The Examination of Peroxidase-Positive Leukocytes in Semen

Özet

This paper presents an economical and efficient protocol for examining peroxidase-positive leukocytes in semen. With the assistance of a computer-assisted semen analysis (CASA) system, the concentration of peroxidase-positive leukocytes in semen can be obtained within a total of 60 min, effectively improving the efficiency of andrology laboratory and andrologists.

Abstract

Leukocytospermia can lead to decreased spermatozoa motility, increased spermatozoa morphological abnormalities, elevated spermatozoa DNA fragmentation index, impairment of the spermatozoa acrosome function, and even affected embryonic development. It is a common andrological disease in clinical practice and one of the important causes of male infertility. When determining whether male reproductive tract inflammation exists, andrologists often choose to examine round cells or seminal plasma elastase in the semen as a clinical diagnostic basis. However, the examination of round cells is easily influenced by sloughed spermatogenic cells and reproductive tract epithelial cells, which do not contribute to reducing the indiscriminate and unnecessary use of antibiotics. At the same time, the detection process of elastase is relatively complicated, time-consuming, and slow in reporting results, which is not beneficial for early diagnosis and treatment of diseases such as male genital tract infections (MGTIs). We have innovatively applied the examination of peroxidase-positive leukocytes in semen assisted by a computer-assisted semen analysis (CASA) system as a diagnostic criterion for leukocytospermia, successfully solving these problems. This examination only requires the addition of the operating fluid consisting of four reagents into the specimen, and the total reaction time at room temperature can be controlled within 20-30 min. With the subsequent smear and microscopic examination, the concentration of peroxidase-positive leukocytes in semen can be obtained within a total of 60 min, which can be used to diagnose whether the inflammation of the male reproductive tract existed.

Introduction

Infertility has emerged as a global public health issue affecting approximately 15% of couples in their reproductive age. Male factors are contributed by around 50% of total subfertility cases, and nearly 20% to 30% can be attributed solely to male factors^1,2,3. Male genital tract infections (MGTIs) constitute one of the significant causes of male infertility, accounting for about 15% of cases^4,5.

Most individuals have leukocytes in their semen, constituting 13% of non-spermatozoan cells, with differential proportions being neutrophils 12%, macrophages 0.9%, and lymphocytes 0.1%^6,7. According to the World Health Organization (WHO) laboratory manual for the examination and processing of human semen (5th ed.), leukocytospermia is usually defined as the presence of >1 × 10⁶ cells/mL leucocytes in semen⁸. This condition can be caused by numerous factors such as environmental toxins, substance abuse, varicoceles, MGTIs, etc., all potentially leading to an abnormal increase in leukocyte concentration in semen⁹. Leukocytes can elevate reactive oxygen species (ROS) levels in semen, causing lipid peroxidation and oxidative damage to protein and DNA, which results in decreased spermatozoa motility, increased spermatozoa morphological abnormalities, elevated spermatozoa DNA fragmentation index, impairment of the spermatozoa acrosome function, and even negative impacts on embryo development^10,11.

Currently, andrologists commonly choose to examine round cells in semen or elastase in seminal plasma when identifying genital tract inflammation. However, it's often challenging to distinguish sloughed spermatogenic cells and reproductive tract epithelial cells, which do not contribute to reducing the indiscriminate and unnecessary use of antibiotics⁶. The latter examination method is relatively complicated and time-consuming, with slow result reporting, which is not beneficial for early diagnosis and treatment of diseases such as MGTIs. The American Urological Association (AUA) suggests further differentiation between leukocytes and sloughed cells from the genital tract should be made when the round cell concentration in semen analysis is greater than 1 × 10⁶ cells/mL¹². Some andrology labs utilize flow cytometry¹³ or leukocyte antigen (e.g., CD45) immunocytochemistry¹⁴ to examine leukocytes. While these methods are precise, they are expensive and time-consuming, making large-scale clinical implementation difficult, especially in developing countries.

Peroxidase is widely distributed in various types of cells and plays a crucial role in the resistance to oxidative stress damage. Myeloperoxidase (MPO) is a member of the peroxidase subfamily that is generally expressed in immune cells. It is most highly expressed in neutrophils' azurophilic granules^15,16 and is also expressed in lymphocytes^17,18, monocytes, and macrophages¹⁹. The concentration of leukocytes, especially neutrophils, in semen can be obtained by examining round cells that are peroxidase-positive^7,20. To make the examination process of leukocytes in semen economical, convenient, and efficient, we reengineered the examination method. Assisted by the computer-assisted semen analysis (CASA) system, the concentration of leukocytes can be obtained within 60 min. This new method reduces the patients' examination costs and waiting time for obtaining results, alleviates the workload of lab technicians, and shortens the doctor's diagnosis and treatment waiting period.

Protocol

This study has been reviewed and approved by the Medical Ethics Committee of The Third Affiliated Hospital of Sun Yat-sen University.

1. Preparation of the working solution

1. Gather the following reagents essential for preparing the working solution- ortho-toluidine substrate solution, saturated ammonium chloride (NH₄Cl) solution, 148 mmol/L ethylenediaminetetraacetic acid disodium salt (Na₂EDTA) solution, and 6% (v/v) hydrogen peroxide solution. Place them on a room temperature (RT) lab bench along with the necessary tools, such as transfer pipettes (1000 µL, 200 µL, 10 µL) and test tube racks.
   CAUTION: Read the safety data sheet (SDS) before handling the above-mentioned reagents.
2. Prepare an opaque brown reagent bottle to store and protect the prepared working solution from exposure to light.
3. Transfer the reagents into the opaque brown reagent bottle as per the proportions specified below:
   1. Utilize the 200 µL transfer pipette and adjust the scale to 100 µL by turning the knob. Then, transfer 100 µL of the NH₄Cl solution into the opaque brown reagent bottle.
   2. Replace the pipette tip and transfer 100 µL of the 148 mmol/L Na₂EDTA solution into the opaque brown reagent bottle using the 200 µL transfer pipette mentioned above. Then, gently blow by pipetting and thoroughly homogenize the solution.
   3. Utilize the 1000 µL transfer pipette and adjust the scale to 900 µL by turning the knob. Then, transfer 900 µL of the ortho-toluidine substrate solution into the opaque brown reagent bottle, gently blow by pipetting, and thoroughly homogenize the solution.
   4. Utilize the 10 µL transfer pipette and adjust the scale to 5 µL by turning the knob. Then, transfer 5 µL of the 6% (v/v) hydrogen peroxide solution into the opaque brown reagent bottle.
4. Utilize the 1000 µL transfer pipette and adjust the scale to 1000 µL by turning the knob. Replace the pipette tip, gently blow by pipetting, and thoroughly homogenize the solution in the opaque brown reagent bottle.
5. Store the ready-to-utilize working solution in the opaque brown reagent bottle at RT, away from light. The working solution is reactive for 24 h.
6. Each semen sample requires 900 µL of the working solution. Ensure that the quantity of the working solution prepared each day corresponds to the number of samples examined daily.

2. Preparation of semen sample

1. Before collecting the semen sample, ensure the patient has abstained from ejaculation for 2 to 7 days as required by the World Health Organization (WHO) laboratory manual for examining and processing human semen (5th ed.)⁸. Masturbation is the preferred method for obtaining semen samples. To improve the accuracy of the examination, instruct the patient to collect all ejaculated semen.
2. Turn on the constant temperature stand and heat up to 37 °C in advance so that the subsequent liquefaction of the semen sample can proceed smoothly.
3. Upon receipt of the patient's semen sample, meticulously record detailed information, including identification code, collection time, abstinence duration, and other pertinent information, on the outer wall of the polymer-based receptacle.
4. Place the polymer-based receptacle housing the semen sample on the constant temperature stand preheated to 37 °C, which is beneficial for the semen liquefaction.
5. Draw the semen sample into a polymer-based pipette and observe the state of liquefaction every 5 min until the semen sample is fully liquefied.
6. If the semen sample has not been fully liquefied within 30 min, do not perform the examination yet; continue to wait for another 30 min. If the semen is still not fully liquefied after 60 min, add an equal volume of phosphate buffer solution for a 1:1 dilution and gently agitate to promote liquefaction.
7. After thorough agitation and consistent homogenizing, retain the fully liquefied semen sample for the subsequent examination procedures.

3. Staining of the peroxidase-positive leukocytes and analyzing the original semen sample

1. Prior to sampling, utilize a polymer-based pipette to repeatedly blow by pipetting and thoroughly homogenize the semen sample under examination.
2. Utilize the 200 µL transfer pipette and adjust the scale to 100 µL by turning the knob. Replace the pipette tip, and then transfer 100 µL of the semen sample into a centrifuge tube.
3. Utilize the 1000 µL transfer pipette and adjust the scale to 900 µL by turning the knob. Replace the pipette tip, then transfer 900 µL of the above-mentioned working solution into the centrifuge tube.
4. Repeatedly blow by pipetting and thoroughly homogenize the reactant, which consists of the semen sample and working solution, using the 1000 µL transfer pipette.
5. Position the centrifuge tube into a test-tube rack upon a two-dimensional shaker and subject it to continuous agitation at 200 rpm for 30 min at RT.
6. While waiting for the staining reaction, analyze the semen sample utilizing the CASA system to determine the concentration of spermatozoa in the original semen sample in the polymer-based receptacle.
   1. Turn on the power switch of the CASA system and double-click the SCA SCOPE icon to open the application software.
   2. Before sampling, repeatedly blow by pipetting and thoroughly homogenize the original semen sample again with a polymer-based pipette.
   3. Utilize the 10 µL transfer pipette and adjust the scale to 10 µL by turning the knob. Replace the pipette tip, pipette up 10 µL of the original semen sample, and place it into the SCA counting chamber. Wait for 60 s for the semen sample to stop drifting.
   4. Position the SCA counting chamber onto the sample holder of the CASA system and set the patient's information in the dialog box of the application software to prepare for the formal examination.
   5. Examine the original semen sample in the SCA counting chamber with the CASA system by clicking the Start button and record the necessary data, such as the concentration of spermatozoa.

4. Observation of the peroxidase-positive leukocytes and spermatozoa

1. Utilize the 1000 µL transfer pipette and adjust the scale to 1000 µL by turning the knob. Replace the pipette tip, then repeatedly blow by pipetting and thoroughly homogenize the reactant, which consists of semen sample and working solution in the centrifuge tube again at the end of the 30 min reaction process.
2. Utilize the 10 µL transfer pipette and adjust the scale to 10 µL by turning the knob. Replace the pipette tip, pipette up 10 µL of the reactant mentioned above, and place it into the sperm counting chamber. Finally, cover the sperm counting chamber with a dedicated glass coverslip.
3. Place the sperm counting chamber onto the microscopic stage and turn on the power switch of the optical microscope with two 10x eyepieces. Then, wait for 60 s for the reactant sample to stop drifting.
4. Switch the microscopic converter to the 10x objective lens during the waiting time. Then adjust the coarse adjustment knob and the fine adjustment knob in sequence. Finally, observe the spermatozoa and round cells under a magnification of 10×10.
5. Switch the microscopic converter to the 40x objective lens after determining the field of view to be analyzed, and observe the spermatozoa and brown peroxidase-positive leukocytes under a magnification of 10 × 40.
6. Utilize the electronic counter system to count at least 200 spermatozoa and the number of brown peroxidase-positive leukocytes within the corresponding fields.

5. Calculation of the concentration of peroxidase-positive leukocytes

1. Calculate the concentration of peroxidase-positive leukocytes through the using the following formula:
   

Representative Results

Upon implementing the aforementioned procedure, the reactant in the centrifuge tube frequently manifests a subtranslucent, opalescent appearance (Figure 1). Detecting an obviously brown color discloses potential degranulation of leukocytes, which may result in liquid staining. Consequently, the staining process could fail, rendering it necessary to either re-stain or collect a new semen sample for retesting²¹.

Peroxidase-positive leukocytes displayed a brown hue, contrasting with the unstained ordinary round cells (Figure 2, ordinary slide). Subsequently, every spermatozoon and brown leukocyte under the current microscope field was counted before shifting to an entirely new field for continuous observation and tallying. To ensure optimal accuracy, it is necessary to count a minimum of 200 spermatozoa and the leukocytes present in the corresponding field of view.

Figure 1: Reactant solution. The reactant that completes the reaction process is a subtranslucent opalescent liquid. Please click here to view a larger version of this figure.

Figure 2: Observation under optical microscope (ordinary slide). (A) Peroxidase-positive leukocyte and (B) ordinary round cell observed at 10 x 40 magnification optical microscope. Please click here to view a larger version of this figure.

Discussion

Ortho-toluidine and hydrogen peroxide have photosensitive properties and can decompose when exposed to light. To ensure the effectiveness of the test reagents, it is recommended that the prepared working fluid be stored in a dark place.

Semen samples naturally exhibit heterogeneous characteristics. In the preparation process of semen samples, correctly judging the liquefaction state of semen and thoroughly homogenizing the semen samples before each sampling are crucial for improving the reliability of the test results. Semen samples liquefy completely within 15 min. Mechanical homogenizing or enzymatic digestion should be considered if they have not completely liquefied after 60 min. More homogeneous semen samples can be obtained by gently shaking the sample container continuously during the liquefaction process using a two-dimensional shaker⁸.

The examination method presented in this paper possesses the advantages of simplicity, speed, and cost-effectiveness. However, it may not be advantageous under certain circumstances: peroxidase is stored in azurophilic granules that will be secreted into the intercellular space through degranulation or cytopempsis upon activation^21,22. Consequently, neutrophils may fail to stain and cannot be identified. In such cases, repeating the staining process or even requiring the patient to provide a new semen sample for re-examination becomes necessary. To minimize this situation, it is recommended to utilize fresh semen samples whenever possible with minimal placement time and a gentle homogenizing process. This method can only identify active diseases or infections and cannot identify diseases in later stages, such as chronic infections¹⁶. Additionally, other types of leukocytes with lower peroxidase content, such as lymphocytes, macrophages, and monocytes, pose difficulties in staining using this method; hence, obtaining reliable results can be achieved by employing leukocyte antigen (e.g., CD45) immunohistochemical staining.

As required by the 5th edition of the WHO manual, traditional positive myeloperoxidase staining (Endtz test) requires microscopic counting of at least 200 peroxidase-positive leukocytes in a blood cell count plate to calculate their concentration⁸. Compared to the traditional manual Endtz test, this protocol introduces the spermatozoa concentration data obtained by CASA detection, combining manual and automatic methods. To calculate the concentration of peroxidase-positive leukocytes, at least 200 spermatozoa and leukocytes in the corresponding field of view were counted under a microscope. Since the concentration of spermatozoa in semen is much greater than that of leukocytes, less time was required to count 200 spermatozoa compared to counting 200 peroxidase-positive leukocytes.

Patients with severe oligospermia, cryptozoospermia, and azoospermia often face challenges in counting a sufficient number of spermatozoa. In such cases, the concentration of peroxidase-positive leukocytes can be directly calculated using a blood cell counting plate as described in the manual for the plate. Additionally, for patients who do not require CASA or laboratories without CASA equipment, a blood cell counting plate can also be utilized to determine the concentration of peroxidase-positive leukocytes.

Materials

148 mmol/L ethylenediamine tetraacetic acid disodium (Na2EDTA) solution	ShenZhen HuaKang Biomedical Engineering Co.,LTD	20230301	Agentia B
6% (v/v) hydrogen peroxide solution	ShenZhen HuaKang Biomedical Engineering Co.,LTD	20230301	Agentia D
Automatic Sperm Class Analyzer – SCA SCOPE	Microptic S.L.	1222996; Model type: SCA-SCOPE-H	Computer-Assisted Semen Analysis (CASA) equipment
Disposable centrifugal tube (1.5 mL)	Zhejiang Gongdong Medical Equipment Co., LTD	2210009	Centrifugal tube for Staining process
Disposable transfer pipette (3 mL)	Jiangsu Kangjian Medical Supplies Co., LTD	20221101	Polymer-based pipette
Electronic counter	None	None	A multichannel counter
Electronic scales	Shanghai Liangping Instrument Co., LTD	D9008084	MAX = 100 g, e = 10 d, d = 0.01 g
Makler counting chamber	Makler	MQ30004	0.01 sq.mm, 10 μm deep
Optical microscope	Olympus	3G41067201307	CX31, 10×40
Ortho-toluidine substrate solution	ShenZhen HuaKang Biomedical Engineering Co.,LTD	20230301	Agentia C
Pipet tips(1 mL)	Coming Life Sciences (Wujiang) Co.,Ltd	02923205	1000 tips/unit, 5 units/case
Pipet tips(10 µL)	Coming Life Sciences (Wujiang) Co.,Ltd	00323961	1000 tips/unit, 20 units/case
Pipet tips(200 µL)	Coming Life Sciences (Wujiang) Co.,Ltd	32822810	1000 tips/unit, 20 units/case
Saturated ammonium chloride (NH4Cl) solution	ShenZhen HuaKang Biomedical Engineering Co.,LTD	20230301	Agentia A
SCA counting chamber	Microptic S.L.	102230608	10 µm, 2 Chambers
The container for semen sample	Jiangsu Kangjian Medical Supplies Co., LTD	20230701	Polymer-based receptacle for semen sample
Thermostatic table	Shenzhen MIAOQUAN Instrument Co., LTD	MQ30004	MQ-300
Transfer pipette (10 µL)	Eppendoff	3121000.015
Transfer pipette (1000 µL)	Eppendoff	3121000.12
Transfer pipette (200 µL)	Eppendoff	3121000.082
Two-dimensional shaker	DragonLab	822000010000	VC5A002205