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Computer Assisted Semen Analysis

This System Utilize Modern Advance Image Processing Techniques

   Sperm Motility

   Morphology

   Vitality

   DNA Fragmentation

Based on the sperm test standards of the WHO critera
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Sperm Motility

Sperm concentration assessment (M/ml) and motility analysis are performed on native samples by recording AVI clips (acquired live from your imaging device to PC memory or prerecorded to disk). The analysis strictly follows the requirements of the “WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction”. The analysis is based on frame by frame detection of sperm heads on video clips and building precise tracks which reveal the nature of sperm movement and provide sperm concentration value. The total number of sperms is also calculated automatically. One frame with tracks is automatically saved to database record to serve as visual support in the report or you can save a suitable frame yourself.

The following parameters are calculated:

  • VCL = curvilinear velocity (micron/s). Time-average velocity of a sperm head along its actual curvilinear path, as perceived in two dimensions in the microscope.
  • VSL = straight line velocity (micron/s). Time-average velocity of a sperm head along the straight line between its first detected position and its last.
  • VAP = average path velocity (micron/s). Time-average velocity of a sperm head along its average path. This path is computed by smoothing the actual path.
  • LIN = linearity. The linearity of a curvilinear path.
  • STR straightness. The linearity of the average path.
  • BCF = beat cross frequency (beats/s). The average rate at which the sperm's curvilinear path crosses its average path.
  • ALH = amplitude of lateral head displacement. Magnitude of lateral displacement of a sperm head about its average path.
  • WOB = wobble. A measure of oscillation of the actual path about the average path, VAP/VCL.
  • MAD mean angular displacement of the sperm head.
  • Elongation of sperm head.

Pointing with your mouse cursor to a track, you can see the exact values of the parameters for the selected spermatozoon and classification result in a pop-up window. The clip can be played again, rewound, played frame by frame for detailed analysis. You can save clips to your hard disc uncompressed without loss of quality or by using virtually any codec installed on your PC (xVid codec is recommended). You can adjust detection threshold and also change the classification limits if required.

Based on the parameters mentioned above, the motility of each spermatozoon is graded A, B, C or D (WHO), according to whether it shows:

  • A = rapid progressive motility
  • B = slow or sluggish progressive motility
  • C = nonprogressive motility
  • D = immotility
  • A+B = progressive motility
  • A+B+C = total motility

Additionally, the software enables the user to assess the concentration of:

  • White blood cells
  • Immature germ cells
  • Round cells

Flexible tool for adjustment of sperm head detection will let you work with both bright field and phase contrast (negative phase contrast yields the best results) and not only human sperms but also many animal species.

Morphology

Morphology of the sperm head is an important criterion for the correct diagnosis. The software is set up to analyze still images of smears stained with the Diff-Quik stain according to strict Krueger’s criteria. We have selected Diff-Quik as worldwide recognized leader in rapid staining of sperm. With Diff-Quik, the head is stained pale blue in the acrosomal region and dark blue in the post-acrosomal region which is a good basis for precise image analysis. The following parameters are assessed for every spermatozoon:

  • Area of the head.
  • FFC = form factor circle. The degree of similarity of the sperm head to a circle.
  • Perimeter of the head
  • Brightness.
  • ELL_B = Big axis of ellipse outlining the sperm head, the length of the sperm head.
  • ELL_S = Small axis of ellipse outlining the sperm head, the width of the sperm head.
  • Elng = elongation of the sperm head.
  • FFE = form factor ellipse. The degree of similarity of the sperm head to an ellipse.
  • Acrosome = Percentage of the acrosomal region.

The software classifies spermatozoa into Norm and Head Pathology classes automatically based on head parameters. You can easily correct the results manually and also specify other anomalies (Tail Pathology, Neck Pathology). An extended morphology classifier is available which allows you to specify the following spermatozoa structure abnormalities indicating potential infertility: tapered, pyriform, round, amorphous, vacuolated, small acrosome, double head, pinhead, bent neck, asymmetrical neck, thick insertion, thin neck, short tail, bent tail, coiled tail, excess residual cytoplasm (ERC).

In case you can not receive a good image of stained smear suitable for automated detection (e.g. because of incorrect sample preparation), there is a special tool which allows you to outline the cells manually.

Software is able to detect both samples prepared according to WHO recommendations for CASA including centrifugation (recommended) but also can be adjusted to easier procedures which provide higher background staining. For complicated cases, there is an option of manual drawing and deleting of objects.

It is now customary to record the number of morphological sperm defects divided by the number of defective spermatozoa, a measure called the teratozoospermia index (TZI). After the analysis is finished, the TZI is calculated automatically. The teratozoospermic index values should read between 1.00 (each abnormal spermatozoon has only one defect) to 3.00 (each abnormal spermatozoon has head, neck and tail defects).

Vitality

Sperm vitality is an important test, especially for samples with less than about 40% progressively motile sperms. The percentage of live spermatozoa is assessed by identifying those with an intact cell membrane, from dye exclusion or by hypotonic swelling. The dye exclusion method is based on the principle that damaged plasma membranes, such as those found in non-vital (dead) cells, allow entry of membrane-impermeant stains. The hypo-osmotic swelling test presumes that only cells with intact membranes (live cells) will swell in hypotonic solutions. Sperm vitality should be assessed as soon as possible after liquefaction of the semen sample, preferably at 30 minutes, but in any case, within 1 hour of ejaculation, to prevent observation of deleterious effects of dehydration or of changes in temperature on vitality. It is clinically important to know whether immotile spermatozoa are alive or dead. Vitality results should be assessed in conjunction with motility results from the same semen sample. The presence of a large proportion of vital but immotile cells may be indicative of structural defects in the flagellum. A high percentage of immotile and non-viable cells (necrozoospermia) may indicate epididymal pathology.

DNA Fragmentation

Sperm DNA fragmentation test provides additional information on semen fertility potential that can not be achieved by means of standard motility, morphology and vitality tests. We have selected the Sperm Chromatin Dispersion (SCD) method as simple, fast, accurate, and highly reproducible method for the analysis of sperm DNA fragmentation in semen and processed sperm. The SCD test utilizes the idea that after acid denaturation and removal of nuclear proteins sperm with non-fragmented DNA produce the well seen halo of dispersed DNA loops while spermatozoa with fragmented DNA fail to produce such halo. When somatic cells or spermatozoa with nonfragmented DNA are immersed in an agarose matrix and directly exposed to lysing solutions, the resulting deproteinized nuclei show extended halos of DNA dispersion. The halos correspond to relaxed DNA loops attached to the residual nuclear structure. These deproteinized nuclei are called "nucleoids". The presence of DNA breaks promotes the expansion of the halo of the nucleoid and is the basis for the halo test to detect DNA damage when sperm are treated with an acid solution prior to lysis buffer, the DNA dispersion halos that are observed in sperm nuclei with nonfragmented DNA after the removal of nuclear proteins are either minimally present or not produced at all in sperm nuclei with fragmented DNA.


Big Halo

Medium Halo

Small Halo

Without Halo

Degraded

slide immersion tray

For perfect cell lysing in the coated semen sample slide.

For immerse Lyse 1, Lyse 2 and DI water

Specially made for DNA fragmentation assay procedure.

semen sample incubator & dna warmer

For Incubate the Semen samples in the temperature of 37oC and helps to liquify the Semen sample

We can able to adjust the temperature from 1oC to 70oC.

slide warmer

To dry the stained slides while preparing the Morphology, Vitality abd DNA fragmentation specimens.

We can able to adjust the temperature from 1oC to 70oC.


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