This technique presents a practical approach to estimate multiple human spermatozoa functional biomarkers by flow cytometry without substantial compromise of experiment quality induced by prolonged waiting times. This technique can be a practical and reliable toolkit for the diagnosis of infertility and evaluation of sperm function at both bench and bed, and complement the routine sperm examination. This procedure is carried out with Accuri C6 platform and it could be applied to other commercial platforms with minor modifications.
To begin, incubate the semen sample at 37 degrees Celsius and completely liquefy the sample for one hour. To count the sperm cells, mix the liquified semen sample thoroughly, then add 10 microliters of semen into a sperm counting chamber and scan the slide in a sperm class analyzer. Count at least six areas and 400 spermatozoa to estimate sperm concentration.
For detection of sperm apoptosis, using annexin-5 FITC PI staining, add 10 to the sixth sperm cells to a 1.5 milliliter centrifuge tube. Wash the cells with 500 microliters of cold PBS. Then, centrifuge the sperm cell suspension and discard the supernatant before re-suspending the cells in 200 microliters of binding buffer.
Next, add two microliters of FITC annexin-5 and two microliters of PI.Gently vortex the cells and incubate for 15 minutes at room temperature in the dark before placing them in the flow cytometer for analysis. For analysis of mitochondrial membrane potential, or MMP, using JC-1 probe, add two times 10 to the sixth sperm cells to a 1.5 milliliter tube and centrifuge. After centrifugation, re-suspend the sperm cell suspension in a one milliliter JC-1 working solution.
Gently vortex the cells and incubate for 20 minutes at 37 degrees Celsius in the dark. Again, centrifuge the suspension, discard the supernatant and wash the pellet twice with one milliliter of PBS. Then, re-suspend the dyed sperm cells in one milliliter of PBS in a flow cytometer tube, and immediately place the tube in the flow cytometer for analysis.
Next, use Carbonyl Cyanide m-Chlorophenylhydrazone, or CCCP as a positive control. Re-suspend the sperm cells in one milliliter of CCCP working solution and gently vortex before incubating at room temperature for 20 minutes. After incubation, centrifuge the suspension, discard the supernatant, and perform the steps to analyze MMP using the JC-1 probe as demonstrated.
For sperm chromatin structure assay, or SCSA, thaw the semen sample at a 37 degree Celsius water bath and dilute with TNE Buffer to a concentration of one to two times 10 to the sixth cells per milliliter. Next, add 200 microliters of the diluted sample to a flow cytometer test tube and mix it with 400 microliters of acid solution. Stain the sample with 1.2 milliliters of acridine orange solution.
Use a reference sample as an internal standard to flow cytometer setup and calibrations. Dilute the sample with four degrees Celsius TNE Buffer to a concentration of one to two times 10 to the sixth cells per milliliter, and perform the steps for SCSA, as demonstrated. Open the flow cytometer software and start the cytometer.
To collect sample data, load a control sample onto the flow cytometer and click Run to start data collection. Create dot plots for viewing data. Select the XY axis parameters and display linear to specify data.
Then, select and apply a polygonal gate to delineate the sperm population for analysis. For sperm apoptosis, set the FL1 as the X axis and FL2 as the Y axis. Then, to isolate the live apoptotic or necrotic cells, tap and drag the quadrant gate to subset the sperm populations down to four specific populations.
For MMP, set FL1 as the X axis and FL2 as the Y axis. Then, create a polygonal gate to subset the sperm populations down to two specific populations. For SCSA, set the FL4 as the X axis and FL1 as the Y axis.
Draw the gate, set a 45-degree angle to exclude cellular debris signals. Calculate 10, 000 sperm cells for each sample for sperm apoptosis, MMP and SCSA analyses, set a run limit to indicate when to stop collecting data, fluidics rate, depending on the cell numbers and threshold to eliminate debris and noise. Apply these settings for all samples.
If necessary, set the color compensation to correct fluorescent spillover. Gently back pipette the sample before loading it onto the flow cytometer. Then, enter the sample name and click Run.
Once the run is complete, save the sample data as FCS file by giving a file name for further analysis. The measurement of sperm apoptosis using annexin-five FITC PI staining is shown here. This analysis included negative control of unstained sperm cells to set compensation and quadrants.
In a sample with sperm apoptosis, the results were expressed as the percentages of viable or live early apoptotic or apoptotic, late apoptotic and necrotic cells. The sperm subpopulation with high and low MMP is shown here. A good quality semen sample showed high orange and low green fluorescence.
Conversely, a poor quality semen sample demonstrated low orange and high green fluorescence. The SCSA cytograms from a fertile and infertile man are shown here. The sperm cell population was identified as normal with high DNA stability, DNA fragmentation index, and cell debris.
The semen sample must be incubated at 37 degrees Celsius and completely liquefied for up to one hour.
