Name: Staining of Peroxidase-Positive Leukocytes in Semen Samples
Uploaded: 2024-01-19T14:15:00
Description: Watch this Scientific Journal Video about Staining of Peroxidase-Positive Leukocytes in Semen Samples at JoVE.com

staining of peroxidase-positive leukocytes in semen samples

Analyse van sperma voor peroxidase-positieve leukocytencellen

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.

Auteur in de schijnwerpers: een eenvoudige en kosteneffectieve methode voor leukocytenanalyse in sperma 

Het onderzoek van peroxidase-positieve leukocyten in sperma

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.

Watch this Scientific Journal Video about Staining of Peroxidase-Positive Leukocytes in Semen Samples at JoVE.com

Staining of Peroxidase-Positive Leukocytes in Semen Samples  

Kleuring van peroxidase-positieve leukocyten in spermamonsters

Maak om te beginnen de verzamelde menselijke spermamonsters vloeibaar en homogeniseer ze grondig met een pasteurpipet. Breng vervolgens 100 microliter van het spermamonster over in een centrifugebuis. Voeg aan deze buis 900 microliter van de kleuroplossing toe.Herhaaldelijk mengen door pipetteren en de reactant grondig homogeniseren. Plaats de buis op een tweedimensionale schudder voor continu roeren bij 200 RPM gedurende 30 minuten bij kamertemperatuur. Terwijl u wacht op de kleuringsreactie, gaat u verder met de analyse van de spermatozoaconcentratie met behulp van het CASA-systeem.Dubbelklik na het inschakelen van het systeem op het SCAscope-pictogram om de applicatiesoftware te openen. Meng vervolgens het spermamonster grondig door te pipetteren en breng maximaal 10 microliter van het monster over op de SCA-telkamer. Wacht 60 seconden totdat het spermamonster stopt met drijven.Plaats vervolgens de SCA-telkamer op de monsterhouder van het CASA-systeem. Voer in de applicatiesoftware de gegevens van de patiënt in het dialoogvenster in. Klik op de startknop om de concentratie spermatozoa in het sperma vast te leggen.Stop vervolgens de shaker wanneer de kleuringsreactie van 30 minuten is voltooid. Meng vóór de analyse de inhoud van de centrifugebuis door te pipetteren. Breng tot 10 microliter van de reactant over in de spermatelkamer en dek deze af met een speciale glazen afdekglaasje.Plaats de spermatelkamer op de microscopische tafel en zet de optische microscoop aan. Wacht vervolgens 60 seconden totdat het reactantmonster stopt met afdrijven. Pas onder de 10x objectieflens de koers- en fijnafstellingsknoppen aan om een gezichtsveld te selecteren.Schakel vervolgens over naar een 40x objectieflens en observeer de spermatozoa en peroxidase-positieve leukocyten. Gebruik het elektronische tellersysteem om ten minste 200 spermatozoa en het aantal peroxidase-positieve leukocyten in bruin te tellen. Met behulp van deze techniek werden peroxidase-positieve leukocyten gekleurd in het spermamonster.De peroxidase-positieve leukocyten vertoonden een bruine tint die contrasteerde met de ongekleurde, gewone, ronde cellen.

Research

JoVE Journal

Medicine

Staining of Peroxidase-Positive Leukocytes in Semen Samples

The Examination of Peroxidase-Positive Leukocytes in Semen

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