B

Fig. 2. Examples of in situ PRINS labeling of human sperm nuclei. (A) Direct three-color PRINS labeling on chromosome 1 (yellow), chromosome 9 (red), and chromosome 7 (green) using the FITC/TRITC/FITC labeling order. (B) Direct four-color PRINS labeling on chromosome 1 (yellow), chromosome 9 (red), chromosome 16 (green), and chromosome 18 (blue) using the labeling order FITC/TRITC/FITC/Cas-cade Blue. (Please see color insert following p. 48.)

4. The use of NaOH solution allows the simultaneous denaturation and decondensation of sperm nuclei, with the possibility of a rapid control of the degree of nucleus decondensation under the microscope. The success of the technique depends to a high degree on the quality and efficiency of the decondensing protocol. Initially, we used 3 M NaOH, but numerous experiments and a comparison of the results in terms of quality of the preparation obtained, led us to adopt a 0.5 MNaOH solution. Combined with PRINS, this method provides homogeneous sperm decondensation, and subsequently a high level of sperm in situ labeling. The duration of 0.5 MNaOH treatment depends on the age of the sperm preparation slides. The longer the slides are aged, the longer they need 0.5 M NaOH treatment: 2-d-old, 4 min; 3-d-old, 5 min; 4-d-old, 6 min. The NaOH treatment induces uniform swelling of the sperm nucleus 1.5 to 2 times its normal size, and maintains the characteristics and shape of the sperm nucleus, including the tail, allowing one to differentiate between spermatozoa and other cells, such as leukocytes or immature germ cells, present in the ejaculate.

5. For the three-color PRINS reactions, the combination order FITC/TRITC/FITC gives the best results, with distinct red, green, and yellow spots. When using the reversed labeling combination TRITC/FITC/TRITC, there is no pure yellow color; instead, a mixed orange color is obtained for the first labeled chromosome. Indeed, a mean ratio of green to red color of 70%:30% must be respected to obtain well-defined green, red, and yellow signals. In the four-color procedure, the addition of the blue label does not affect the final coloring of the three previously labeled targets. The Cascade Blue dye provides a color that contrasts well with the longer-wavelength green and red fluorophores.

6. In the conventional PRINS protocol, the labeling reaction consists of two programmed steps corresponding to (1) the annealing of the primer to the DNA target sequence and (2) the primer elongation catalyzed by the Taq DNA polymerase. Because the range for optimal activity of Taq DNA polymerases is between 55 and 75°C, we decided to use a unique one-temperature step for both annealing and elongation, which allows us to simplify the protocol and shorten the duration of the reaction. The optimal primer annealing temperature is determined empirically, and slight adjustments may be necessary according to the thermal cycler used. The conditions described here must be taken as indications only, and not as definitive values.

7. Strict scoring criteria are required to ensure accurate aneuploidy estimates on human sperm samples. Sperm nuclei are scored as haploid when they display distinct signals in different colors corresponding to each labeled chromosome. Nuclei are considered disomic when they show two signals of the same color, are comparable in size and intensity, and are separated by the diameter of at least one fluorescent spot. Overlapped spermatozoa or sperm heads without a well-defined boundary should not be counted. In human sperm, the frequency of aneuploidy for a given chromosome can be extremely low. Consequently, a large number of spermatozoa must be scored to ensure that reliable disomy estimates are obtained. The statistical pitfalls associated with scoring small numbers of cells are obvi ous. There is a high risk of error in estimates if fewer than 2000 spermatozoa are scored. In this case, scoring one or two more or less cells can significantly change the frequency of disomy. Consequently, a minimum of 5000 spermatozoa per individual and per chromosome must be scored.

Acknowledgments

The experiments performed for elaborating this protocol were supported by the Projet Hospitalier de Recherche Clinique, a French research project (no.

7732), from the Centre Hospitalo-Universitaire of Montpellier and a European

INTAS project no. 03-51-4060.

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