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 av sæd for peroksidase-positive leukocyttceller

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.

Forfatter Spotlight: En enkel og kostnadseffektiv metode for leukocyttanalyse i sæd 

Undersøkelsen av peroksidase-positive leukocytter i sæd

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  

Farging av peroksidase-positive leukocytter i sædprøver

For å begynne, fortynn de innsamlede menneskelige sædprøvene og homogeniser dem grundig ved hjelp av en pasteurpipette. Overfør deretter 100 mikroliter av sædprøven til et sentrifugerør. Til dette røret, tilsett 900 mikroliter av fargeløsningen.Bland gjentatte ganger ved pipettering og homogeniser reaktanten grundig. Plasser røret på en todimensjonal shaker for kontinuerlig omrøring ved 200 o / min i 30 minutter ved romtemperatur. Mens du venter på fargereaksjonen, fortsett for analyse av spermatozoakonsentrasjon ved hjelp av CASA-systemet.Etter at du har slått på systemet, dobbeltklikker du på SCAscope-ikonet for å åpne programmet. Bland deretter sædprøven grundig ved pipettering, og overfør opptil 10 mikroliter av prøven på SCA-tellekammeret. Vent i 60 sekunder til sædprøven slutter å drive.Deretter plasserer du SCA-tellekammeret på prøveholderen til CASA-systemet. I applikasjonsprogramvaren skriver du inn pasientens informasjon i dialogboksen. Klikk på startknappen for å registrere konsentrasjonen av spermatozoa i sæden.Deretter stopper du shakeren når den 30 minutter lange fargereaksjonen er fullført. Før analyse, bland innholdet i sentrifugerøret ved pipettering. Overfør opptil 10 mikroliter av reaktanten inn i sædtellekammeret og dekk det med et dedikert glassdeksel.Plasser sædtellekammeret på det mikroskopiske stadiet og slå på det optiske mikroskopet. Vent deretter i 60 sekunder til reaktantprøven slutter å drive. Under 10x objektivlinse justerer du kursen og finjusteringsknappene for å velge et synsfelt.Bytt deretter til 40x objektivlinse og observer spermatozoa og peroksidase positive leukocytter. Bruk det elektroniske tellersystemet til å telle ut minst 200 spermatozoer og antall peroksidase positive leukocytter i brunt. Ved hjelp av denne teknikken ble peroksidasepositive leukocytter farget i sædprøven.Peroksidase-positive leukocytter viste en brun nyanse som kontrasterte med de ufargede, vanlige, runde cellene.

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 ...