Human Sperm DNA Fragmentation Test 2

Androlog Mail

Regarding alternative methods to measure sperm DNA fragmentation. We
have used flow cytometry, COMET, TUNEL, morphometry with toluidine
based dyes and acridine orange, and Annexin V over last 5 years.
The advantages and technological problems of each method as well as
concerns about clinical and prognostic utility of assays based on
acridine orange and flow cytometry have been extensively reported by
others. We have reported in couple abstracts and publication in
Fertility and Sterility last year, our experience with TUNEL assay -
which is our test of choice.

TUNEL assays has an advantage of direct measurement of nicks in DNA
which can be identified by terminal deoxynucleotidyl transferase, an
enzyme that will catalyze the addition of dUTPs. We use commercially
available, high fidelity, transferase and typically the dUTPs are
linked to fluorochrome. We have tried flow cytometry and plate method
- and although fast, neither of them is useful in severely
oligospermic men or in complex cellular specimens from testicular
biopsy or men with immature germ cells. In those specimens the
replication of DNA with condensation and normally occurring nicks in
DNA, as a result of telomerase action, lead to high rate of false
positive results. Thus we now exclusively use cytopathological method
of assessing DNA damage on the microscope slides using TUNEL. The
advantages of this method is high reliability, we have previously
shown that inter and intraassay variability is < 5% in severely
oligospermic specimens, and less than 3 % in ejaculated sperm. Thus
we need just 200 sperm to make statistically valid results.
We agree with previously published cut off of 10 % as a normal result
in ejaculated sperm. So far no large study assessing sperm DNA
integrity in normal testicular specimens have been published.

The fluorescent method has couple advantages - it is easy to train
the imaging software and write and algorithm for automatic counting -
we use latest ImagePro with Deconvolution package (Mediacybernetics).
In complex specimens this is the only method where one can actually
differentiate between sperm, spermatocytes, and other cells. We use
cyanide based nuclear staining together with standard array of
nuclear stains. We found out that the specialized blue laser cube is
critical since it allows for very clear identification of highly
condensed DNA of sperm in complex specimens. However for ejaculated
DAPI or Texas Red cubes are sufficient. We use two-channel
acquisition - for DNA selective dyes first (like DAPI) and then we
detect dUTP using either FITC or Texas Red channel. The DAPI channel
is used to count the number of sperm, the FITC is used to count the
TUNEL positive sperm and results are expressed as fraction of
FITC/DAPI (or other stain). In addition, we comment on the shape of
the head and stainability, and presence of immature germ cells. There
is clear association between decreased stainability, increase size of
sperm head - fragmentation and intensity of FITC staining.
Quality control is achieved by preparing 4 slides = 2 are counted.
We always include positive and negative controls. Positive controls
are achieved by treating stock semen slides with DNAse I.
Overall it takes 4 hours from the time the specimen is obtained to
the time it is ready for analysis. Advantages of TUNEL are many: it
uses equipment ready available in every embryology lab- like high
quality microscope, it allows for on site analysis hence may be
especially useful in male factor infertility and IVF/ICSI cases since
it can be done in embryology lab. It is the only viable method for
analysis of testicular biopsy, it has binary characteristic of signal
(not like Comet or AO based assay), it has a low CV and can be
automated.
Disadvantages are rather high cost of enzyme - but having a high
quality of transferase is critical. It is cytopathology test hence
require manual processing and proficiency in microscopy. However
learning curve is short - all fellows who went through our lab were
able to do it.
We are about to submit our 5 years experience with TUNEL to RBMonline
this week. The manuscript has detailed description of methods,
microscope and software settings and performance characteristics.
I hope it will help to implement this relatively simple test in the
most of embryology labs.

-- 
Darius A. Paduch, MD., PhD
Assistant Professor of Urology and Reproductive Medicine
Staff Scientist, Population Council
Department of Urology
Weill Cornell Medical College
525 E 68th St., F -924A
New York, NY 10021
office: 212-746-5309
lab: 212-327-8740
fax: 212-746-7287