Aneuploidy is the most common class of chromosome abnormalities in humans, accounting for a significant proportion of miscarriages and multiple congenital malformation syndromes in liveborns. Direct chromosomal analysis of spermatozoa constitutes an essential approach for the investigation of the occurrence and etiology of chromosomal abnormalities in humans under a wide variety of clinical conditions. The advent of molecular genetic techniques has brought forth new procedures for in situ chromosomal analysis and has opened the way for comprehensive studies on the occurrence of aneuploidy in human spermatozoa. To date, numerous chromosomal analyses on human sperm have been performed using fluorescence in situ hybridization (FISH [1,2]). These reports have demonstrated the efficiency of the in situ labeling procedure on male gametes but also have pointed out the limitations of FISH on this biological material, which essentially are linked to the size of the probes and the reliability of the associated sperm decondensation treatments (3,4).

From: Methods in Molecular Biology, vol. 334: PRINS and In Situ PCR Protocols, Second Ed.

Edited by: F. Pellestor © Humana Press Inc., Totowa, NJ

The primed in situ (PRINS) reaction offers an efficient alternative approach for the direct chromosome analysis of human spermatozoa. Based on the use of chromosome-specific primers, the PRINS method combines the high sensitivity of the polymerase chain reaction with the cytological localization of DNA sequences. Since its introduction (5), the PRINS protocol has been continuously optimized, and several studies have demonstrated that PRINS labeling was more effective and exhibited higher specificity than FISH on human sperm (6-8).

In the conventional PRINS procedure, the identification of several chromosomes is performed by in situ sequential labeling of each targeted chromosome by using chromosome-specific primers and different reporter molecules or fluo-rochromes. This initial multicolor PRINS procedure was adapted successfully to human spermatozoa, and has been shown to be very well adapted to this material because of the small size of the PRINS primers and the requirement of a moderate sperm nucleus decondensation (9). However, a blocking step based on the use of ddNTP is used between each labeling reaction to avoid the mixing of the labeling. This intermediate reaction significantly extends the total duration of the multi-PRINS procedure and may lead to a prejudicial decrease in the intensity of the labeling of the first targeted chromosomes.

Recently, Yan et al. (10) have reported a new multicolor PRINS protocol based only on in situ mixing of two fluorochromes for generating the distinct and specific labeling of three chromosomes. Because this sequential procedure without blocking steps significantly simplifies the multicolor PRINS protocol and improves the efficiency of PRINS labeling, we have adapted this new method on human sperm samples. Improvements in the timing and the procedure have been introduced, leading to the elaboration of a new ultra-rapid three-or four-color PRINS protocol for sperm chromosomal screening, described in this chapter. The principles of this new PRINS labeling procedure are illustrated in Fig. 1.

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