The authors are grateful to the Andrology Laboratory of the Center for Reproductive Medicine, the Third Affiliated Hospital of Sun Yat-sen University, for providing the facilities for this study. No funding was involved in the preparation of this article.

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
<ol>
	<li>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 &#181;L, 200 &#181;L, 10 &#181;L) and test tube racks. 
	CAUTION: Read the safety data sheet (SDS) before handling the above-mentioned reagents.</li>
	<li>Prepare an opaque brown reagent bottle to store and protect the prepared working solution from exposure to light.</li>
	<li>Transfer the reagents into the opaque brown reagent bottle as per the proportions specified below:
	<ol>
		<li>Utilize the 200 &#181;L transfer pipette and adjust the scale to 100 &#181;L by turning the knob. Then, transfer 100 &#181;L of the NH4Cl solution into the opaque brown reagent bottle.</li>
		<li>Replace the pipette tip and transfer 100 &#181;L of the 148 mmol/L Na2EDTA solution into the opaque brown reagent bottle using the 200 &#181;L transfer pipette mentioned above. Then, gently blow by pipetting and thoroughly homogenize the solution.</li>
		<li>Utilize the 1000 &#181;L transfer pipette and adjust the scale to 900 &#181;L by turning the knob. Then, transfer 900 &#181;L of the ortho-toluidine substrate solution into the opaque brown reagent bottle, gently blow by pipetting, and thoroughly homogenize the solution.</li>
		<li>Utilize the 10 &#181;L transfer pipette and adjust the scale to 5 &#181;L by turning the knob. Then, transfer 5 &#181;L of the 6% (v/v) hydrogen peroxide solution into the opaque brown reagent bottle.</li>
	</ol>
	</li>
	<li>Utilize the 1000 &#181;L transfer pipette and adjust the scale to 1000 &#181;L by turning the knob. Replace the pipette tip, gently blow by pipetting, and thoroughly homogenize the solution in the opaque brown reagent bottle.</li>
	<li>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.</li>
	<li>Each semen sample requires 900 &#181;L of the working solution. Ensure that the quantity of the working solution prepared each day corresponds to the number of samples examined daily.</li>
</ol>
2. Preparation of semen sample
<ol>
	<li>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.</li>
	<li>Turn on the constant temperature stand and heat up to 37 &#176;C in advance so that the subsequent liquefaction of the semen sample can proceed smoothly.</li>
	<li>Upon receipt of the patient&#39;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.</li>
	<li>Place the polymer-based receptacle housing the semen sample on the constant temperature stand preheated to 37 &#176;C, which is beneficial for the semen liquefaction.</li>
	<li>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.</li>
	<li>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.</li>
	<li>After thorough agitation and consistent homogenizing, retain the fully liquefied semen sample for the subsequent examination procedures.</li>
</ol>
3. Staining of the peroxidase-positive leukocytes and analyzing the original semen sample
<ol>
	<li>Prior to sampling, utilize a polymer-based pipette to repeatedly blow by pipetting and thoroughly homogenize the semen sample under examination.</li>
	<li>Utilize the 200 &#181;L transfer pipette and adjust the scale to 100 &#181;L by turning the knob. Replace the pipette tip, and then transfer 100 &#181;L of the semen sample into a centrifuge tube.</li>
	<li>Utilize the 1000 &#181;L transfer pipette and adjust the scale to 900 &#181;L by turning the knob. Replace the pipette tip, then transfer 900 &#181;L of the above-mentioned working solution into the centrifuge tube.</li>
	<li>Repeatedly blow by pipetting and thoroughly homogenize the reactant, which consists of the semen sample and working solution, using the 1000 &#181;L transfer pipette.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>Turn on the power switch of the CASA system and double-click the SCA SCOPE icon to open the application software.</li>
		<li>Before sampling, repeatedly blow by pipetting and thoroughly homogenize the original semen sample again with a polymer-based pipette.</li>
		<li>Utilize the 10 &#181;L transfer pipette and adjust the scale to 10 &#181;L by turning the knob. Replace the pipette tip, pipette up 10 &#181;L of the original semen sample, and place it into the SCA counting chamber. Wait for 60 s for the semen sample to stop drifting.</li>
		<li>Position the SCA counting chamber onto the sample holder of the CASA system and set the patient&#39;s information in the dialog box of the application software to prepare for the formal examination.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Observation of the peroxidase-positive leukocytes and spermatozoa
<ol>
	<li>Utilize the 1000 &#181;L transfer pipette and adjust the scale to 1000 &#181;L 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.</li>
	<li>Utilize the 10 &#181;L transfer pipette and adjust the scale to 10 &#181;L by turning the knob. Replace the pipette tip, pipette up 10 &#181;L of the reactant mentioned above, and place it into the sperm counting chamber. Finally, cover the sperm counting chamber with a dedicated glass coverslip.</li>
	<li>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.</li>
	<li>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&#215;10.</li>
	<li>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 &#215; 40.</li>
	<li>Utilize the electronic counter system to count at least 200 spermatozoa and the number of brown peroxidase-positive leukocytes within the corresponding fields.</li>
</ol>
5. Calculation of the concentration of peroxidase-positive leukocytes
<ol>
	<li>Calculate the concentration of peroxidase-positive leukocytes through the using the following formula: 
	<img alt="Equation 1" src="/files/ftp_upload/66211/66211eq01v2.jpg" style="margin: auto;" /></li>
</ol>

Analysis of Semen for Peroxidase-Positive Leukocyte Cells

<ol>
	<li>Agarwal, A., Mulgund, A., Hamada, A., Chyatte, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+view+on+male+infertility+around+the+globe.">A unique view on male infertility around the globe.</a> Reprod Biol Endocrinol. 13, 37 (2015).</li><li>Sharlip, I. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Best+practice+policies+for+male+infertility.">Best practice policies for male infertility.</a> Fertil Steril. 77 (5), 873-882 (2002).</li><li>Choy, J. T., Eisenberg, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+infertility+as+a+window+to+health.">Male infertility as a window to health.</a> Fertil Steril. 110 (5), 810-814 (2018).</li><li>Pellati, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genital+tract+infections+and+infertility.">Genital tract infections and infertility.</a> Eur J Obstet Gynecol Reprod Biol. 140 (1), 3-11 (2008).</li><li>Rivero, M. J., Kulkarni, N., Thirumavalavan, N., Ramasamy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+management+of+male+genital+tract+infections+in+the+setting+of+male+infertility:+an+updated+review.">Evaluation and management of male genital tract infections in the setting of male infertility: an updated review.</a> Curr Opin Urol. 33 (3), 180-186 (2023).</li><li>Long, S., Kenworthy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Round+cells+in+diagnostic+semen+analysis:+A+guide+for+laboratories+and+clinicians.">Round cells in diagnostic semen analysis: A guide for laboratories and clinicians.</a> Br J Biomed Sci. 79, 10129 (2022).</li><li>Johanisson, E., Campana, A., Luthi, R., de Agostini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+'round+cells'+in+semen+analysis:+a+comparative+study.">Evaluation of 'round cells' in semen analysis: a comparative study.</a> Hum Reprod Update. 6 (4), 404-412 (2000).</li><li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WHO+Laboratory+Manual+for+the+Examination+and+Processing+of+Human+Semen.+5th+edn.">WHO Laboratory Manual for the Examination and Processing of Human Semen. 5th edn.</a> World Health Organization. , (2010).</li><li>Domes, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+incidence+and+effect+of+bacteriospermia+and+elevated+seminal+leukocytes+on+semen+parameters.">The incidence and effect of bacteriospermia and elevated seminal leukocytes on semen parameters.</a> Fertil Steril. 97 (5), 1050-1055 (2012).</li><li>Aziz, N., Agarwal, A., Lewis-Jones, I., Sharma, R. K., Thomas, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+associations+between+specific+sperm+morphological+defects+and+leukocytospermia.">Novel associations between specific sperm morphological defects and leukocytospermia.</a> Fertil Steril. 82 (3), 621-627 (2004).</li><li>Alahmar, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+oxidative+stress+in+male+infertility:+An+updated+review.">Role of oxidative stress in male infertility: An updated review.</a> J Hum Reprod Sci. 12 (1), 4-18 (2019).</li><li>Schlegel, P. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+treatment+of+infertility+in+men:+AUA/ASRM+guideline+Part+I.">Diagnosis and treatment of infertility in men: AUA/ASRM guideline Part I.</a> J Urol. 205 (1), 36-43 (2021).</li><li>Ricci, G., Presani, G., Guaschino, S., Simeone, R., Perticarari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+detection+in+human+semen+using+flow+cytometry.">Leukocyte detection in human semen using flow cytometry.</a> Hum Reprod. 15 (6), 1329-1337 (2000).</li><li>Brunner, R. J., Demeter, J. H., Sindhwani, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+guidelines+for+the+evaluation+and+treatment+of+leukocytospermia+in+male+infertility.">Review of guidelines for the evaluation and treatment of leukocytospermia in male infertility.</a> World J Mens Health. 37 (2), 128-137 (2019).</li><li>Amulic, B., Cazalet, C., Hayes, G. L., Metzler, K. D., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+function:+from+mechanisms+to+disease.">Neutrophil function: from mechanisms to disease.</a> Annu Rev Immunol. 30, 459-489 (2012).</li><li>Khan, A. A., Alsahli, M. A., Rahmani, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloperoxidase+as+an+active+disease+biomarker:+recent+biochemical+and+pathological+perspectives.">Myeloperoxidase as an active disease biomarker: recent biochemical and pathological perspectives.</a> Med Sci (Basel). 6 (2), 33 (2018).</li><li>Khan, A. A., Rahmani, A. H., Aldebasi, Y. H., Aly, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+pathological+studies+on+peroxidases+-an+updated+review).">Biochemical and pathological studies on peroxidases -an updated review).</a> Glob J Health Sci. 6 (5), 87-98 (2014).</li><li>Liu, W. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloperoxidase-derived+hypochlorous+acid+promotes+ox-LDL-induced+senescence+of+endothelial+cells+through+a+mechanism+involving+beta-catenin+signaling+in+hyperlipidemia.">Myeloperoxidase-derived hypochlorous acid promotes ox-LDL-induced senescence of endothelial cells through a mechanism involving beta-catenin signaling in hyperlipidemia.</a> Biochem Biophys Res Commun. 467 (4), 859-865 (2015).</li><li>Nicholls, S. J., Hazen, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloperoxidase+and+cardiovascular+disease.">Myeloperoxidase and cardiovascular disease.</a> Arterioscler Thromb Vasc Biol. 25 (6), 1102-1111 (2005).</li><li>Wolff, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biologic+significance+of+white+blood+cells+in+semen.">The biologic significance of white blood cells in semen.</a> Fertil Steril. 63 (6), 1143-1157 (1995).</li><li>Chen, Y., Hashiguchi, N., Yip, L., Junger, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertonic+saline+enhances+neutrophil+elastase+release+through+activation+of+P2+and+A3+receptors.">Hypertonic saline enhances neutrophil elastase release through activation of P2 and A3 receptors.</a> Am J Physiol Cell Physiol. 290 (4), C1051-C1059 (2006).</li><li>Weiss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+destruction+by+neutrophils.">Tissue destruction by neutrophils.</a> N Engl J Med. 320 (6), 365-376 (1989).</li></ol>

The authors have nothing to disclose.

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...

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 factors1,2,3. Male genital tract infections (MGTIs) constitute one of the significant causes of male infertility, accounting for about 15% of cases4,5.
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 &#62;1 &#215; 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 development10,11.
Currently, andrologists commonly choose to examine round cells in semen or elastase in seminal plasma when identifying genital tract inflammation. However, it&#39;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 &#215; 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&#39; azurophilic granules15,16 and is also expressed in lymphocytes17,18, monocytes, and macrophages19. The concentration of leukocytes, especially neutrophils, in semen can be obtained by examining round cells that are peroxidase-positive7,20. 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&#39; examination costs and waiting time for obtaining results, alleviates the workload of lab technicians, and shortens the doctor&#39;s diagnosis and treatment waiting period.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>148 mmol/L ethylenediamine tetraacetic acid disodium (Na2EDTA) solution</td>
			<td>ShenZhen HuaKang Biomedical Engineering Co.,LTD</td>
			<td>20230301</td>
			<td>Agentia B</td>
		</tr>
		<tr>
			<td>6% (v/v) hydrogen peroxide solution</td>
			<td>ShenZhen HuaKang Biomedical Engineering Co.,LTD</td>
			<td>20230301</td>
			<td>Agentia D</td>
		</tr>
		<tr>
			<td>Automatic Sperm Class Analyzer &#8211; SCA SCOPE&#160;</td>
			<td>Microptic S.L.</td>
			<td>1222996; Model type: SCA-SCOPE-H</td>
			<td>Computer-Assisted Semen Analysis (CASA) equipment</td>
		</tr>
		<tr>
			<td>Disposable centrifugal tube (1.5 mL)</td>
			<td>Zhejiang Gongdong Medical Equipment Co., LTD</td>
			<td>2210009</td>
			<td>Centrifugal tube for Staining process</td>
		</tr>
		<tr>
			<td>Disposable transfer pipette (3 mL)</td>
			<td>Jiangsu Kangjian Medical Supplies Co., LTD</td>
			<td>20221101</td>
			<td>Polymer-based pipette</td>
		</tr>
		<tr>
			<td>Electronic counter</td>
			<td>None</td>
			<td>None</td>
			<td>A multichannel counter</td>
		</tr>
		<tr>
			<td>Electronic scales</td>
			<td>Shanghai Liangping Instrument Co., LTD</td>
			<td>D9008084</td>
			<td>MAX = 100 g, e = 10 d, d = 0.01 g</td>
		</tr>
		<tr>
			<td>Makler counting chamber</td>
			<td>Makler</td>
			<td>MQ30004</td>
			<td>0.01 sq.mm, 10 &#956;m deep</td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>Olympus</td>
			<td>3G41067201307</td>
			<td>CX31, 10&#215;40</td>
		</tr>
		<tr>
			<td>Ortho-toluidine substrate solution</td>
			<td>ShenZhen HuaKang Biomedical Engineering Co.,LTD</td>
			<td>20230301</td>
			<td>Agentia C</td>
		</tr>
		<tr>
			<td>Pipet tips(1 mL)</td>
			<td>Coming Life Sciences (Wujiang) Co.,Ltd</td>
			<td>02923205</td>
			<td>1000 tips/unit, 5 units/case</td>
		</tr>
		<tr>
			<td>Pipet tips(10 &#181;L)</td>
			<td>Coming Life Sciences (Wujiang) Co.,Ltd</td>
			<td>00323961</td>
			<td>1000 tips/unit, 20 units/case</td>
		</tr>
		<tr>
			<td>Pipet tips(200 &#181;L)</td>
			<td>Coming Life Sciences (Wujiang) Co.,Ltd</td>
			<td>32822810</td>
			<td>1000 tips/unit, 20 units/case</td>
		</tr>
		<tr>
			<td>Saturated ammonium chloride (NH4Cl) solution</td>
			<td>ShenZhen HuaKang Biomedical Engineering Co.,LTD</td>
			<td>20230301</td>
			<td>Agentia A</td>
		</tr>
		<tr>
			<td>SCA counting chamber</td>
			<td>Microptic S.L.</td>
			<td>102230608</td>
			<td>10 &#181;m, 2 Chambers</td>
		</tr>
		<tr>
			<td>The container for semen sample</td>
			<td>Jiangsu Kangjian Medical Supplies Co., LTD</td>
			<td>20230701</td>
			<td>Polymer-based receptacle for semen sample</td>
		</tr>
		<tr>
			<td>Thermostatic table</td>
			<td>Shenzhen MIAOQUAN Instrument Co., LTD</td>
			<td>MQ30004</td>
			<td>MQ-300</td>
		</tr>
		<tr>
			<td>Transfer pipette (10 &#181;L)</td>
			<td>Eppendoff</td>
			<td>3121000.015</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette (1000 &#181;L)</td>
			<td>Eppendoff</td>
			<td>3121000.12</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette (200 &#181;L)</td>
			<td>Eppendoff</td>
			<td>3121000.082</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-dimensional shaker</td>
			<td>DragonLab</td>
			<td>822000010000</td>
			<td>VC5A002205</td>
		</tr>
	</tbody>
</table>

the examination of peroxidase-positive leukocytes in semen

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.

Author Spotlight: A Simple and Cost-Effective Method for Leukocyte Analysis in Semen 

The Examination of Peroxidase-Positive Leukocytes in Semen

Male reproductive medicine is my primary research focus, and our protocol primarily addresses the issues of speed and cost in analyzing peroxidase-positive leukocytes in semen. Semen is inherently heterogeneous, which greatly affects the stability of semen analysis results. Therefore, it is essential to ensure that semen is liquified and thoroughly mixed before examination.Our protocol combines manual and automatic methods to calculate the concentration of leukocytes by utilizing sperm concentration, which saves time and is more cost-effective compared to the ready counting leukocytes.

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...

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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.

Leukocytospermia can lead to decreased spermatozoa motility, increased spermatozoa morphological abnormalities, elevated spermatozoa DNA fragmentation ...

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JoVE Journal

Medicine

Preparation of Working Solution and Semen Sample for Leukocyte Analysis

Staining of Peroxidase-Positive Leukocytes in Semen Samples

2.8K Views.  The Third Affiliated Hospital of Sun Yat-sen University.  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.

Analysis of Semen for Peroxidase-Positive Leukocyte Cells

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