norwalk, Lange Medical Publications, 1981, p 407 Aitken RJ, West KM: Analysis of the relationship between reactive oxygen species production and leukocyte infiltration in fractions of human semen separated on Percoll gradients. lnt J Androl 13:433, 1990 Barratt CLR, Bolton AE, Cooke ID: Functional significance of white blood cells in the male and female reproductive tract. Hum Reprod 5:639, 1990 Alvarez JG, Touchstone JC, Blasco L, Storey TS: Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major major enzyme protectant against oxygen toxicity. J Androl 8:338, 1987
Kovalski et al.
Neutrophil concentration in semen
953