Sperm quality measures for fertility assessment

This material was originally published in the

Purdue Cytometry CD-ROM Series,volume 2


Donald Evenson
Olson Biochemistry Laboratories
South Dakota State University
Department of Chemistry and Biochemistry
Brookings, SD 57007 USA


SPERM CHROMATIN STRUCTURE ASSAY (SCSA;10): A Measure of Nuclear DNA/Protein Structure Related to Fertility Potential

Abnormal chromatin structure is defined by the SCSA as an increased susceptibility to acid (pH 1.2, 30 sec) induced denaturation. When acridine orange (AO) stained sperm are exposed to 488 nm laser light, AO intercalated into ds DNA fluoresces green and AO bound to ss DNA fluoresces red. The SCSA measures the shift from green (native, ds DNA) to red (denatured, ss DNA) fluorescence in each of 5000 cells per sample and the extent of this denaturation is quantified by alpha t [alpha t, = red/(red+green) fluorescence]. Normal chromatin remains structurally sound at low pH, producing minimal red fluorescence and giving a narrow at alpha distribution. Usually, DNA with abnormal chromatin structure partially denatures under the acid conditions of the SCSA, yielding increased red fluorescence and a broader alpha t distribution; i.e. higher mean channel (X alpha t) and increased % of Cells Outside the Main Population (COMP alpha t), The standard deviation of (alpha t (SD alpha t) describes the extent of chromatin structure abnormality (1,2,5,7,9-12). Correlations between at variables and fertility potential have been as high as 0.94 (p -.0l).


Figure 1: Green versus red fluorescence cytograms and corresponding alpha t frequency histograms of Acridine Orange (AO) stained bull semen samples prepared and measured by the Sperm Chromatin Structure Assay (SCSA). In cytograms (A & B), green fluorescence (Y-Axis) corresponds to native, ds DNA and red fluorescence (X-Axis) to denatured, ss DNA. A & C represent semen analyzed from a bull of known high fertility, and B & D from a bull of very low fertility. Note the higher proportion of sperm with increased levels of red fluorescence in cytogram B compared to A and the resulting shift in the alpha t distributions (compare C to D).


Figure 2: The SCSA can be done concomitantly with a sperm count determination by admixing a known number of fluorescent beads with the sperm sample (13). The methodology is simple, highly accurate and a logical extension of this assay. The gate in the bottom left hand corner excludes debris and only sperm and beads are included in the analysis. Since the bead concentration is known, a simple calculation determines the sperm concentration, an important parameter of fertility potential.


The R123/Pl assay provides a quantitative measurement of mitochondrial membrane potential (cell motility) and cell membrane viability. After staining with R123, fluorescence intensity in the sperm midpiece is related to the mitochondrial membrane potential and motility. Red fluorescence from the PI stained nuclear DNA results from compromised cell membranes and is indicative of dead or dying cells. Bright green fluorescence was correlated with vigorous sperm motility and dull green fluorescence correlated with slow motility. Bright red and bright green fluorescence are mutually exclusive.


Figure 3: Two parameter isometric displays of green and red fluorescence from 5000 bull sperm stained with Rhodamine 123 and Propidium Iodide (RI23/Pl). Sperm samples from known fertile (A) and less fertile (B) donors are shown.

II. SYBR14/Pl (15,16)

Co-staining sperm with SYBRI4 and PI readily identifies cells with intact membranes versus membrane compromised cells. SYBRI4, a membrane permeable DNA stain (green fluorescence) requires an intact cell membrane for optimal fluorescence. PI, a membrane impermeable stain (red fluorescence) stains dead or dying cells with breaks in the cell membranes.


Figure 4: Two parameter green (log) versus red (linear) fluorescence isometric displays of 10,000 cells collected from fertile (A) and less fertile (B) bull sperm stained with SYBRI4 and counterstained with Propidium Iodide (PI). Population I contains all red (PI, dead) sperm and population 3 contains bright green (SYBRI 4, viable) sperm; population 2 are sperm in transition from live to dead.

TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE ASSAY (TdTA;18,19,21,22): Detection of DNA Strand Bmaks in Sperm Nuclei

DNA strand breaks in fertilizing sperm nuclei have potentially serious consequences for developing embryos. DNA strand breaks can be detected by incubating fixed sperm in the presence of avidintagged DUTP and TDTA, which adds the base to the ends of the DNA strand breaks. These tagged additions can be quantitated by incubation in the presence of FITC tagged biotin followed by flow cytometric measurement of the sperm.


Figure 5: Flow cytometric data from TDTA incubated sperm from the same bull collected during a fertile period (A,D) and collected 3 to 3.5 years later (B,C,E,F), during periods of decreasing fertility. The panels on the left (A,C) show green fluorescence frequency histograms from the TdT control samples. The panels on the right (D-F) show green fluorescence frequency histograms depicting the subtraction of the TdT control green (upper curve) from the TdT-positive green (lower curve). Shaded areas in D-F depict sperm with increased presence of endogenous DNA strand breaks. A correlation has been observed (19,21) between percent of sperm with susceptibility to DNA denaturation and percent sperm demonstrating DNA strand breaks.

SPERM IMAGING (17,23,24)

Sperm morphology and morphometry have been shown to relate to fertility potential.(23). Most fertility clinics utilize light microscopy of stained semen smears on slides for assessment of sperm morphology. Recently, some sperm motion analyzers have been fitted with very basic morphology measures, e.g., length, width, circumference. Our laboratory uses a cooled CCD camera and ONCOR imaging software to measure a variety of morphology and morphometry parameters. Sixteen parameters of Feulgen stained sperm were utilized in a recent study. A regression model for fertility rankings incorporating the standard deviation of the imaging variables area, bending energy, nmac, eccentricity, condensity, light blobs and dark blobs was highly significant (r 2=0.999, P- 0.05). These results indicate that variation of morphometry measurements is likely a sensitive biomarker related to fertility potential and abnormal chromatin structure (23).


Figure 6: Display of sperm nuclei, illustrating various states of normal/abnormal head morphology. Nuclei A & B are slightly different from each other, both in length and width, yet both would be visually classified as being morphologically normal. Nucleus C is shorter and wider than normal. D & E are tapered near their base which will alter the curvature measurements and length and width of the nuclei. F is wider than normal and illustrates staining alterations seen in several samples. Dark staining covers the base of the nucleus in most examples but in F, is skewed to one side. Nucleus G is smaller than normal in all respects, and H is highly rounded, indicative of immature sperm. Nucleus I is representative of a special case of sperm head abnormality; the ring of lightly stained areas midway across the nucleus represent "crater" or "diadem" defects.



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