Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects

Xi Ling; Peng Zou; Lin Ao; Niya Zhou; Xiaogang Wang; Lei Sun; Huan Yang; Jinyi Liu; Jia Cao; Qing Chen

doi:10.3791/63790

Medicine

Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects

Published: April 21, 2022 doi: 10.3791/63790

Xi Ling¹, Peng Zou¹, Lin Ao¹, Niya Zhou¹, Xiaogang Wang², Lei Sun¹, Huan Yang¹, Jinyi Liu¹, Jia Cao¹, Qing Chen¹

¹Institute of Toxicology, College of Preventive Medicine, Army Medical University (Third Military Medical University), ²Department of Chemical Defense Medicine, College of Military Preventive Medicine, Army Medical University (Third Military Medical University)

Summary

The present protocol provides a practical solution that allows for the measurement of apoptosis, mitochondrial membrane potential, and DNA damage in human sperm with a single cytometer.

Abstract

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no abnormalities in conventional semen parameters. Additional technologies are needed to explain the idiopathic infertility and detect subtle sperm defects. Currently, biomarkers of sperm function, including sperm apoptosis, mitochondrial membrane potential (MMP), and DNA damage, reveal sperm physiology at the molecular level and are capable of predicting male fertility.

With flow cytometry (FCM) techniques, each of these markers can be rapidly, accurately, and precisely measured in human semen samples, but time costs substantially increase and results could be obstructed if all the biomarkers need to be tested with a single cytometer. In this protocol, after collection and immediate incubation at 37 °C for liquefication, semen samples were further analyzed for sperm apoptosis using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The MMP was labeled with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) probe, and DNA damage was assessed using the sperm chromatin structure assay (SCSA) with acridine orange (AO) staining. Thus, flow cytometric analysis of sperm function markers can be a practical and reliable toolkit for the diagnosis of infertility and evaluation of sperm function at both bench and bed.

Introduction

Infertility has become a public health problem, with male infertility contributing to 40%-50% of all cases¹^,². Although conventional semen quality analysis plays an important role in determining male fertility potential, approximately 15% of patients with infertility have normal sperm parameters such as sperm count, motility, and morphology³. Moreover, routine sperm examinations have poor repeatability, provide limited information on sperm function, and cannot give an accurate evaluation of male fertility or reflect the subtle defects of sperm. Multiple techniques have been developed to test sperm function, such as the hemizona assay (HZA), the sperm penetration assay, the hypo-osmotic swelling test, the antisperm antibody testing, but these methods are time-consuming and vulnerable to subjective influences of the operators. Therefore, it is necessary to develop rapid and accurate methods for sperm function analysis.

FCM is a rapid, single-cell analysis technology developed in the 1970s, which has been widely used in various fields of cell biology and medicine. FCM is a robust tool for the evaluation of sperm function that analyzes spermatozoa labeled with a specific fluorescence probe⁴. The spermatozoa pass through single- or multi-channel lasers, and scattered light and emitted fluorescence are produced by a laser beam. The scattered light includes forward scattered light (FSC) and side scattered light (SSC), which reflects the size of the tested cell and the cell granularity or internal structure. These signals are collected, displayed, and analyzed by the computer system, and, consequently, a series of characteristics from the spermatozoa are measured quickly and accurately. Therefore, FCM is a rapid, objective, multi-dimensional, and high-throughput technique that has gained increasing attention in the field of sperm analysis. The application of FCM can make up for shortcomings in traditional methods and provides a new approach for the detection of sperm internal structure and function.

Sperm apoptosis is closely related to male fertility⁵. Detection of sperm apoptosis is an important index to evaluate sperm function at a molecular level. FCM is widely recognized as a reliable and sensitive method to detect sperm apoptosis using Annexin V-FITC/PI staining. The basic principle is that phosphatidylserine (PS) is transferred from the inner layer of the cell membrane to its outer layer in the early stage of apoptosis. Annexin V is a Ca²⁺-dependent phospholipid protein (usually labeled by FITC) that has a high affinity for PS, thus, detects PS exposed to the outer surface of the cell membrane⁶. Necrosis and apoptosis can be distinguished in combination with Pl staining. Therefore, the method of Annexin V-FITC/PI double staining is widely used, as it is rapid, simple, and easy to detect different apoptotic sperm cells.

A mature mammalian sperm contains approximately 72-80 mitochondria⁷, suggesting a biological reason for the retention of sperm mitochondria⁸. Sperm mitochondria have been found to play important roles in sustaining sperm motility and fertility⁹. The JC-1 stain, which can be used as an indicator of MMP in a variety of cell types, is one of the most popular fluorochromes for sperm mitochondrial activity assessment¹⁰. JC-1 is a cationic dye that potential-dependently accumulates in the mitochondria. JC-1 has a maximum fluorescence emission at 525 nm (green) and forms J-aggregates¹¹ when binding to membranes with a high potential (ΔΨm, 80-100 mV), resulting in a shift in fluorescence emission to ~590 nm (orange-red). Consequently, sperm mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio, and FCM can be used to detect MMP levels in human semen samples.

The SCSA methodology was invented by Evenson et al.¹², and is considered a precise and repeatable test with unique dual-parameter data (red vs green fluorescence) on a 1,024 × 1,024 channel scale¹³. In damaged sperm, DNA and altered proteins in sperm nuclei are labeled by AO, and breakages of the DNA strand are measured by red fluorescence, while intact double strands emit green fluorescence in FCM¹⁴. Nowadays, many methods have been developed to estimate sperm DNA integrity. However, unlike SCSA, these assays are often labor-intensive and lack the ability for a diagnosis of male infertility. The SCSA test results significantly corelate with human DNA pregnancy and miscarriage, provide compelling evidence that SCSA may be useful in human semen analysis, and may serve as a valuable tool to infertility clinicians¹⁵.

Chromatin integrity, MMP, and apoptosis reflect different aspects of sperm function, and thus, the combination of these biomarkers may provide a more comprehensive insight into sperm status. FCM can be used for separate measurements of either chromatin integrity, MMP, or apoptosis in human semen specimens. Although the expense of FCM and the cost of dyes have limited the wide application of these techniques in clinical laboratories for reproductive health, their value for fecundity estimation is being accepted.

However, as each of the experiments requires pretreatment of sperm samples, and all pretreated samples need to be tested as soon as possible, simultaneous measurement of all the three biomarkers for a sample with a single FCM may lead to a long waiting time for some of the experiments and obstruct the reliability of the results. This is because the sperms receive additional damage during the waiting process, if the protocol is not properly arranged taking all the three experiments entirely into consideration. This paper presents a protocol that realizes the fluent measurement of all the three biomarkers in a single cytometry without substantial compromise of experiment quality induced by prolonged waiting times.

Protocol

NOTE: Workflow for the detection of biomarkers for sperm function damage by flow cytometry includes (1) cell preparation, (2) staining with fluorescent reagents, and (3) flow cytometric analysis and data interpretation (Figure 1). The protocol follows the guidelines of the Ethics Committees of the Army Medical University (1.0/2013.4-12).

1. Cell preparation

Obtain human semen samples by masturbation in plastic clinical specimen jars, preferably after 3-5 days of abstinence.
Immediately incubate the semen samples in a 37 °C incubator and completely liquefy the samples (up to 1 h).

2. Sperm cells counted using computer-aided sperm analysis (CASA)

Mix the liquefied semen samples thoroughly.
Add 10 µL of semen into a sperm counting chamber (see the Table of Materials).
Scan the slides in the Sperm Class Analyzer (see the Table of Materials), and count at least six areas and 400 spermatozoa for the estimation of sperm concentration.
NOTE: Semen samples must be fresh, not frozen, for sperm apoptosis and MMP analysis.

3. Staining sperm cells

Detection of sperm apoptosis using Annexin V-FITC/PI staining
1. Add 1 × 10⁶ sperm cells to a 1.5 mL centrifuge tube.
2. Wash the cells with 500 µL of cold phosphate-buffered saline (PBS).
3. Centrifuge the sperm cell suspension at 300 × g for 7 min and discard the supernatant.
4. Resuspend the sperm cells in 200 µL of 1x Binding Buffer.
5. Add 2 µL of FITC Annexin V and 2 µL of PI (see the Table of Materials).
6. Gently vortex the cells, incubate for 15 min at room temperature (25 °C) in the dark, and place them in the flow cytometer immediately for analysis.
Analysis of mitochondrial membrane potential using JC-1 probe
1. Add 2 × 10⁶ sperm cells to a 1.5 mL centrifuge tube and centrifuge at 600 × g for 5 min.
2. Resuspend the sperm cell suspension in 1 mL of JC-1 working solution (see the Table of Materials).
3. Gently vortex the cells and incubate for 20 min at 37 °C in the dark.
4. Centrifuge the suspension at 600 × g for 5 min and discard the supernatant.
5. Wash the pellet twice with 1 mL of PBS at a speed of 600 × g for 5 min each.
6. Resuspend the dyed sperm cells in 1 mL of PBS in flow cytometer tubes, and place the tubes in the flow cytometer immediately for analysis.
7. Use carbonyl cyanide m-chlorophenyl hydrazone (CCCP) as a positive control. Resuspend the sperm cells (as previously described in step 3.2.1) in 1 mL of CCCP working solution (see the Table of Materials), and gently vortex the cells and incubate for 20 min at room temperature. Centrifuge the suspension at 600 × g for 5 min and discard the supernatant. Repeat steps 3.2.2-3.2.6.
Sperm chromatin structure assay
1. Place an aliquot of liquefied raw semen (0.25 mL) into a cryotube, and immediately freeze the semen samples in liquid nitrogen (-196 °C) until ready for the SCSA.
2. Thaw the semen samples in a 37 °C water bath and dilute them with TNE buffer (see the Table of Materials) to a concentration of 1-2 × 10⁶ cells/mL.
3. Add 200 µL of the diluted sample to a flow cytometer test tube and mix it with 400 µL of acid solution (see the Table of Materials) for 30 s.
4. Stain the samples with 1.2 mL of AO staining solution (see the Table of Materials) and place in the flow cytometer immediately for analysis.
5. Use a reference sample as an internal standard to flow cytometer set-up and calibrations. Dilute the reference sample with 4 °C TNE buffer to a concentration of 1-2 × 10⁶ cells/mL. Repeat steps 3.3.1-3.3.4.
  NOTES: All solutions and buffers are stored at 4 °C. (1) The semen should not be diluted before flash freezing. Raw semen samples should be thawed and diluted with TNE buffer at the time of flow cytometry measurements. (2) For infertility clinics, ~0.25 mL of raw semen should be flash-frozen in an ultracold freezer or a LN2 tank. These frozen aliquots can then be sent to an SCSA diagnostic laboratory on dry ice or in a LN2 dry shipper. (3) A human ejaculate sample that demonstrates heterogeneity of DNA integrity (e.g., 15% DNA fragmentation index [DFI]) was used as an internal standard reference.

4. Flow cytometer setup

NOTE: Before starting the flow cytometer, the instrument's system check should be performed to verify analytical performance. Run the samples after all checks have been completed and passed.

Open the flow cytometer software and start the cytometer.
Collect sample data.
1. First, load a control sample onto the flow cytometer, and click RUN to start data collection (adjust settings if needed). Wait for the instrument to begin aspirating the sample and provide a real-time preview of detected events.
  NOTE: The control sample: a negative control (unstained sperm cells) for sperm apoptosis; a positive control (CCCP-treated cells) for MMP; a reference sample for SCSA.
2. Create dot plots for viewing data and select the x/y-axis parameters (such as FSC, SSC) and linear to specify data are displayed. Select and apply a polygonal gate to delineate the sperm population for analysis, and so that the instrument plots detected events in real-time.
3. Analyze the sperm events within the region.
  1. For sperm apoptosis, set the FL1 as the x-axis and the FL2 as the x-axis. To isolate the live, apoptotic, or necrotic cells, tap and drag the quadrant gate to subset the sperm populations down to four specific populations.
  2. For MMP, set the FL1 as the x-axis and the FL2 as the y-axis. Create a polygonal gate to subset the sperm populations down to two specific populations.
  3. For SCSA, set the FL4 as the x-axis (red fluorescence, 125/1,024 flow cytometry channels) and the FL1 as the y-axis (green fluorescence, 475/1,024 flow cytometry channels). Draw the gates at a 45° angle to exclude cellular debris signals.
4. Set a run limit to indicate when to stop collecting data. Calculate a total of 10,000 sperm cells for each sample for sperm apoptosis, MMP, and SCSA analyses.
5. Set the fluidics rate (slow, medium, and fast, or a custom fluidics rate), depending on the cell numbers.
  NOTES: For SCSA analysis, to determine sperm concentrations, thaw the frozen raw sample and run it on the FCM. If the flow rate is >250/s, dilute the sample to the correct flow rate. Measure all samples independently twice, and calculate the mean values. A reference sample was tested when every 10-15 samples were measured to ensure standardization and stability of the instrument from day-to-day.
6. Set the threshold to eliminate debris and noise from cell samples. Apply these settings for all samples in the experiment.
7. If needed, set the color compensation to correct fluorescence spillover.
8. Gently back-pipette the samples before loading them onto the flow cytometer, name the sample, and run the samples.
9. Once all samples have been run, save the sample data as FCS files by giving them a file name for further software analysis. Save the work template of the current experiment for retrieval in future runs (optional).

5. Data analysis

Import the data into the data analysis software for the flow cytometer (see the Table of Materials).
Double-click the first sample in workspace. Wait until a Graph window opens, showing a plot of events along FSC versus SSC parameters. Create a gate that isolates the sperm population included within the gate, producing a 'child' population from the parent set of events.
Double-click within the gated area, and open a new Graph window containing only the sperm events. Set the x- and y-axis parameters to select fluorescent channel.
Select the Gate tool. Click within the plot to make the gates appear, so that the created gates isolate the different cell populations.
Right-click (or control-click) any of the highlighted rows, and select Copy analysis to group. Apply the gating tree to all samples within the group.
Save and export the data table.

Representative Results

Figure 2 shows the measurement of sperm apoptosis using Annexin V-FITC/PI staining. The FITC signal (green fluorescence) was measured in the FL1 channel, and the PI signal (red fluorescence) was measured in the FL2 channel. In the Annexin V/PI bivariate analysis, the quadrant makers identified four distinctive sperm populations. The results were expressed as the percentages of Annexin V^-/PI⁻ sperm (viable or live cells), Annexin V⁺/PI⁻ sperm (early apoptotic cells or apoptotic cells), Annexin V⁺/PI⁺ sperm (late apoptotic cells), and Annexin V⁻/PI⁺ sperm (necrotic cells)¹⁶^,¹⁷ (Figure 2B). A negative control (unstained sperm cells) was also used to set up compensation and quadrants (Figure 2A).

Figure 3 shows the measurement of MMP using JC-1 probe in human sperm. MMP % is presented as the orange-red/(green + orange-red) fluorescence ratio by analyzing the cytogram. A good-quality semen sample usually shows high orange-red fluorescence and low green fluorescence (active mitochondria, upper-left quadrant) (Figure 3A). Conversely, a poor-quality semen sample usually shows low orange-red fluorescence and high green fluorescence (inactive mitochondria, lower-right quadrant), possibly due to mitochondrial disruption (Figure 3B).

Figure 4 shows the data of SCSA¹³^,¹⁴. These typical SCSA cytograms were obtained from two individual samples. Sample A came from a fertile man and sample B from an infertile man. Analysis of the flow cytometric data was carried out using FlowJo software. Cytograms show the source of each component of SCSA data. The x-axis (red fluorescence with 1,024 gradations of red fluorescence) indicates fragmented DNA; the y-axis (green fluorescence with 1,024 gradations) indicates native DNA stainability. The dotted line at y = 750 represents the boundary of normal sperm. Above that line of y = 750 are sperm with partially uncondensed chromatin, which were determined as immature sperm.

Figure 1: Workflow for the detection of biomarkers for sperm function damage by flow cytometry. Semen samples were obtained and pretreated as described in protocol step 1. (1) Cell preparation, (2) staining with fluorescent reagents, and (3) flow cytometric analysis and data interpretation. Abbreviations: MMP = mitochondrial membrane potential; SCSA = sperm chromatin structure assay; PBS = phosphate-buffered saline; AO = acridine orange. Please click here to view a larger version of this figure.

Figure 2: Representative results of sperm apoptosis using Annexin V-FITC/PI staining. The lower left quadrant contains Annexin V^-/PI⁻ sperm (viable or live cells), the lower right quadrant shows Annexin V⁺/PI⁻ sperm (early apoptotic cells or apoptotic cells), the upper right quadrant represents Annexin V⁺/PI⁺ sperm (late apoptotic cells), and the upper left quadrant contains Annexin V⁻/PI⁺ sperm (necrotic cells). (A) Negative control (unstained sperm cells); (B) a sample with sperm apoptosis. Abbreviations: FITC = fluorescein isothiocyanate; PI = propidium iodide. Please click here to view a larger version of this figure.

Figure 3: Measurement of mitochondrial membrane potential using JC-1 probe. (A) The sperm subpopulation with high MMP. (B) The sperm subpopulation with low MMP. Set FL1-A and FL2-A as the x axis (green fluorescence) and the y-axis (orange-red fluorescence), respectively. Abbreviations: MMP = mitochondrial membrane potential. Please click here to view a larger version of this figure.

Figure 4: Detecting DNA damage in human sperm using sperm chromatin structure assay. (A) A semen sample with normal chromatin structure. (B) A semen sample with high proportion of spermatozoa with abnormal chromatin structure. FlowJo software was used to make computer gates around the cell populations identified as: 1) normal sperm, 2) HDS sperm, 3) DFI sperm, and 4) cell debris, and percentages of HDS and DFI sperm were calculated. Abbreviation: SCSA = sperm chromatin structure assay; HDS = high DNA stainability; DFI = DNA fragmentation index. Please click here to view a larger version of this figure.

Discussion

Chromatin integrity, MMP, and apoptosis of human spermatozoa have been found to be valuable predictors of reproductive outcomes. The latest-released WHO laboratory manual for the examination and processing of human semen (sixth edition) also highlighted some of these indicators (chromatin integrity and apoptosis) as extended examinations beyond the basic examination of routine parameters such as sperm count¹⁸. Recent publications also suggest that these indicators may be more sensitive markers of response to external hazardous exposures, indicating their potential for the identification of reproductive damage at an earlier stage¹⁹^,²⁰. Combining these indicators with routine examinations may also provide a more comprehensive understanding about the profile of sperm dysfunction and the complexity of male reproductive damage in the population.

There are several key issues that substantially determine the success of sperm analysis with FCM. First, the measurement of sperm MMP and apoptosis requires immediate examination after the semen is liquefied. To minimize the time cost, two technicians can separately take charge of the pretreatment of MMP and apoptosis analysis of a sample. As the pretreatment of apoptosis is simpler, it would transfer to the flow-cytometric step faster than the MMP analysis. For the SCSA analysis, fresh semen samples should be cryopreserved immediately upon availability after liquefaction. The sperm samples can then be stored in liquid nitrogen for a relatively long period and do not need immediate examination.

Accordingly, the in-site time cost of the experiment can be compressed to 40 min, which is the duration of MMP analysis plus the flow-cytometric step of sperm analysis. Second, the setup of the gating in the flow cytometric platform must be decided for each batch of samples separately. The sperm samples with clear cell subgroups in the scatter plot can be selected as the reference to draw the gating area. Third, as there are no widely accepted clinical reference values for the indicators of this experiment, historic results may be used as an important tool for quality control. Positive and negative controls are also encouraged to be set in each batch of measurements, especially for SCSA and MMP.

The representative results of sperm analysis provided here are derived using an Accuri C6. However, the technique could be applied to other commercial platforms with minor modification. Usually, a total of 5 × 10⁶ sperm cells would be needed to meet the requirement for the measurement of all the indicators. If the sperm concentration of a sample is much lower than the average level, the need for the volume of semen would increase.

Similar to routine sperm parameters, there is also noticeable intraindividual variation in the chromatin integrity, MMP, and apoptosis of human spermatozoa²¹. Accordingly, multiple tests of samples collected at different times may provide a more accurate estimation of the sperm function damage level. Although there is no recommended period of abstinence before sperm sampling for the flow cytometric analysis, 2-7 days of ejaculatory abstinence, suggested for routine sperm analysis by WHO, may be adopted. It may help to improve the comparability of the results among laboratories and among different samples collected from the same men.

A major limitation of this method is that two of the three tested biomarkers - the apoptosis and mitochondrial membrane potential - require fresh semen samples. This may limit their application to men who can provide semen samples to the laboratory. For the test of DNA damage (SCSA), reference samples should be prepared before the experiment to calibrate the flow cytometer. Of note, the application of the present protocol requires an FCM instrument in the lab, which is still a considerable challenge for many clinical laboratories, although cooperation with a third-party service may be an alternative choice in some regions.

Additionally, the expense of the dyes and other reagents is also a financial factor for males who may need the examination. Lastly, the protocol may need modification if different brands or versions of FCM are used to examine the biomarkers. In summary, this paper presents a practical approach to estimate three biomarkers of human spermatozoa damage by a single flow cytometry machine. This may provide valuable information about male reproductive health and complement the routine sperm examination.

Disclosures

The authors declare that there are no conflicts of interest.

Acknowledgments

We thank all the fieldworkers for their help and the interviewees for their cooperation.

Materials

Name	Company	Catalog Number	Comments
0.1 M citric acid buffer	Sigma-Aldrich Chemical Co., USA	251275-5G	Add 21.01 g/L citric acid monohydrate (FW = 210.14; 0.10 M) to 1.0 L H₂O. Store up to several months at 4 °C.
0.2 M Na₂PO₄ buffer	Sigma-Aldrich Chemical Co., USA	V900061-500G	Add 28.4 g sodium phosphate dibasic (FW = 141.96; 0.2 M) to 1.0 L H₂O. Store up to several months at 4 °C.
10 mM CCCP stock solution	Sigma-Aldrich Chemical Co., USA	C2759	Add 20.46 mg CCCP to 10.0 mL DMSO,making up to the final of CCCP in a concentration of 10mM, aliquot and store at −80 °C.
10 µM CCCP working solution			Add 10 mL of PBS to a 15 mL polypropylene centrifuge tube and add 10 μL CCCP stock solution, making up to the final of CCCP in a concentration of 10 µM.
1 mM JC-1 stock solution	Sigma-Aldrich Chemical Co., USA	T4069	JC-1 is purchased lyophilized. Add a small quantity of DMSO to the vial and vortex for several minutes until all the dye has dissolved. Transfer the solution to a light-tight tube and rinse the vial with appropriate volume of DMSO, making up to the final of JC-1 in a concentration of 1 mg/mL aliquot and store at −20 °C.
37 °C incubator	Thermo Scientific, USA
5 µM JC-1 working solution			Add 10 mL of PBS to a 15 mL polypropylene centrifuge tube and add 50 μL from a thawed JC-1 stock aliquot. Stir gently to assure a homogenous dilution. This solution must be stored in the dark and used promptly.
Accuri C6 Flow cytometer	BD Pharmingen, San Diego, CA, United States
Acid solution, pH 1.2			Combine 20.0 mL of 2.0 N HCl (0.08 N), 4.39 g of NaCl (0.15 M), and 0.5 mL of Triton X-100 (0.1%) in H₂O for a final volume of 500 mL. Adjust pH to 1.2 with 5 mol/L HCl.
Acridine Orange (AO) stock solution, 1.0 mg/mL	Polysciences, Inc, Warrington, Pa	65-61-2	Dissolve chromatographically purified AO in dd-H₂O at 1.0 mg/mL.
AO staining solution (working solution)			Add 600 μL of AO stock solution to each 100 mL of staining buffer.
Biological safety hood	Airtech, USA
Computer-aided sperm analysis system (CASA )	Microptic, Barcelona, Spain		Sperm Class Analyzer 5.3.00
Sperm Counting Chamber	Goldcyto, Spain
Equipments
FITC Annexin V Apoptosis Detection Kit I	BD Biosciences, San Jose, CA	556547	Included: (1) FITC Annexin V is bottled at 100 ng/µL; (2) Propidium Iodide (PI):The PI Staining Solution is composed of 50 µg PI/mL in PBS (pH 7.4) and is 0.2 µM sterile filtered; (3) 10x Binding Buffer: 0.1 M Hepes (pH 7.4), 1.4 M NaCl, 25 mM CaCl₂. For a 1x working solution, dilute 1 part of the 10x Annexin V Binding Buffer to 9 parts of distilled water. Store at 4 °C and protected from prolonged exposure to light. Do not freeze.
FlowJo 10	Tree Star, Inc., San Carlos, CA, USA
Horizontal centrifuge	Thermo Scientific, USA
liquid nitrogen tank	Thermolyne, USA
Materials
PBS	Beyotime, Shanghai, China	C0221A	Ready-to-use PBS buffers is purchased and stored at room temperature
Staining buffer, pH 6.0			Combine 370 mL of 0.10 M citric acid buffer, 630 mL of 0.20 M Na₂PO₄ buffer, 372 mg of EDTA (disodium, FW = 372.24; 1 mM), and 8.77 g of NaC1 (0.15 M). Mix overnight on a stir plate to insure that the EDTA is entirely in solution. pH to 6.0 with saturated NaOH solution.
The reference sample for SCSA analysis			The reference sample was diluted with cold (4 °C) TNE buffer to a working concentration of 1–2 × 10⁶ cells/mL, and used to set the green at 475/1,024 flow cytometry channels and set the red at 125/1,024 flow cytometry channels.
TNE buffer, 1x, pH 7.4 (working solution)			Combine 60 mL of 10x TNE and 540 mL of H₂O. Check pH (7.4).
TNE buffer, 10x, pH 7.4	Perfemiker, Shanghai, China	PM11733	Ready-to-use buffers is purchased and stored at 4 °C.

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