Male Infertility Testing - SCSA®

Flow Cytometry

FLOW CYTOMETER - the highly technological instrument needed for the SCSA® test.

The name flow cytometer comes from "CYTOMETRY", the study of cells, and "FLOW"; hence, cells are flowing in a liquid medium past a measuring point rather than being fixed on a light microscope slide.

In the following, numbers in (#) refer to specific publications by Dr. Evenson and are found on his personal page.

The essence of a flow cytometer is that cells are stained with a fluorescent dye and then forced through a glass channel in a liquid suspension. When the cells pass through the focal point of the laser beam, the laser light will cause the fluorescent dye to emit fluorescent light of a certain color. For the SCSA® test, green fluorescing sperm have very low levels (non-detectable) of fragmented DNA while red fluorescing sperm have moderate to high levels of fragmented DNA. Moderate to high DNA fragmentation levels found within these red fluorescing sperm are likely beyond the repair capacity of the egg. Five thousand individual sperm are measured to determine the percent of sperm with green and red color, the percent of sperm nuclear immaturity and the extent of DNA fragmentation calculated with SCSA® soft software.

1. WHAT DOES THE SCSA® TEST MEASURE?

The five major populations of sperm identified by the SCSA® test:

Non-detectable - DNA Fragmentation Index (DFI): percentage of mature sperm with non-detectable levels of nuclear DNA fragmentation.
Moderate DFI: percentage of sperm with moderate levels of DNA fragmentation identified by their moderate susceptibility to acid-induced denaturation.
High DFI: percentage of sperm with high levels of DNA fragmentation identified by their high susceptibility to DNA denaturation.
DNA Fragmentation Index (DFI): the percentage of sperm with moderate and high levels of DNA fragmentation (% Moderate DFI + % High DFI).
High DNA Stainability (HDS): the percentage of sperm with immature chromatin. HDS sperm have less chromatin condensation, which leads to increased DNA stainability. These sperm are compromised for lower efficiency of routine IVF fertilization, however, the DNA integrity appears normal and can be used for ICSI.

The DEGREE of normality / abnormality of the entire sperm population:

In addition to providing the percent of each of the four sperm populations described above, the SCSA® test calculates the mean and standard deviation of the DFI based on all sperm analyzed. In some cases, the mean and standard deviation of DFI provides insights into the fertility diagnosis that are not available from the percent DFI alone. This important parameter shown on the center scattergram below and associated number data output are available by our SCSAsoft® software.
Furthermore, SCSA® cytogram patterns may indicate that semen is contaminated with bacteria, leukocytes or cellular debris.

The SCSA® test does not measure:

Mutations and translocations, unless they affect DNA / chromatin integrity
Aneuploidy, unless it represents a significant amount of total DNA content

2. WHAT IS THE RATIONALE FOR USING THE SCSA® TEST IN THE ART LABORATORY?

Incorporating the SCSA® test into conventional semen analysis will likely enhance the ability of clinicians and ART directors to predict male fertility potential.

3. HOW DOES THE SCSA® TEST COMPARE TO OTHER SEMEN QUALITY TESTS?

The SCSA® test has a huge advantage over existing light microscope based clinical assays of sperm quality because:

5000 (or more) individual sperm are evaluated in seconds,
neat (not washed) semen is measured allowing the evaluation of all cell types present in the ejaculate,
data are derived from objective, instrument-defined criteria rather than from potentially biased, human observations,
data have a higher level of repeatability than those of any other currently used semen parameter,
easily prepared, frozen samples can be sent by air courier to SCSA® Diagnostics, Inc. for evaluation,
data may be used to identify men with a significantly increased risk of infertility
The SCSAsoft® custom software provide the unique cytogram of total DNA stainability vs DFI Fragmentation Index. The orientation of the dot placement pattern is uniquely advantageous to help decipher the pathology of the sample. The table columns of mean DFI and SD DFI are unique to SCSAsoft®and provide quantitative and qualitative values that are free from human intervention for derived SCSA numerical output. This parameter is only available from SCSA Diagnostics, Inc. and their international partners.

NO other semen quality test matches all of these criteria.

SOME CONCLUSIONS ABOUT THE SCSA® TEST

The SCSA® test is an independent assessment of sperm quality as shown by the weak correlation between SCSA® data and other semen parameters.

Animal and human studies show a strong correlation between SCSA® data and fertility status.

CHARACTERISTICS OF SPERM CHROMATIN REVEALED BY THE SCSA® TEST

The SCSA® test is the most stable measure over time as compared to classical semen parameters (15).

Stability of SCSA® test data over time

Consecutive monthly semen samples from 50 individuals providing one semen sample per month showed a very high level of repeatability of SCSA® test data over time. The cytograms below show the repeatability of the SCSA® test data for 5 men with samples given once a month for 8 months. Notice that individual men have unique patterns that are extremely consistent from month to month.

Can SCSA® test data change with time? YES! (15)

Note that for donor 50, all monthly samples were similar except month 4 due to prescription drug use for an injured back.

The Nature of DNA Fragmented Sperm

Sperm with high levels of DNA fragmention contain DNA strand breaks that make the DNA more susceptible to acid-induced DNA denaturation (days 18, 25.)

The Nature of High DNA Stainable (HDS) Sperm

Sperm with a high level of DNA stainability (HDS) are immature and contain abnormally condensed chromatin due to the presence of unprocessed P2 protamines (day 33.)

Note the SCSA® test can assess both DNA fragmentation (DFI) and chromatin maturity (HDS) in a single measurement. NO OTHER TEST CAN DO THIS.

4. WHAT IS THE ROLE OF THE SCSA® TEST IN THE HUMAN INFERTILITY CLINIC?

The quality of sperm nuclear DNA affects the expression of the male genome and the overall fertility potential of the man. Therefore, integrating the SCSA® test (a rapid, objective and highly sensitive DNA quality assessment) into conventional semen analyses enhances the diagnosis and treatment of male infertility.

Criteria for whether or not a test is clinically useful

Clinically useful tests have a threshold that provides discriminatory and predictive capabilities, with little overlap between unaffected and affected groups, e.g., fertile and infertile men. Conventional semen parameters often do not meet these standards. For example, a 'normal' sample has a concentration of >20 million sperm/ml; however, this was described from a study where the median sperm concentrations in the fertile and infertile groups were 90 and 74 million/ml, respectively. Obviously, sperm concentration, like many other conventional parameters, has little power to identify potentially infertile men.

Predictive status of the SCSA® test from a major in vivo fertility study (29)

When infertile couples seek treatment, the male partner is evaluated to gather information about his fertility potential through history, physical exam and diagnostic testing. As described above, the most valuable diagnostic tests identify thresholds that discriminate between fertile and infertile men. The SCSA® test provides the 30% DNA Fragmentation Index (DFI) threshold, which predicts an increased risk for a negative pregnancy outcome in patients trying to conceive naturally or through ART.

Following the Science article showing a very significant difference in DNA fragmenation between fertile and infertile men, the DFI threshold for infertility was first established in the comprehensive "Georgetown Male Factor Infertility Study" (29) including 200 couples attempting to conceive naturally. Data from this study were used to establish the statistical thresholds of >30% DFI for 'significant lack of', 15-30% DFI for 'reasonable' and <15% DFI for 'high' fertility status. As seen in the following figure, the SCSA® test data demonstrate a relationship between DFI values and infertility.

Example data from the "Georgetown Fertility Study"

Note the significant variation between men. The letters designate the pregnancy outcome.

Men that initiated a pregnancy during the first 3 months of the study had a significantly lower DNA Fragmentation Index (%DFI) than the 40 men initiating a pregnancy during the 4-12 months (P<0.01) and the 31 men not initiating a pregnancy during the 12 month study (P<0.001).
Of the men that initiated a pregnancy in the first 3 months of the study, 84% (n=73) had a DFI of <15%.
Based on logistic regression, DFI was the best predictor of infertility.
Using selected cut-off values for DNA Fragmentation (DFI) and High DNA Stainability (HDS), SCSA® test data predicted seven of 18 miscarriages (39%). This is close to 50%, the percentage of miscarrages thought to be attributable to male factors.

What is the etiology or cause of abnormal SCSA® test results?

Fever, illness, drugs, chemicals, pollution, radiation, heat, leukocytospermia and trauma to testis

Although the SCSA® test is generally stable within individual men over time, results may fluctuate due to various stressors including infections involving leukocytospermia (49).
The direct correlation of leukocyte concentration to DNA fragmentation in immature and mature sperm indicates that the presence of contaminating leukocytes during periods of infection may alter the regulation of spermatogenesis and compromise male fertility.
High fever (39.9°C or 103.8°F) was associated with both DNA fragmentation (DFI) as well as abnormal DNA packaging (High DNA Stainability, HDS) in a case study of a man with influenza (34).
Testicular cancer (6,8,26) leukemia and Hodgkins disease and other undefined conditions have a moderate to severe influence on SCSA® test results.

Effects of environmental pollution (8,42,59)

The sensitivity of the SCSA® test has been utilized to measure the effects of high levels of environmental pollution on male fertility potential. Eighteen year old Czech Republic army inductees living in a region with high levels of air pollution produced sperm with high levels of DNA fragmentation (%DFI). This level of fragmentation may explain the relationship between air pollution and the increased incidence of miscarriage and congenital abnormalities in children conceived by men from this region. Twenty-five percent of these eighteen year old men had a DFI>30%. Of great interest, only the SCSA® test was sensitive enough to detect time-dose relationships to abnormal DFI; six semen samples/man were collected over two years during seasons of high and low pollution levels. The classical semen parameter did not show significant dose related change except for sperm motility that moved in the opposite direction i.e. high pollution increased sperm motility. Of great interest, preliminary studies on the genotype of these Czech men showed that men positive for glutathion s-transferase (GST) had less DNA damage than men at the same exposure level who were GST negative. GST is an antioxidant related enzyme that likely reduces the ROS damage to sperm DNA.

Therefore, the SCSA® test data may be used as biomarkers of genotoxicant exposure, and men with high DFI may have both a reduced fertility potential and an increased risk of conceiving children predisposed to malformations, cancers and genetic diseases.

5. WHAT ARE THE ADVANTAGES AND DISADVANTAGES OF THE SCSA® TEST FOR ART CLINICS?

Advantages of the SCSA® test:

Sample preparation and transport is simple and convenient.
Samples may be collected at home or at an infertility clinic.
Samples should be frozen after liquifaction.
Cryoprotectant is NOT needed for storing samples in liquid nitrogen.
Frozen samples may be shipped to SCSA® Diagnostics, Inc. by overnight courier. If a sample is sent by Federal Express by late afternoon and has a high priority handling ($50 surcharge) we can provide clinical data by noon the next day.

Disadvantages of the SCSA® test:

The instrumentation necessary to perform the SCSA® test is expensive and highly complex. Of greater importance, the SCSA® test uses techniques and calibration standards that are entirely different from other flow cytometry protocols. A high level of expertise and experience in SCSA® testing is required for proper clinical diagnosis and prognosis. Some clinics without the necessary experience and expertise have given false clinical results to infertile couples. For example, known errors have lead inexperienced laboratories to declare men infertile while SCSA® Diagnostics, Inc. results indicated the fertility potential was excellent.

CONCLUSION

The SCSA® test provided by SCSA® Diagnostics, Inc. is a stable, sensitive measure of DNA fragmentation in ejaculated sperm. SCSA® Diagnostics, Inc. pioneered, developed and patented the SCSA® test and is the only company that can be explicitly trusted to provide accurate and timely prognostic and diagnostic information to infertile couples with regard to DNA integrity based male fertility potential (15).