Valor de los estudios cromosómicos en espermatozoides mediante la técnica de hibridación in situ fluorescente (FISH) en parejas estériles

Los varones estériles presentan una frecuencia elevada de anomalías citogenéticas, algunas de ellas detectables mediante el cariotipo, pero otras limitadas a las células germinales como resultado de meiosis anormales. Con las nuevas técnicas de reproducción asistida como la Inyección Intracitoplásmica de Espermatozoides (ICSI), la fecundación por espermatozoides cromosómicamente anormales puede dar lugar a fallos de implantación, abortos de repetición y a la transmisión de anomalías cromosómicas a la descendencia. La hibridación in situ fluorescente (FISH) de espermatozoides es una técnica de análisis citogenético que, mediante el marcado de los cromosomas con moléculas de ADN fluorescentes, permite valorar la presencia de aneuploidías y diploidías en los espermatozoides en estadio de interfase. El objetivo general de esta tesis es determinar los factores que podrían estar asociados con un incremento de anomalías cromosómicas en espermatozoides de varones con cariotipo normal, y evaluar cómo afecta este incremento al éxito reproductivo en parejas con problemas de infertilidad. Hemos observado una incidencia basal de espermatozoides anormales procedentes de testículo del orden de 2 veces superior a la de los espermatozoides de eyaculado de varones fértiles normozoospérmicos, lo que justifica el uso de controles específicos de espermatozoides de testículo para identificar con más rigor los varones azoospérmicos con peor pronóstico reproductivo. El 14,6% de las muestras procedentes de eyaculado de varones infértiles ha mostrado un resultado de FISH anormal comparado con el grupo control. El 42,1% de los varones con azoospermia secretora ha presentado un resultado de FISH anormal comparado con el control de testículo, siendo este porcentaje sólo del 6,3% en varones con azoospermia obstructiva. El único parámetro seminal que se ha asociado con el resultado del FISH ha sido la concentración espermática, aumentando de forma gradual el porcentaje de varones con un resultado de FISH anormal a medida que disminuye la concentración de espermatozoides en el eyaculado. Ninguna de las indicaciones por las que se solicitó el estudio de FISH en espermatozoides (aborto de repetición, fallo repetido de implantación, factor masculino severo, causas mixtas) ha presentado una probabilidad superior a las restantes de por sí sola asociarse a un resultado de FISH anormal. Adicionalmente hemos observado que la presencia de una incidencia elevada de anomalías cromosómicas en los espermatozoides afecta negativamente al éxito reproductivo de la pareja tras ciclos de FIV/ICSI convencional. Sin embargo, la selección de embriones cromosómicamente normales mediante Diagnóstico Genético Preimplantatorio (DGP) para el estudio de aneuploidías de 9 cromosomas mediante FISH ha mejorado los resultados reproductivos en parejas con FISH anormal en espermatozoides. Así pues, el análisis de los espermatozoides mediante FISH previo a un tratamiento de reproducción asistida podría ser muy útil para seleccionar a los pacientes con mayor riesgo de generar embriones con anomalías cromosómicas, y ayudaría al clínico evaluar los tratamientos con mejores posibilidades reproductivas para la pareja infértil.Infertile males show a high frequency of cytogenetic abnormalities , some of which are detectable by the karyotype , but other limited to the germ cells as a result of abnormal meiosis . With new assisted reproductive techniques such as Intracytoplasmic Sperm Injenction (ICSI), the fertilization of chromosomally abnormal sperm may lead to implantation failure, recurrent miscarriages and transmission of chromosome abnormalities to the offspring. Fluorescence in situ hybridization (FISH) in sperm as cytogenetic analysis technique, can assess the presence of aneuploidy and diploidy by labelling the sperm chromosomes with fluorescent DNA probes. The overall objective of this thesis is to identify factors that might be associated with increased chromosomal abnormalities in sperm of males with normal karyotype , and evaluate how this increase affects to the reproductive success in couples with infertility problems . We have observed a baseline incidence of abnormal sperm from testis of about 2 times higher than that of ejaculated sperm from normozoospermic fertile men, justifying the use of specific testicular sperm controls to more rigorously identify azoospermic men with worse reproductive prognosis. The 14.6% of the ejaculate samples from infertile male has shown an abnormal FISH result compared to the control group. The 42.1% of men with non-obstructive azoospermia showed an abnormal FISH result compared to the testis control, with the rate being only 6.3% in men with obstructive azoospermia. Sperm concentration has been the only seminal parameter associated with the FISH result, gradually increasing the percentage of men with abnormal FISH result with decreasing the concentration of sperm in the ejaculate . None of the indications to requested the FISH analysis in sperm (recurrent miscarriage, repeated implantation failure, severe male factor, mixed causes) had a higher probability than the others to be associated with an abnormal FISH result. Additionally, we observed that the presence of a high incidence of chromosomal abnormalities in sperm negatively affects the reproductive success of the couple after conventional IVF/ICSI cycles. However, the selection of chromosomally normal embryos using Preimplantation Genetic Diagnosis (PGD) for aneuploidy screening of 9 chromosomes by FISH has improved reproductive outcomes in couples with abnormal FISH in sperm. Thus, sperm FISH analysis before assisted reproduction treatment may be useful for selecting patients most at risk of generating embryos with chromosomal abnormalities, and help clinicians to choose the best reproductive option in infertility couples

High incidence of chromosomal abnormalities in large-headed and multiple-tailed spermatozoa

Rodrigo Vivo, Lorena, [email protected] ; Prados Dodd, Nicolas, [email protected] ; Gil Salom, Manuel Luis, [email protected] ; Remohi Gimenez, Jose Alejandro, [email protected]

False positive rate of an arrayCGH platform for single-cell preimplantation genetic screening and subsequent clinical application on day-3

In this work, false positive rate of an arrayCGH platform for its use in day-3 single-blastomere analysis was calculated. For this purpose, 38 embryos diagnosed as abnormal on day-3 by FISH were re-biopsied on day-4. Single-cell day-4 arrayCGH diagnosis was then performed. A successful amplification was obtained in 97.4 % (37/38) of the day-4 cells analysed by arrayCGH. Day-3 FISH and day-4 arrayCGH diagnosis were concordant in 35/37 cases. The two discordant embryos were spread and all the cells from each embryo were re-analysed by FISH on day 5. The same error rate (2.7 %) for day-3 FISH and day-4 arrayCGH was obtained when comparing day-5 FISH re-analysis. After this pre-clinical phase, the platform was used for day-3 arrayCGH clinical application in 320 patients (1,760 embryos). Day-3 amplification rate was 98.6 %. An optimal reproductive outcome was obtained when applying arrayCGH to a clinical program: clinical pregnancy rate per cycle of 38.4 % and 60.3 % per transference were obtained, with an implantation rate of 53.5 %. Overall miscarriage rate was 10.6 %. Additionally, day-5 FISH re-analysis was performed in 42 of the embryos from the clinical phase, obtaining a concordance rate of 97.6 % with day-3 arrayCGH.Electronic supplementary materialThe online version of this article (doi:10.1007/s10815-012-9918-4) contains supplementary material, which is available to authorized users

New Tools for Embryo Selection: Comprehensive Chromosome Screening by Array Comparative Genomic Hybridization

The objective of this study was to evaluate the usefulness of comprehensive chromosome screening (CCS) using array comparative genomic hybridization (aCGH). The study included 1420 CCS cycles for recurrent miscarriage (n = 203); repetitive implantation failure (n = 188); severe male factor (n = 116); previous trisomic pregnancy (n = 33); and advanced maternal age (n = 880). CCS was performed in cycles with fresh oocytes and embryos (n = 774); mixed cycles with fresh and vitrified oocytes (n = 320); mixed cycles with fresh and vitrified day-2 embryos (n = 235); and mixed cycles with fresh and vitrified day-3 embryos (n = 91). Day-3 embryo biopsy was performed and analyzed by aCGH followed by day-5 embryo transfer. Consistent implantation (range: 40.5–54.2%) and pregnancy rates per transfer (range: 46.0–62.9%) were obtained for all the indications and independently of the origin of the oocytes or embryos. However, a lower delivery rate per cycle was achieved in women aged over 40 years (18.1%) due to the higher percentage of aneuploid embryos (85.3%) and lower number of cycles with at least one euploid embryo available per transfer (40.3%). We concluded that aneuploidy is one of the major factors which affect embryo implantation