Types of Samples for ROS Measurement

ROS measurement can be performed in various types of samples such as:

1. Neat or unprocessed, whole seminal ejaculate. In this method, the seminal plasma is not removed. All components of the seminal ejaculate are intact and ROS levels indicate the actual de novo levels in the ejaculate. This is the ideal setup for assessing the clinical relevance of ROS in the seminal ejaculate and is indicative of oxidative stress and sperm dysfunction.

2. Processed sample: Liquefied semen specimens are centrifuged at 300 x g for 7 min and seminal plasma is removed. The sperm pellet is washed and resus-pended to 1 mL volume in PBS. This is also called the simple "wash and resuspend" method. With this method, the seminal plasma that confers protection to the sperm and other dissolved components is removed. However, all the cellular components such as debris, round cells, white blood cells, and leukocytes are still present in the sample.

3. Spermpreparation by the swim-upprocedure: This method is used to measure ROS in highly motile sperm prepared by swim up. After liquefaction, an aliquot of specimen is mixed with sperm wash media using a sterile Pasteur pipette. It is centrifuged at 330 x g for 10 min. The supernatant is carefully aspirated and the pellet resuspended in 3 mL of fresh sperm wash media. The resuspended sample is carefully transferred in equal parts to two 15-mL sterile round-bottom test tubes and centrifuged at 330 x g for 5 min. Motile sperm are allowed to swim up during the incubation of test tubes at a 45° angle in 5% CO2 at 37°C for 1 h. Supernatant is aspirated into a clean test tube and centrifuged at 330 xg for 7 min. The final supernatant is aspirated and the sperm pellet is resuspended in sperm wash media and is used for measurement of ROS.

4. Spermpreparation by density gradients: This method is used to measure ROS in immature (morphologically abnormal, poor motility) and mature (highly motile and morphologically normal) sperm. A double density gradient (40% "Upper phase" and 80% "Lower phase") is used. Both the density gradient and the sperm wash media is brought to 37°C or room temperature. Using a sterile pipette, 2.0 mL of the "Lower phase" is transferred into a 15-mL conical centrifuge tube.

Similarly, 2.0 mL of the "Upper phase" is carefully placed on the top of the lower layer. The liquefied semen sample (1-2 mL) is placed on top of the upper layer, and the tube is centrifuged for 20 min at 330 x g or 1,600 revolutions per minute (rpm). The uppermost layer containing clear seminal plasma and dead white cells and other debris are discarded. For separating the immature sperm, the interphase between the upper and lower layer is carefully transferred to a conical tube. For separating the mature sperm, the pellet is carefully aspirated and transferred into another centrifuge tube. Using a transfer pipette, 2-3 mL of sperm wash media is added, and the immature and mature fractions are centrifuged for 7 min at 330 xg or 1,600 rpm. The supernatant is discarded, and the pellet is suspended in 1.0 mL of sperm wash media. Sperm count, motility, and ROS levels are measured in the recovered fractions.

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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