The true panel of cystic fibrosis mutations in the Sicilian population.

BACKGROUND: The aim was to establish the true risk of having an affected child with Cystic Fibrosis (CF) in the Sicilian infertile population. METHODS: A longitudinal CFTR screening of 1279 Sicilian infertile patients for all CFTR mutations sequencing the entire gene by Next Generation Sequencing (NGS) was performed from patient's blood. RESULTS: One patient out of 16 was a carrier of a CFTR mutation. Twenty-four mutations were found. Theoretically one couple out of 256 was at risk of CF transmission. CONCLUSIONS: The risk of CF transmission is unexpectedly high in Sicily and with a high heterogeneity. Sequencing an entire and long gene such as CFTR makes accessible the true panel of mutations in a specific population and helps better to understand the true risk of having an affected child.

Universal strategy for preimplantation genetic testing for cystic fibrosis based on next generation sequencing.

PURPOSE: We developed and applied a universal strategy for preimplantation genetic testing for all cystic fibrosis gene mutations (PGT-CF) based on next-generation sequencing (NGS). METHODS: A molecular protocol was designed to diagnose all CF mutations at preimplantation stage. The detection of CF mutations was performed by direct gene sequencing and linkage strategy testing 38 specific SNPs located upstream and inside the gene for PGT-CF. Seventeen couples at risk of CF transmission decided to undergo PGT-CF. Trophectoderm cell biopsies were performed on day 5-6 blastocysts. PGT for aneuploidy (PGT-A) was performed from the same samples. Tested embryos were transferred on further natural cycles. RESULTS: PGT was performed on 109 embryos. Fifteen CF mutations were tested. PGT-CF and PGT-A were conclusive for respectively 92.7% and 95.3% of the samples. A mean of 24.1 SNPs was informative per couple. After a single embryo transfer on natural cycle, 81.3% of the transferred tested embryos were implanted. CONCLUSIONS: The present protocol based on the entire CFTR gene together with informative SNPs outside and inside the gene can be applied to diagnose all CF mutations at preimplantation stage.

Universal strategy for preimplantation genetic testing for cystic fibrosis based on next-generation sequencing.

PURPOSE: We developed and applied a universal strategy for preimplantation genetic testing for all cystic fibrosis gene mutations (PGT-CF) based on next-generation sequencing (NGS). METHODS: A molecular protocol was designed to diagnose all CF mutations at preimplantation stage. The detection of CF mutations was performed by direct gene sequencing and linkage strategy testing 38 specific SNPs located upstream and inside the gene for PGT-CF. Seventeen couples at risk of CF transmission decided to undergo PGT-CF. Trophectoderm cell biopsies were performed on days 5-6 blastocysts. PGT for aneuploidy (PGT-A) was performed from the same samples. Tested embryos were transferred on further natural cycles. RESULTS: PGT was performed on 109 embryos. Fifteen CF mutations were tested. PGT-CF and PGT-A were conclusive for, respectively, 92.7% and 95.3% of the samples. A mean of 24.1 SNPs was informative per couple. After single embryo transfer on natural cycle, 81.3% of the transferred tested embryos implanted. CONCLUSIONS: The present protocol based on the entire CFTR gene sequencing together with informative SNPs outside and inside the gene can be applied to diagnose all CF mutations at preimplantation stage.

The decision on the embryo to transfer after Preimplantation Genetic Diagnosis for X-autosome reciprocal translocation in male carrier.

BACKGROUND: The aim of Preimplantation Genetic Diagnosis (PGD) on embryos produced in vitro is to identify the embryos without genetic or chromosomal defect from those embryos that will develop the genetic disease or are chromosomally abnormal. In case of PGD for structural chromosome indication (PGR-SR), the normal/balanced embryos are transferred in the maternal uterus. This protocol is valid and widely applied for autosomal chromosome translocation. But which embryo should be transferred after preimplantation genetic diagnosis (PGD-SR) for X-3 reciprocal translocation in male patient? CASE PRESENTATION: The female patient was 26 years old with normal 46,XX karyotype. The male patient had a karyotype with balanced translocation 46,Y,t(X;3)(p11.2;p14)mat, inherited from the mother. The female patient underwent two cycles of ovarian stimulation. In the first cycle, the metaphase II oocytes were vitrified, while in the second cycle they were used as fresh. ICSI was performed on vitrified/warmed and fresh oocytes. Embryos were biopsied at blastocyst stage. Chromosomal analysis was performed by Next Generation Sequencing.Eleven blastocysts were biopsied from 23 vitrified/warmed and fresh metaphase II oocytes. Two embryos were diagnosed 46,XY; two embryos were diagnosed 46,XX; four embryos were diagnosed with unbalanced translocations and three embryos were diagnosed aneuploid. We knew that the two embryos diagnosed as 46,XX inherited the balanced translocation from the father and the two embryos diagnosed as 46,XY had a normal karyotype. It was explain to the couple that the phenotype of balanced translocated female embryos cannot be predicted because of the random inactivation of X chromosome and that could also occur on the der(X). The couple asked to have a 46,XY embryo transferred. Clinical pregnancy was obtained and non invasive prenatal test confirmed PGD-SR result. CONCLUSIONS: Proposing PGD-SR for gonosome-autosome reciprocal translocation implies the risk to exclude balanced translocated female embryos with a normal phenotype for transfer because the early and late normal development at post-natal stage cannot be predicted based on the only chromosomal analysis.

The accumulation of vitrified oocytes is a strategy to increase the number of euploid available blastocysts for transfer after preimplantation genetic testing.

PURPOSE: In a preimplantation genetic diagnosis for aneuploidy (PGD-A) program, the more embryos available for biopsy, consequently increases the chances of obtaining euploid embryos to transfer. The aim was to increase the number of viable euploid blastocysts in patients undergoing PGD-A using fresh oocytes together with previously accumulated vitrified oocytes. METHODS: Sixty-nine patients with normal ovarian reserve underwent PGD-A for repeated implantation failure or recurrent pregnancy loss indication. After several cycles of ovarian stimulation, 591 accumulated vitrified oocytes and 463 fresh oocytes were micro-injected with the same partner's semen sample. PGD-A was completed on 134 blastocysts from vitrified/warmed oocytes and 130 blastocysts from fresh oocytes. RESULTS: A mean of 9.6% euploid blastocyst per micro-injected vitrified/warmed oocytes and 11.4% euploid blastocyst per micro-injected fresh oocyte were obtained (p > 0.05). The euploidy and aneuploidy rates were comparable in blastocysts obtained from micro-injected vitrified/warmed oocytes and fresh oocytes (42.5 versus 40.8% and 57.5 versus 59.2%, p > 0.05). Implantation rates of euploid blastocysts were comparable between the two sources of oocytes (56.0% from vitrified/warmed oocytes versus 60.9% from fresh oocytes, p > 0.05). CONCLUSIONS: Oocyte vitrification and warming do not generate aneuploidy in blastocysts. The number of viable euploid embryos for transfer can be increased by using accumulated vitrified oocytes together with fresh oocytes in ICSI. TRIAL REGISTRATION: NCT02820415 ClinicalTrials.gov.