Detection of numerical abnormalities of chromosome 9 and p16/CDKN2A gene alterations in ovarian cancer with fish analysis.

BACKGROUND: The molecular events leading to the development of ovarian cancer are not well-established. Defects of the retinoblastoma protein (pRb)/cyclin-D1/p16 pathway have been shown to play a critical role in the development of human malignancies. In particular, the p16/cyclin-dependent kinase inhibitor 2A (CDKN2A) gene located on chromosomal region 9p21 frequently is altered in several types of cancer. MATERIALS AND METHODS: To investigate both the presence of numerical abnormalities of chromosome 9 and p16 gene alterations in ovarian cancer, we studied 28 cases by the fluorescence in situ hybridization (FISH) technique using a DNA p16 probe and an a-satellite probe specific for chromosome 9. RESULTS: Numerical abnormalities of chromosome 9 were found in all studied cases. Polysomy 9 was detected in 10 cases while monosomy 9 in seven cases. In 11 cases, there were two cell populations, one with polysomy 9 and the other with monosomy 9. In all cases, the p16 gene deletion was observed. Among them, 25 cases presented deletion of p16 gene in 21.43%-86.3% of the examined cells. Three cases carried deletion of the p16 gene in a lower proportion (12.04%-19.49%). In five cases with p16 gene deletion, homozygous deletion was detected. CONCLUSION: Numerical aberrations of chromosome 9 and p16 gene deletion are common findings in ovarian cancer. Data suggest that the p16 gene, located in the short arms of chromosome 9, may play a role in ovarian carcinogenesis. In addition, polysomy 9 could lead to activation of a number of oncogenes, thus participating in the neoplastic process in the ovaries.

Genetic skeletal disorders of the fetus and infant: pathologic and molecular findings in a series of 41 cases.

BACKGROUND: Genetic skeletal disorders of the fetus and infant are a large group of genetic disorders, comprising the groups formerly assigned as skeletal dysplasias (osteochondrodysplasias), dysostoses, and malformation syndromes with a skeletal component. Genetic skeletal disorders may be prenatally detected by ultrasonography or result in intrauterine or early postnatal death, constituting one difficult diagnostic field met by the pathologist who performs the perinatal autopsy. METHODS: In this retrospective study, we have gathered radiologic, physical, histopathologic, and molecular data regarding 41 cases of genetic skeletal disorders diagnosed among 1980 fetal and perinatal autopsies over a 10-year period. RESULTS: Our series of cases were classified according to the 2006 Nosology and Classification of Genetic Skeletal Disorders. The overall frequency of genetic skeletal disorders was 1:48 autopsies. The FGFR3 group and osteogenesis imperfecta type 2 were the more frequently encountered disorders. The mean gestational age at autopsy was 21.9 weeks (range, 12-37 weeks). A final diagnosis was obtained in 95% of cases. Genetic skeletal disorders were detected by prenatal ultrasound in 90% of cases, with a correct typing of the disorder achieved in only 34%. Molecular analysis was confirmative in 5 cases. CONCLUSIONS: The central role of the perinatal pathologist in collaboration with specialized services is essential for the correct interpretation of the radiologic, physical, and histopathologic findings, to accurately classify specific types of genetic skeletal disorders and enable genetic counseling.

Effect of PRL on in vitro follicle growth, in vitro oocyte maturation, fertilization and early embryonic development in mice.

Prolactin (PRL), along with other hormones, plays a role in oocyte maturation, fertilization, and early embryonic development in mammals. In order to investigate the role of PRL on in vitro oocyte maturation from early follicular growth stages, as well as on fertilization and early embryonic development, we cultured preantral mouse follicles with and without PRL, followed by fertilization of the in vitro matured oocytes. Prolactin significantly improved the rate of oocyte maturation, fertilization, and early embryo development. Four isoforms of PRL-Receptor (R) have been found in whole ovaries of mice: one long (PRL-RL) and three short (-RS(1), -RS(2), and -RS(3)). We examined expression of the four PRL-R isoforms in preantral follicles, in cumulus-oocyte complexes (COCs) and in germinal vesicle GV stage oocytes by RT-PCR. Prolactin-RL, -RS(2) and -RS(3) mRNA, but not -RS(1), were expressed in preantral follicles, COCs, and GV stage oocytes. Our results indicate the prolactin pathway is functional in early preantral follicles, in COCs and in GV stage oocytes, and promotes oocyte maturation, meiosis, fertilization, and early embryonic development.

Detection of circulating fetal cells utilizing automated microscopy: potential for noninvasive prenatal diagnosis of chromosomal aneuploidies.

OBJECTIVE: As fetal cells can be indisputably identified through detection of Y FISH signals, we utilized an automated microscopy system developed to identify and enumerate cells bearing X and Y FISH signals. We further investigated the potential of fetal hemoglobin expression as a gender independent marker for automated identification of fetal cells. METHOD: For FISH-based scanning, verified fetal cells were identified based on the presence of a single X-signal and individual signals for each of the two Y FISH probes. For cell identification based on fetal hemoglobin expression, putative fetal cells were verified based on the presence of signals for anti-gamma or anti-epsilon globin antibody, and FISH signals for the X- and Y- chromosomes. RESULTS: Fetal cells were identified, by FISH-based scanning, in 28 of the 29 maternal samples from pregnancies with male fetuses. Simple density gradient centrifugation achieved a 3- to 5-fold increase in the number of fetal cells detected. CONCLUSION: Automated microscopy identified fetal cells in both first and second trimester maternal blood samples. Although we were unable to detect fetal erythroblasts in numbers sufficient for clinical diagnosis, the ability to reliably detect fetal cells by FISH-based scanning opens the possibility for prenatal detection of chromosomal aberrations utilizing circulating fetal cells.

FSH receptor gene polymorphisms have a role for different ovarian response to stimulation in patients entering IVF/ICSI-ET programs.

PURPOSE: To examine the frequency distribution of the Ser680Asn polymorphism of the follicle-stimulating hormone receptor (FSHR) gene in ovarian dysfunction (OD) infertile women, "poor responders" (PR) and "good responders" (GR). METHODS: The hormonal profiles and treatment of all patients were analyzed and FSHR polymorphism was examined by PCR and RFLP. Women from all groups were classified as Asn/Asn, Asn/Ser, and Ser/Ser genotypes. RESULTS: The frequency distribution of Ser/Ser, Asn/Ser and Asn/Asn variants in OD patients was 45.5, 22.7, and 31.8%, respectively. Day 3 FSH levels in OD and GR patients were higher in Ser/Ser and Asn/Asn subgroups. Asn/Ser carriers from OD and GR groups provided more follicles and oocytes compared to other allelic variants. CONCLUSIONS: GR patients carry more often the Asn/Ser genotype. The latter is correlated with more follicles and oocytes in both OD and GR patients. The Ser/Ser variant might be related to higher serum FSH levels, while the Asn/Ser with lower.

Prolactin receptor mRNA expression in oocytes and preimplantation mouse embryos.

Prolactin was first identified as an anterior pituitary lobe hormone, responsible for the regulation of mammary gland growth and development. Prolactin receptors have been localized in a number of peripheral tissues, including tissues involved in reproduction. Studies with knockout animals have shown that prolactin receptor deficient mice present reproductive defects, whereas prolactin promotes the developmental potential of preimplantation mouse and rat embryos in vitro. To better understand the role of prolactin in the process of reproduction and early embryo development in mice, the expression of the four transcript variants of prolactin receptor was examined in the first stages of mouse embryo development. Prolactin long receptor mRNA was expressed in all stages examined, that is in cumulus cells, oocytes, zygotes, 2-cell embryos, 4-cell embryos, morulae and blastocysts. Prolactin receptor type S1 mRNA was observed only in cumulus cells, while S2 mRNA was present in cumulus cells, oocytes, zygotes and 2-cell embryos. S3 mRNA was expressed only in cumulus cells and oocytes. These results indicate that different isoforms of prolactin receptors may be present in the various stages of mouse preimplantation embryo and may play an important role in the control of its growth and development.

Automated detection of rare fetal cells in maternal blood: eliminating the false-positive XY signals in XX pregnancies.

OBJECTIVE: The purpose of this study was to develop a new method to help differentiate XX from XY signals in maternal blood from women carrying XY fetuses. STUDY DESIGN: We have developed a system to scan automatically for cells that bear X and Y fluorescence in situ hybridization signals. These XY target cells are identified by scans at low (x20) magnification, and all identified targets are revisited and verified at high (x100) magnification. The viewer software component of the system displays x20 images of all cells and intracellular fluorescence in situ hybridization signals that are present in each of the 4000 optical fields per slide, along with x100 images of automatically detected target cells. RESULTS: We initially examined 36,000 fields from 18 slides in 12 pregnancies (6 male and 6 female) using our system that is based on fluorescence in situ hybridization with a single probe for the X-chromosome and a single probe for the Y-chromosome and found XY nuclei in all samples, regardless of fetal gender. In the second phase of the study, a refinement of the approach that incorporated 2 independent probes for the Y-chromosome resulted in a false-positive rate for detection of XY nuclei in XX cases <0.00005%. CONCLUSION: Our data suggest that this system may allow for excellent "signal to noise" separation, which is required absolutely for fetal cell methods to differentiate aneuploid from normal pregnancies. Quantitation of fetal cells in the maternal circulation and standardization of processes that have been developed for their enrichment are crucial to moving fetal cell assessment from esoteric basic science to applied new technology.