Assisted Reproduction and Infertility Figure 1: Clinical Sperm Chromatin Structure Assay Data 100
High DNA stainability
75 50 25 0 0 Patient 7272–113
Moderate population
75 50 } 25 50 75 Fragmented DNA
Measurement 1
2
Mean SD
DFI
563.7 561.4 562.6 1.2
DFI = DNA fragmentation index; HDS = high DNA stainability; SD = standard deviation.
beam, this crystal produces a metachromatic shift from green to red fluorescence. Thus, a flow cytometric measurement of the amount of green and red fluorescence in each sample of 5,000 sperm determines the DFI.
Sperm Chromatin Structure Assay Clinical Data The left scattergram in Figure 1 shows 5,000 dots, with each dot representing a single sperm. The amount of double-stranded DNA per cell is shown on the vertical axis. The extent of DNA damage corresponds to the positions of the dots on the horizontal axis. Each axis has a range from 0 to 1,024 units of green or red fluorescence. The SCSA is so highly quantitative that, when a sample is run for a second time, the dots repeat at the same position. No other DNA fragmentation test provides such a precise, two-parameter determination.9,10
Monthly
semen samples taken from 45 men over eight months showed dramatic cytogram repeatability at each month.11
When DNA has a strand break, it will lose an approximate unit of green fluorescence and gain a unit of red fluorescence. Thus, the classic pattern of various levels of DNA damage per cell will show a 45-degree angle (see Figure 1 left panel).
SCSAsoft® Software
SCSA Diagnostics’ proprietary SCSAsoft® software very importantly transforms the dot pattern seen on the left panel to one that is orientated in a vertical and horizontal pattern (center panel). Rather than trying to draw computer gates around the cells (dots) with increased red fluorescence in the left cytogram, as attempted by some laboratories, SCSAsoft, importantly, produces a frequency histogram from the data in the middle panel to make a highly accurate determination of the proportion of sperm with damaged DNA from the frequency histogram (see Figure 1 right panel).9,10 human intervention.
This is done without 90 Sperm Nuclear Proteins
Abnormalities in sperm nuclear proteins are also a negative factor for pregnancy outcomes. An improper lack of exchange of somatic to protamine proteins results in an increased amount of green fluorescing DNA (see Figure 1, left panel, top horizontal line).9,10,12
High population 100 0 0 25 50 75 Fragmented DNA
SD DFI 307
304.8 305.9 1.1
% DFI 64.9
64.9 64.9 0
100 0 0 0.5 DNA fragmentation index
% HDS 6.4
7.2 6.8 0.4
1 25 75 Non-detectable 50 25 Moderate High 100
100 125
This population is
the high DNA stainability fraction. Some studies have shown that, when more than 35 % of the sperm has this defect, no pregnancies occur. These immature sperm also produce reactive oxygen species (ROS) activity, adding to the DNA damage of the sperm population.13
Replicate
measurements show an extremely high repeatability of the scattergrams between the first measurement and the replicate measurement, with standard deviation from 0 to 0.1. If the small protamine molecules that are intended to protect DNA have abnormal ratios (P1 and P2 protamines), this could lead to sites being susceptible to DNA breaks from ROS. ROS is thought to be the primary mechanism for sperm DNA damage.13
Does High DNA Fragmentation Index Lead to Birth Defects?
One of the obvious and major questions for couples is, if the sperm DNA is damaged, will this lead to birth defects in our child? The evidence strongly suggests that, when sperm has a high DFI, this results primarily in lack of pregnancy or miscarriage. There is no evidence to suggest that a high DFI has resulted in a significantly increased level of birth defects compared with what occurs naturally.
Other Sperm DNA Fragmentation Tests Since the introduction of the SCSA in 1980, other sperm DNA fragmentation tests—Tunel, Comet, and Halo sperm kits—have been introduced. Compared with these other tests, the SCSA is the most widely used test for sperm DNA damage. As recently stated,14
“the
SCSA® remains the most robust test, and the one for which most clinical data are available and, indeed, many of the current indications for
US OBSTETRICS & GYNECOLOGY
Native DNA stainability
Total DNA stainability
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