PubMed Health⌕ Search

Biomedical subjects

David S Cram

Publications and source records attributed to David S Cram.

5 recordsLinked to original sources
PubMed2007

Optimization of a microarray based approach for deriving representative gene expression profiles from human oocytes.

The purpose of the present study was to optimize a protocol for deriving reproducible and representative gene expression profiles from very rare research samples of human oocytes using microarrays. Immature oocytes produced as a result of administration of gonadotrophins for the treatment of infertility were donated to research. Linear amplification (L-amp) and exponential amplification (E-amp) were both capable of generating sufficient product for hybridization to the microarrays even from the low amount of template mRNA present in a single human oocyte. Slightly higher numbers of transcripts were detected by microarray following linear rather than E-amp but both techniques generated a product with reliably reproducible sensitivity and fidelity providing oocytes were pooled in minimum numbers of three. The majority of the variance associated with amplification and hybridization to arrays comes from the molecular processing. Slightly greater additional variance is associated with biological differences in immature oocytes from the same or different donors. The findings suggest that representative gene expression profiles can be generated from human oocytes for comparative purposes following L-amp and hybridization to microarray. However, oocytes must be pooled for the starting template for each array and sufficient independent microarray experiments performed to minimize the variance associated with molecular processing.

Female↗
PubMed2005

Chromosome 21 mosaic human preimplantation embryos predominantly arise from diploid conceptions.

OBJECTIVE: High rates of chromosomal mosaicism in human IVF embryos question the accuracy of preimplantation genetic diagnosis, and, with the majority of embryo transfers still resulting in no pregnancy, chromosomal mosaicism is likely to be a contributing factor to human IVF failure. The aim of this study was to investigate the origin and nature of chromosome 21 (Ch21) cell division errors in human IVF embryos. DESIGN: Perform single cell Ch21 allelic profiling on human IVF embryos. SETTING: Academic research environment. PATIENT(S): Women of advanced maternal age (> 35 yrs) (n = 65) undergoing infertility treatment; and amniocytes/chorionic cells from trisomy 21 pregnancies (n = 28). INTERVENTION(S): Cells were analyzed by single cell allelic profiling, MAIN OUTCOME MEASURE(S): The origin and nature of cell division errors. RESULT(S): The vast majority of Ch21 mosaic embryos (approximately 80%) originated from diploid conceptions. In contrast, all fetal trisomy 21 originated from aneuploid conceptions. Increasing maternal age was significantly associated with aneuploid conceptions, meiotic cell division error, and adverse pregnancy outcome (P < .05). The mean daily FSH dose that produced embryos with normal Ch21 cell division was significantly lower than the mean daily FSH dose that produced embryos with mitotic Ch21 cell division errors (P < .01) and embryos with meiotic cell division errors (P < .05). CONCLUSION(S): Chromosomal mosaicism of Ch21 in human IVF embryos predominantly originate from diploid conceptions. Further understanding of chromosomal mosaicism with respect to IVF parameters, such as daily FSH dose, may eventually lead to improvements in IVF outcomes.

Adult↗
PubMed2005

DNA identification of fetal cells isolated from cervical mucus: potential for early non-invasive prenatal diagnosis.

OBJECTIVES: To develop a reliable method to isolate fetal cells for genetic diagnosis. DESIGN: Aspiration of cervical mucus from pregnant women in the first trimester. SETTING: Pregnant women were recruited before an elective termination of pregnancy. POPULATION: Sixty pregnant women (7-10 weeks of gestation). METHODS: Fetal cells were isolated from aspirated cervical mucus of pregnant women using a combination of enzymatic digestion, fluorescent immunohistochemistry, micromanipulation and single-cell DNA allelic profiling. MAIN OUTCOME MEASURES: The isolation and identification of fetal cells. RESULTS: The transformation of the tenacious cervical mucus into a single-cell suspension enabled the isolation and identification of fetal cells by fluorescent immunohistochemistry. Confirmation of fetal origin was accomplished by single-cell DNA allelic profiling alongside known maternal cells. CONCLUSIONS: This novel non-invasive method is rapid and efficient with results attainable within 24 hours as early as seven weeks of gestation. The technique would offer earlier reassurance and the option of first trimester therapeutic abortions to both high and low risk pregnant women.

Cervix Mucus↗
PubMed2002

Issues and concerns of couples presenting for preimplantation genetic diagnosis (PGD).

BACKGROUND: The use of preimplantation genetic diagnosis (PGD) to select genetically 'normal' human embryos and to transfer them to the uterus of a woman has generated considerable controversy. Debate has occurred over the implications of PGD, sex selection, safety of embryonic manipulation and eugenics. This study evaluates a range of social and moral concerns of couples towards PGD and assisted reproductive technologies (ART) prior to treatment to obtain unbiased authentic attitudes independent of the treatment cycle and the outcome. METHODS: A total of 121 subjects were administered a structured questionnaire after each couple's in vitro fertilization (IVF) or genetic counselling session. Group A consisted of 41 subjects presenting for PGD of single gene disorders (PGD-SG) and group B consisted of 48 subjects undertaking PGD for aneuploidy screening (PGD-AS). A control group consisted of 32 subjects that were about to commence their first IVF cycle. RESULTS AND DISCUSSION: All groups found PGD to be a highly acceptable treatment. They expressed little concern about its extension to testing non-disease states such as sex and they were strongly in favour of a shared decision-making model in which couples have considerable autonomy over decisions about the embryo(s) to transfer. Differences between the groups included issues surrounding the transfer of embryos, restrictions to PGD and the destruction of embryos.

Adult↗