Estimation of age-dependent decrease in blastocyst euploidy by next generation sequencing: development of a novel prediction model.

BACKGROUND: We developed a model to estimate the female age-dependent decrease in blastocyst euploidy and the impact of blastocyst cohort size on the likelihood of having at least one euploid blastocyst for transfer. METHODS: Retrospective analysis of 1296 trophectoderm biopsies by next-generation sequencing analysis from 436 infertile couples undergoing intracytoplasmic sperm injection and preimplantation genetic testing for aneuploidy. A logistic regression model was fit to the data. The dependent and independent variables were embryo genetic status and female age, respectively. The method of fitting was quadratic on age, and the model was validated with cross validation by a data splitting technique. RESULTS: The decrease in the probability of blastocyst euploidy follows an age-dependent binomial distribution, progressing with every year of female age, from 1.2% to 24.5% in 28-44 years-old women (P<0.0001). The minimum number of blastocysts needed to obtain at least one euploid blastocyst for transfer was computed for different probabilities and female ages. At the age of 28 years, a total of three blastocysts is required to obtain at least one euploid blastocyst with 90% probability, whereas it is 4, 5, 6, 9, 16 and 29 for ages 35, 37, 39, 41, 43, and 45, respectively. CONCLUSIONS: A novel prediction model estimates the probability of blastocyst euploidy and the number of blastocysts required to obtain at least one euploid embryo for transfer. This new resource based on f emale age and blastocyst cohort size will aid clinicians counsel and plan treatment of infertile couples undergoing IVF/ICSI.

The first South American case of pre-implantation genetic diagnosis to select compatible embryo for cord blood transplantation as treatment for sickle cell anemia.

Sickle cell anemia is an inherited systemic hemoglobinopathy that affects hemoglobin production in red blood cells, leading to early morbidity and mortality. It is caused by a homozygous nucleotide substitution (c.20A>T) in the ß-globin gene (HBB) that changes a glutamic acid to a valine in the protein. We present a case report of a fertile couple, both carriers of the sickle cell anemia mutation, with one affected daughter. Six cycles of assisted reproductive techniques were performed, resulting in 53 embryos in cleavage stage. Each embryo was biopsied and analyzed for pre-implantation genetic diagnosis (PGD) by fluorescent polymerase chain reaction, using polymorphic markers of the region of interest followed by capillary electrophoresis in an automated genetic analyzer. HLA Compatible and normal embryos for the mutation represented 3 (5.66%); while the carriers and compatible 6 (11.32%); therefore, embryos matching those of the affected daughter represented 9 (16.98%). A selected embryo in blastocyst stage was transferred, resulting in a healthy male newborn, who had the umbilical cord blood cells collected and stored. The affected daughter was immunosuppressed and received transplanted cells from the umbilical cord blood of her brother; the treatment was successful. Embryo selection using PGD technologies represent the most effective treatment plan for parents who want to have a healthy child, and it could cure another child already affected by inherited hemoglobinopathy.

Characterization of vitamin K-dependent carboxylase mutations that cause bleeding and nonbleeding disorders.

Vitamin K-dependent coagulation factors deficiency is a bleeding disorder mainly associated with mutations in γ-glutamyl carboxylase (GGCX) that often has fatal outcomes. Some patients with nonbleeding syndromes linked to GGCX mutations, however, show no coagulation abnormalities. The correlation between GGCX genotypes and their clinical phenotypes has been previously unknown. Here we report the identification and characterization of novel GGCX mutations in a patient with both severe cerebral bleeding disorder and comorbid Keutel syndrome, a nonbleeding malady caused by functional defects of matrix γ-carboxyglutamate protein (MGP). To characterize GGCX mutants in a cellular milieu, we established a cell-based assay by stably expressing 2 reporter proteins (a chimeric coagulation factor and MGP) in HEK293 cells. The endogenous GGCX gene in these cells was knocked out by CRISPR-Cas9-mediated genome editing. Our results show that, compared with wild-type GGCX, the patient's GGCX D153G mutant significantly decreased coagulation factor carboxylation and abolished MGP carboxylation at the physiological concentration of vitamin K. Higher vitamin K concentrations can restore up to 60% of coagulation factor carboxylation but do not ameliorate MGP carboxylation. These results are consistent with the clinical results obtained from the patient treated with vitamin K, suggesting that the D153G alteration in GGCX is the causative mutation for both the bleeding and nonbleeding disorders in our patient. These findings provide the first evidence of a GGCX mutation resulting in 2 distinct clinical phenotypes; the established cell-based assay provides a powerful tool for studying the clinical consequences of naturally occurring GGCX mutations in vivo.

Live births after polar body biopsy and frozen-thawed cleavage stage embryo transfer: case report.

Pre-implantation genetic diagnosis (PGD) or screening (PGS) technology, has emerged and developed in the past few years, benefiting couples as it allows the selection and transfer of healthy embryos during IVF treatments. These techniques can be performed in oocytes (polar-body biopsy) or embryos (blastomere or trophectoderm biopsy). In this case report, we describe the first two live births to be published in Brazil after a polar-body (PB) biopsy. In case 1, a 42-year-old was submitted to PB biopsy with PGS due to advanced maternal age and poor ovarian reserve. Five MII oocytes underwent first and second polar body biopsy and four cleavage embryos were cryopreserved. The PGS analysis resulted in two euploid embryos (next generation sequence). A frozen-thawed embryo transfer (FET) was performed after endometrial priming and a healthy baby was delivered after a cesarean section (37 weeks, female, 3390g, 47.5 cm). In case 2, a 40-year old patient with balanced translocation and poor ovarian response was submitted to PB biopsy. Two MII oocytes underwent first and second polar body biopsy and two embryos were cryopreserved in cleavage stage. The analysis resulted in one euploid embryo that was transferred after endometrial priming. A preterm healthy baby (34 weeks, female, 2100g, 40 cm) was delivered via cesarean section. In conclusion, although the blastocyst biopsy is the norm when performing PGS/PGD during IVF treatments, other alternatives (as PB biopsy) should be considered in some specific situations.

Sperm organelle morphologic abnormalities: contributing factors and effects on intracytoplasmic sperm injection cycles outcomes.

OBJECTIVE: To (1) analyze possible relationships between motile sperm organelle morphology examination (MSOME) and sperm chromatin status, aneuploidy incidence, and patient's age; (2) determine the effects of sperm morphologic abnormalities on intracytoplasmic sperm injection (ICSI) outcomes; and (3) identify the benefits of intracytoplasmic morphologically selected sperm injection (IMSI) in patients with high DNA fragmentation rate. METHODS: The study was performed in 50 patients undergoing ICSI cycles. The MSOME, sperm DNA fragmentation, and sperm aneuploidy incidence were performed in 200 sperm cells of each patient. Regression models were used to assess the relationships among sperm morphology and sperm aneuploidy, sperm DNA fragmentation, patient's age, and ICSI outcomes. In cycles with patients showing a high incidence of DNA fragmentation, oocytes were split into 2 groups according to the sperm selection method: Standard-ICSI (n = 82) and IMSI (n = 79). Fertilization and high-quality embryo rates were compared between the groups. RESULTS: A close relationship between sperm DNA fragmentation and the presence of vacuoles in the MSOME was noted. The patient's age was correlated to the presence of vacuoles. No correlation between sperm aneuploidy and IMSI was observed. Vacuolated cells were negatively correlated with fertilization, pregnancy, and implantation. In patients with a high incidence of sperm DNA fragmentation, fertilization and high-quality embryo rates were similar when comparing IMSI and Standard-ICSI. CONCLUSIONS: Our data demonstrate a correlation between paternal age and the incidence of nuclear vacuoles, as well as an effect of large and small vacuoles on late embryo development.