Computer Assisted Semen Analysis – Dr. IRA

CASA (computer assisted semen analysis)

 

CASA or computer assisted semen analysis is a sophisticated electronic imaging system to visualize the sperm. An advanced software program is used to evaluate dozens of individual sperm parameters. The overall clinical value of semen analysis varies from center to center, and despite efforts in more recent years to introduce standardization and quality control, manual semen analysis remains a time-consuming and poorly reproducible exercise

A potential solution, therefore, has to be to provide either full or partial automation to allow the evaluation of a statistically valid number of sperm and remove much of the subjectivity of a manual analysis. Automated methods have been around for a number of years but are still not widely adopted. The often prohibitively high cost coupled with often weak validation in the human clinical setting means that despite the advantages in terms of rapidity and even reproducibility, clinics are reluctant to abandon their slightly flawed manual analyses.

Inconsistencies in computer-assisted sperm analysis (CASA) analysis appear to be due to a number of factors which appear to include the methods used to determine pixel coordinates, how they manage collisions and near misses, dealing with other bright particles and other nonsperm cells, and, to a degree, the technical expertise of the CASA user.

The semen specimen is placed on the stage of the microscope. This microscope has a high resolution video camera attached. The video camera feeds data into the computer where it undergoes analysis by software. A computerized semen analysis will give many more parameters that are useful to the fertility specialist. More importantly, the results are accurate and reproducible

The CASA hardware included a basic PC running Windows XP, Fire-I 400 Firewire camera (Unibrain, Athens, Greece), Firewire capture card, Olympus (BX-50) microscope, and heated stage set to 37°C. Each CASA reading was performed on the 20-μm Leja slide (Leja; Gynotec Malden, NieuwVennep, The Netherlands). The Leja slide was filled with approximately 7 μL semen (or washed sperm), and the chamber-filling time was recorded in seconds to give an estimate of viscosity. Average compensation factors were calculated to account for the Segre-Silberberg (SS) effect. The SS effect is a phenomenon in which fluid flow in thin capillary-loaded slides results in the sperm suspension being forced transversely toward the walls, causing uneven cell dispersion. The correction factor which makes an allowance for changes in sample viscosity and appears to improve the performance of such slides regarding agreement with the hemocytometer both manually and using CASA was was built in to the processing (5, 17). Broad SS correction factors were calculated for high (1.0), medium (1.1), and low (1.15) viscosity as well as for washed prepared sperm (1.2).

Calibration for the assessment of sperm concentration relied heavily on the accuracy of the sperm detection system and the accurate calculation of the volume of fluid being analyzed on each field. Using a fixed-depth chamber (20-μm Leja slide) and by calculation of the microscopic field area using a stage micrometer, the volume of each field analyzed was calculated, and this enabled expression of sperm concentration in million/mL. Dilution of the specimen was not performed for any sample within this cohort. The reason for this was to test its performance across the entire range of concentrations and to determine whether dilution would indeed be required for samples containing a higher number of sperm.

 

The CASA system employed in this study was the CRISMAS system, version 1.0 (Image House A/S, Copenhagen, Denmark). It calculated CASA parameters of sperm motility according to the guidelines from WHO (1992). In addition to these specific motility parameters, the computer also calculated sperm concentration, total sperm count, the percentage of motile spermatozoa in the sample, and a classification of the spermatozoa into immotile (curvilinear velocity, VCL <5 μm/s), locally motile (VCL = 5–25 μm/s) and motile (VCL >25 μm/s) was made. CRISMAS had an edit function that allowed the user to correct wrong sperm tracks calculated by the computer. It was thus possible to correct misinterpretations such as other particles being recognized as immotile spermatozoa or wrong tracking resulting from paths crossing each other. This property minimized the error rate and made the results more valid. The main components of the CRISMAS system were a Nikon microscope with positive phase contrast optics, a charge-coupled device (CCD) video camera and a personal computer with a digital frame grabber and CRISMAS image processing software. The measuring time for CRISMAS was 4×2.6 s and the frame sampling frequency was 25 Hz

 

SPERM DNA FRAGMENTATION

The integrity of genetic material in the sperm is crucial for successful fertilisation and normal embryo development. Sperm DNA fragmentation is a term used to denote abnormal genetic material within the sperm, which in turn may lead to male subfertility, IVF failure and miscarriage. A conventional semen analysis done for sperm concentration, motility analysis and morphology assessment cannot assess the sperm at the molecular level and as result aid the detection of DNA fragmentation.

A number of sperm DNA fragmentation tests are available on the market, but the one used at the Reproductive Health Group, is the SpermComet test which seems to more detailed information compared to the other tests such as SCSA and TUNEL.

Studies in the literature have shown that:

  • High sperm DNA fragmentation affects blastocyst development
  • Higher the DNA fragmentation levels, higher the chances of failed assisted conception treatment and miscarriage
  • Sperm DNA fragmentation is higher in subfertile men with abnormal sperm parameters
  • Men with normal sperm parameters are also found to have high sperm DNA fragmentation

Advantages of the sperm DNA fragmentation test

  • Provides a reliable analysis of sperm DNA integrity that may help to identify men who are at risk of subfertility
  • Provides information that helps in the clinical diagnosis, management and treatment of male fertility
  • Provides prognostic value in assessing the outcome of assisted conception treatment

High rates of sperm DNA fragmentation and pregnancy potential

  • Normal, healthy pregnancies do occur in couples where the male partner has high percentage of sperm with fragmented DNA, although the chances are significantly reduced, as the percentage of sperm bearing low levels of DNA fragmentation is much lower
  • Embryos derived from sperm with highly fragmented DNA have poor prognosis
  • DNA fragmentation could result in initiation of apoptosis (natural cellular death) and mutations resulting in blastocyst arrest, miscarriage and abnormalities in the offspring
  • Sperms with high DNA fragmentation fertilising younger oocytes than older oocytes carry a better prognosis of successful pregnancy, as they are much more efficient at DNA repair of defective sperm

 

Causes of Sperm DNA Fragmentation

In men, the major contributing factor for sperm DNA fragmentation is oxidative stress, which can be associated with one or more of the following:

  • Infection
  • Pyrexia
  • Elevated testicular temperature
  • Recreational drugs
  • Smoking
  • Alcohol
  • Stress
  • Diet
  • Environmental and occupational pollutants
  • Advanced chronological age
  • Varicocoele

Indications for men who may benefit from the Sperm fragmentation test

  • Unexplained infertility
  • Arrested embryo development
  • Poor blastocyst development
  • Multiple failed IVF/ICSI treatments
  • Recurrent miscarriage
  • Advanced chronological age
  • Varicocoele
  • Poor semen parameters
  • Exposure to harmful substances

Treatment of high sperm DNA fragmentation

It depends essentially on the cause. If the damage is caused by free radicals, a change in lifestyle and diet designed to protect against oxidative stress may help reduce the levels of DNA fragmentation. Other treatment options include:

  • Antibiotics in the co-existence of an infection
  • Life style changes – drugs, smoking and occupation
  • Diet – fresh foods, particularly those containing antioxidants and vitamin C & E
  • Varicocoele surgery
  • Testicular aspiration of sperm (DNA damage occurs at the post-testicular level, hence testicular sperm may have a better DNA integrity than ejaculated sperm)
  • ICSI rather than IVF

Initiatives to reduce the levels of fragmentation can be assessed by undertaking a second test three months later.

What is the Sperm fragmentation test (SpermComet)?

It is a second generation sperm DNA test. It detects the sperm with DNA damage and also tells how much DNA damage each sperm have. A SpermComet test result of 45% means that there is an average of 45% DNA damage in each sperm that was assessed. Sperm are stained with a fluorescent probe that interacts with the DNA molecule. The fluorescence signal changes when the DNA is fragmented, and this is monitored using a flow cytometer.

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