The Examination of Peroxidase-Positive Leukocytes in Semen

Summary

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, w....

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.......

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 relia.......

Materials

Name	Company	Catalog Number	Comments
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