ABSTRACT
BACKGROUND: Comparative genomic hybridization (CGH) is a valuable alternative to fluorescence in situ hybridization (FISH) for preimplantation genetic screening (PGS) because it allows full karyotype analysis. However, this approach requires the cryopreservation of biopsied embryos until results are available. The aim of this study is to reduce the hybridization period of CGH, in order to make this short-CGH technique suitable for PGS of Day-3 embryos, avoiding the cryopreservation step. METHODS: Thirty-two fibroblasts from six aneuploid cell lines (Coriell) and 48 blastomeres from 10 Day-4 embryos, discarded after PGS by FISH with 9 probes (9-chr-FISH), were analysed by short-CGH. A reanalysis by the standard 72 h-CGH and FISH using telomeric probes was performed when no concordant results between short-CGH and FISH diagnosis were observed. The short-CGH was subsequently applied in a clinical case of advanced maternal age. RESULTS: In 100% of the fibroblasts analysed, the characteristic aneuploidies of each cell line were detected by short-CGH. The results of the 48 blastomeres screened by short-CGH were supported by both 72 h-CGH results and FISH reanalysis. The chromosomes most frequently involved in aneuploidy were 22 and 16, but aneuploidies for the other chromosomes, excepting 1, 10 and 13, were also detected. Forty-one of the 94 aneuploid events observed (43.6%) corresponded to chromosomes which are not analysed by 9-chr-FISH. CONCLUSIONS: We have performed a preliminary validation of the short-CGH technique, including one clinical case, suggesting this approach may be applied to Day-3 aneuploidy analysis, thereby avoiding embryo cryopreservation and perhaps helping to improve implantation rate after PGS.
Subject(s)
Aneuploidy , Blastomeres/ultrastructure , Comparative Genomic Hybridization/methods , Preimplantation Diagnosis/methods , Adult , Cell Line , Cryopreservation , Fibroblasts/ultrastructure , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Maternal Age , Middle AgedABSTRACT
La aplicación creciente de técnicas de reproducción asistida para el tratamiento de la esterilidad ha generado diversos problemas y efectos adversos. Entre ellos, las gestaciones múltiples han, emergido como el más frecuente y uno de los más graves desde el punto de vista médico. Con el fin de evaluar su importancia en nuestro medio, y con el objetivo de diseñar estrategias correctivas, se revisaron los registros correspondientes a las 324 gestaciones producidas en el programa de fecundación in vitro de la Fundació Puigvert-Hospital de la Santa Creu i Sant Pau durante los años 2000 a 2003. La proporción de gestaciones múltiples fue de 41,4% (31,2% gemelares, 9,6% trillizos y 0,6% cuatrillzos) aunque en los años 2002 y 2003 se observó una tendencia a la reducción, como resultado de la aplicación de criterios más estrictos en el número de embriones transferidos. La indicación masculina mostró mayor frecuencia de gestaciones múltiples que otras indicaciones. Las variables clinicas y embriológicas más influyentes en la producción de gestaciones múltiples fueron la edad de la muer; el número de folículos y la dosis (menor) de gonadotropinas usada durante la estimulación, el grosor endometrial, el número de embriones totales obtenidos y transferidos, y la suma de puntuación de la calidad embrionaria. Un modelo de función discriminante seleccionó tres variables (calidad global de los embriones transferidos, edad y grosor endometrial) como mejores predictores de gestación múltiple (sensibilidad 59%, especificidad 76%). En conclusión, sigue siendo necesario reducir el número de embriones transferidos en ciclos de FIV, aunque la inclusión de ciertas variables clínicas y embriológicas mediante algoritmos predictivos, puede facilitar la elección en cada caso individual, con el fin de reducir la prevalencia de gestaciones múltiples sin disminuir la tasa global de embarazos
The increasing application of techniques of assisted reproduction for the treatment of sterility has generated a number of adverse effects. Among them, multiple pregnancies have emerged as the most frequent and serious problem from the medical point of view. With the purpose of evaluating their importance in OUT environment, and with the objective to design corrective strategies, we reviewed the registries corresponding to the 324 gestations produced in the program of fertilization in vitro of the Fundació Puigvert-Hospital de la Santa Creu i Sant Pau during years 2000 to 2003. The proportion of multiple gestations was of 41.4% (31.2% twins, 9.6% triplets and 0.6% quadruplets) althougth in 2002 and 2003 there was a tendency towards a reduction, as a result of the application of stricter criteria in the number of transferred embryos. Male infertility indication showed greater frequency of multiple gestations that other indications. The clinical and embryologic variables that significantly influenced the production of multiple pregnancies were age of the woman, the number of follicles and the (smaller) dose of gonadotrophins used during the stimulation, the endometrial thickness, the number of embryos obtained and transferred and the sum of the scores showing embryonic quality. A discriminant function model selected three variables (global quality of the transferred embryos, age and endometrial thickness) as the best predictors of multiple pregnancy (sensitivity 59% speciftcity 76%). In conclusion, it is necessary to further reduce the number of embryos transferred in cycles of FIV, although the inclusion of certain clinical and embryologic variables by means of predictive algorithms, can facilitate the election in each individual case, with the purpose of reducing the prevalence of multiple gestations without diminishing the global rate of pregnancies
