|Introduction: Preimplantation genetic diagnosis (PGD) has been offered to carriers of balanced translocations as an alternative to prenatal diagnosis. Fluorescence in-situ hybridisation (FISH) is the method of choice for detecting chromosome rearrangements. The FISH strategy involves the simultaneous use of telomeric probes in combination with centromeric probes (reciprocal translocations), or alpha-satellite/locus-specific enumerator probes (Robertsonian translocations).
Here we present the development of a polymerase chain reaction (PCR)-based PGD approach for detection of chromosomal imbalances on embryos derived from both reciprocal and Robertsonian translocation carriers. The procedure involves testing of single blastomeres by fluorescent multiplex PCR analysis of polymorphic short tandem repeat (STR) markers located along the chromosomes involved by translocation.
Material & methods: STR markers were selected to be located at either side of each breakpoint (reciprocal translocations) or at any point of the chromosomes involved (Robertsonian translocation). STR markers were also included to determine the copy number of chromosomes 13, 14, 15, 16, 18, 21, 22, X, Y in patients of advanced maternal age. Informativity testing of STR markers was performed for both partners of each couple. Only fully informative markers presenting alleles not shared by the partners were selected. In order to avoid misdiagnosis due to possible allele drop-out (ADO) occurrences, at least three STR for each chromosome were included in the protocol. Embryos were diagnosed as “normal-balanced” if PCR results indicated two signals (peaks) for each chromosome tested. Embryos were diagnosed as “unbalanced” if the PCR results showed a deviation from the “normal-balanced” signal pattern, such as trisomies (three peaks), monosomies (one peak) and nullisomies (no PCR signals).
Results: Twelve PGD cycles were carried out for 12 couples carrying six different reciprocal translocations and two Robertsonian translocations. The mean maternal age was 36.4 ± 4.6 years. A total of 204 oocytes were collected, 158 (77.5%) were MII, 126 (79.7%) fertilized and 110 embryos were biopsied on day 3. PCR was successful in 102/110 (92.7%) blastomeres, accounting a positive amplification on a total of 1048/1128 (92.9%) loci. Overall, 102 (92.7%) embryos were successfully diagnosed, 52 of which resulted normal/balanced, 44 were unbalanced and 6 resulted to be haploid. PGS was included in the PGD protocol of five couples, involving testing of 45 embryos, 40 (88.9%) of which were successful diagnosed and 24 (60.0%) showed aneuploidies. Embryos suitable for transfer where identified in 10 cycles. Following transfer of 23 embryos (mean 1.9±1.1), 7 women had a clinical pregnancy confirmed with fetal sacs and heart beat (70.0% pregnancy rate per embryo transfer). A total of 13 embryos implanted (56.5% implantation rate per embryo transferred), for 10 of which heart beat was also detected. Only 2 couples accepted to undergo to prenatal diagnosis, performed by chorion villus sampling (CVS), which confirmed the PGD results. All pregnancies are still ongoing.
Conclusions: The above results demonstrate the feasibility and reliability of our PCR-based PGD protocol for detection of chromosomal imbalances. The present technique has the potential to overcome to several inherent limitation of the FISH procedure, such as suboptimal fixation, overlapping signals, split signals, lack of signals, cross-hybridization, polymorphisms, limited availability of the probes, combination of colours, decreasing of the accuracy with re-probing. This approach has the advantage to be rapid, low expensive, amenable to automation, involving an easy procedure and data interpretation. Unlike FISH, with the presented protocol is also possible to distinguish the parental origin of chromosomes, allowing detection of uniparental disomies and the achievement of a DNA fingerprint for each embryo useful for identification of embryos that have implanted. Finally, because cell fixation is not necessary, the PCR-based protocol represents an easier procedure for management of transport PGD. Considering the encouraging preliminary clinical outcome obtained, this approach has the potential to represent a valuable alternative to FISH-based PGD.