Name: Preparation of Working Solution and Semen Sample for Leukocyte Analysis
Uploaded: 2024-01-19T14:15:00
Description: Watch this Scientific Journal Video about Preparation of Working Solution and Semen Sample for Leukocyte Analysis at JoVE.com

preparation of working solution and semen sample for leukocyte analysis

Analisi dello sperma per le cellule leucocitarie perossidasi-positive

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.

Autore in primo piano: Un metodo semplice ed economico per l'analisi dei leucociti nello sperma 

L'esame dei leucociti positivi alla perossidasi nel liquido seminale

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 Preparation of Working Solution and Semen Sample for Leukocyte Analysis at JoVE.com

Preparation of Working Solution and Semen Sample for Leukocyte Analysis  

Preparazione della soluzione di lavoro e del campione di sperma per l'analisi dei leucociti

Per iniziare, raccogliere i reagenti per la preparazione della soluzione di lavoro e metterli a temperatura ambiente. Inoltre, prepara un flacone opaco per conservare la soluzione di lavoro e proteggerla dalla luce. Quindi trasferire 100 microlitri della soluzione di cloruro di ammonio nel flacone opaco del reagente utilizzando una pipetta di trasferimento da 200 microlitri.Trasferire anche 100 microlitri della soluzione di EDTA di sodio nel flacone del reagente e miscelare mediante pipettaggio. Successivamente, aggiungere 900 microlitri della soluzione di substrato ortotoluidinale nel flacone di reagente opaco e mescolare delicatamente mediante pipettaggio. Quindi trasferire cinque microlitri della soluzione di perossido di idrogeno nel flacone del reagente.Utilizzando una pipetta di trasferimento da 1000 microlitri, miscelare delicatamente la soluzione mediante pipettaggio. Per preparare i campioni di sperma, accendere il supporto a temperatura costante e riscaldarlo fino a 37 gradi Celsius. Inoltre, tarare la bilancia per il peso del recipiente a base di polimero.Al ricevimento del campione di sperma, pesare il campione e registrare informazioni dettagliate come il codice di identificazione, l'ora di raccolta, la durata dell'astinenza sulla parete esterna del recipiente a base polimerica. Successivamente, posizionare il campione di sperma sul supporto a temperatura costante per la liquefazione dello sperma. Prelevare il campione di sperma in una pipetta a base polimerica e controllare la liquefazione ogni cinque minuti per 30 minuti fino a quando il campione di sperma non è completamente liquefatto.

Research

JoVE Journal

Medicine

Preparation of Working Solution and Semen Sample for Leukocyte Analysis

The Examination of Peroxidase-Positive Leukocytes in Semen

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