Mariona Rius

Birth of a healthy boy after a double factor PGD in a couple carrying a genetic disease and at risk for aneuploidy: Case Report

Preimplantation genetic diagnosis (PGD) for monogenic diseases is widely applied, allowing the transfer to the uterus of healthy embryos. PGD is also employed for the detection of chromosome abnormalities for couples at high risk of producing aneuploid embryos, such as advanced maternal (>35 years). A significant number of patients requesting PGD for monogenic diseases are also indicated for chromosome testing. We optimized and clinically applied a PGD protocol permitting both cytogenetic and molecular genetic analysis. A couple, carriers of two cystic fibrosis (CF) mutations (c.3849 + 10 KbC > T and c.3408C > A) with a maternal age of 38 years and two previously failed IVF-PGD cycles, was enrolled in the study. After ovarian stimulation, six oocytes were obtained. To detect abnormalities for all 23 chromosomes of the oocyte, the first polar body (1PB) was biopsied from five of the oocytes and analyzed using comparative genomic hybridization (CGH). CGH analysis showed that 1PB 1 and 1PB 4 were aneuploid (22X,-9,-13,+19 and 22X,-6, respectively), while 1PB 2, 1PB 3 and 1PB 6 were euploid. Blastomere biopsy was only applicable on embryos formed from Oocyte 3 and Oocyte 6. After whole-genome amplification with multiple displacement amplification, a multiplex PCR, amplifying informative short tandem repeats (D7S1799; D7S1817) and DNA fragments encompassing the mutation sites, was performed. MiniSequencing was applied to directly detect each mutation. Genetic diagnosis showed that Embryo 6 was affected by CF and Embryo 3 carried only the c.3849 + 10 KbC > T mutation. Embryo 3 was transferred achieving pregnancy and a healthy boy was born. This strategy may lead to increased pregnancy rates by allowing preferential transfer of euploid embryos.

Comprehensive embryo analysis of advanced maternal age–related aneuploidies and mosaicism by short comparative genomic hybridization

The short comparative genomic hybridization (short-CGH) method was used to perform a comprehensive cytogenetic study of isolated blastomeres from advanced maternal age embryos, discarded after fluorescent in situ hybridization (FISH) preimplantation genetic screening (PGS), detecting aneuploidies (38.5% of which corresponded to chromosomes not screened by 9-chromosome FISH), structural aberrations (31.8%), and mosaicism (77.3%). The short-CGH method was subsequently applied in one PGS, achieving a twin pregnancy.

Reliability of short comparative genomic hybridization in fibroblasts and blastomeres for a comprehensive aneuploidy screening: first clinical application

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.

Detection of unbalanced chromosome segregations in preimplantation genetic diagnosis of translocations by short comparative genomic hibridization.

OBJECTIVE To apply a comprehensive chromosomal screening through short comparative genomic hybridization (CGH) in the preimplantation genetic diagnosis (PGD) of translocations. DESIGN Clinical research study. SETTING A PGD laboratory and two IVF clinics. PATIENT(S) Three Robertsonian translocation carriers, two reciprocal translocation carriers, and a double-translocation carrier. INTERVENTION(S) After using the short-CGH approach in the reanalysis of two unbalanced embryos, discarded from a PGD for a reciprocal translocation carrier, the same method was applied in the PGD of day-3 embryos of translocation carriers. MAIN OUTCOME MEASURE(S) Ability of short CGH to detect partial chromosomal abnormalities in unbalanced embryos, translocation segregation proportions, and proportion of embryos carrying chromosomal abnormalities not related to the translocations. RESULT(S) The short-CGH technique detected errors resulting from the meiotic segregation of the chromosomes involved in the translocations and other abnormalities affecting the remaining chromosomes. Alternate segregation was detected most frequently among Robertsonian translocation cases, whereas unbalanced chromosome segregations were found predominantly in reciprocal ones. Aneuploidy and structural chromosome errors were found more frequently in Robertsonian than in reciprocal translocation carriers. Application of short-CGH PGD achieved pregnancy in two cases. CONCLUSION(S) Short CGH is a reliable approach for PGD of translocations, as it is capable of detecting partial chromosome errors caused by unbalanced segregations simultaneously to the screening of all chromosomes, and it may improve the results after PGD for translocation carriers.

Outcome of twin babies free of Von Hippel-Lindau disease after a double-factor preimplantation genetic diagnosis: monogenetic mutation analysis and comprehensive aneuploidy screening

OBJECTIVE To increase the embryo implantation rate, a double-factor preimplantation genetic diagnosis (DF-PGD) was performed, selecting for transfer potentially euploid evolved embryos free of the mutation responsible for Von Hippel-Lindau syndrome (VHL). DESIGN Case report. SETTINGS Medical university center and a private IVF center. PATIENT(S) A patient carrier of the R161Q mutation on the VHL gene. INTERVENTION(S) After first polar body (1PB) biopsy, it was analyzed using comparative genomic hybridization (1PB-CGH). On day +3, mutation detection using minisequencing and short tandem repeat analysis was performed in multiple displacement amplification products of a single blastomere per embryo. MAIN OUTCOME MEASURE(S) Transfering embryos free of the disease and originating from euploid oocytes. RESULT(S) Nine of the twelve oocytes obtained were successfully analyzed using 1PB-CGH. One of them was aneuploid (1PB #1: 29XX,+2,+10,+12,+17,+19), and the rest were euploid. All of the oocytes were fertilized and became evolved embryos. Six of the embryos were VHL unaffected and had good quality. Five (83%) of them were potentially euploid. According to cytogenetic results, two of the evolved and healthy embryos were transfered, achieving the birth of healthy twin babies. CONCLUSION(S) The DF-PGD can be a useful tool to increase implantation of transfered embryos in PGD for monogenic diseases.

Errors at mitotic segregation early in oogenesis and at first meiotic division in oocytes from donor females: Comparative genomic hybridization analyses in metaphase II oocytes and their first polar body

The aim of this work is to analyze, using the comparative genomic hybridization technique, the frequencies and the mechanisms involved in the production of aneuploidy events in donor oocytes. The results showed that 32.1% of them were aneuploid, with 51.7% of those originating from first meiotic division errors and 48.3% from the presence of aneuploid oogonium.

First successful double‐factor PGD for Lynch syndrome: monogenic analysis and comprehensive aneuploidy screening

Preimplantation genetic diagnosis (PGD) has been applied worldwide for a great variety of single‐gene disorders over the last 20 years. The aim of this work was to perform a double‐factor preimplantation genetic diagnosis (DF‐PGD) protocol in a family at risk for Lynch syndrome. The family underwent a DF‐PGD approach in which two blastomeres from each cleavage‐stage embryo were biopsied and used for monogenic and comprehensive cytogenetic analysis, respectively. Fourteen embryos were biopsied for the monogenic disease and after multiple displacement amplification (MDA), 12 embryos were diagnosed; 5 being non‐affected and 7 affected by the disease. Thirteen were biopsied to perform the aneuploidy screening by short‐comparative genomic hybridization (CGH). The improved DF‐PGD approach permitted the selection of not only healthy but also euploid embryos for transfer. This has been the first time a double analysis of embryos has been performed in a family affected by Lynch syndrome, resulting in the birth of two healthy children. The protocol described in this work offers a reliable alternative for single‐gene disorder assessment together with a comprehensive aneuploidy screening of the embryos that may increase the chances of pregnancy and birth of transferred embryos.

Whole-Chromosome Aneuploidy Analysis in Human Oocytes: Focus on Comparative Genomic Hybridization

The study of aneuploidy in human oocytes, discarded from IVF cycles, has provided a better understanding of the incidence of aneuploidy of female origin and the responsible mechanisms. Comparative genomic hybridization (CGH) is an established technique that allows for the detection of aneuploidy in all chromosomes avoiding artifactual chromosome losses. In this review, results obtained using CGH in single cells (1PB and/or MII oocytes) are included. The results of oocyte aneuploidy rates obtained by CGH from discarded oocytes of IVF patients and of oocyte donors are summarized. Moreover, the mechanisms involved in the aneuploid events, e.g. whether alterations occurred due to first meiotic errors or germ-line mitotic errors are also discussed. Finally, the incidence of aneuploid oocyte production due to first meiotic errors and germ-line mitotic errors observed in oocytes coming from IVF patients and IVF oocyte donors was assessed.

Does the S phase have an impact on the accuracy of comparative genomic hybridization profiles in single fibroblasts and human blastomeres

OBJECTIVE To investigate if there is an association between single-cell replicative stage and the segmental chromosome imbalances detected by comparative genomic hybridization (CGH). DESIGN First, 135 fibroblasts from cell-line GM03184 (Coriell) at three cell stages (G0/G1, S, and G2/M) were amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) or Sureplex and blindly analyzed by CGH. Second, 85 human blastomeres at the interphase and the metaphase stages, from 30 donated human cryopreserved embryos, were amplified by Sureplex and analyzed by CGH. SETTING Academic center for reproductive medicine. PATIENT(S) None. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Incidence of aneuploidy and segmental imbalances detected at the different cell stages. RESULT(S) In DOP-PCR amplifications of fibroblasts, an increased incidence of segmental abnormalities was detected in the S phase. In Sureplex amplifications of fibroblasts and blastomeres, no differences were detected between the different cell stages. A significantly increased incidence of structural abnormalities was seen in the aneuploid blastomeres. CONCLUSION(S) The segmental imbalances detected after Sureplex amplification in 73.3% of the cryopreserved embryos analyzed are mainly nontransitory. They correspond to segmental imbalances present in the cells due to chromosome instability, rather than to replicative DNA segments.

Non-meiotic chromosome instability in human immature oocytes.

Aneuploidy has been a major issue in human gametes and is closely related to fertility problems, as it is known to be present in cleavage stage embryos and gestational losses. Pre-meiotic chromosome abnormalities in women have been previously described. The aim of this study is to assess the whole-chromosome complement in immature oocytes to find those abnormalities caused by mitotic instability. For this purpose, a total of 157 oocytes at the germinal vesicle or metaphase I stage, and discarded from IVF cycles, were analysed by CGH. Fifty-six women, between 18 and 45 years old (mean 32.5 years), including 32 IVF patients (25–45 years of age) and 24 IVF oocyte donors (18–33 years of age), were included in the study. A total of 25/157 (15.9%) of the oocytes analysed, obtained from three IVF clinics, contained chromosome abnormalities, including both aneuploidy (24/157) and structural aberrations (9/157). Independently of the maternal age, the incidence of abnormal oocytes which originated before meiosis is 15.9%, and these imbalances were found in 33.9% of the females studied. This work sheds light on the relevance of mitotic instability responsible for the generation of the abnormalities present in human oocytes.