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postgraduate thesis: Isolation of human leukocyte antigen G/cytokeratin 7 positive fetal cells from transcervical samples for potential use in prenatal genetic diagnosis

TitleIsolation of human leukocyte antigen G/cytokeratin 7 positive fetal cells from transcervical samples for potential use in prenatal genetic diagnosis
Authors
Issue Date2015
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Wong, H. V. [王愷曦]. (2015). Isolation of human leukocyte antigen G/cytokeratin 7 positive fetal cells from transcervical samples for potential use in prenatal genetic diagnosis. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5388026
AbstractThere has been an increase in rates of chromosomal abnormalities in newborns as a result of reproductive aging. For the past decades, a lot of effort has been placed on identifying pregnancies at risk of genetic defects. Conventional prenatal genetic diagnosis is achieved by invasive procedures that have been associated with an increased risk of pregnancy loss. This has led the researchers to explore the use of non-/minimally invasive techniques for prenatal diagnosis. Trophoblasts are known to be shed from regressing chorionic villi into the lower uterine pole of pregnant women during the first trimester. These cells are trapped within cervical mucus, which can be retrieved with a cytobrush. By using human leukocyte antigen G (HLA-G) and cytokeratin-7 (CK7) as trophoblast markers, this study aims to investigate the possibility of isolating individual fetal trophoblast from transcervical samples for genetic diagnosis. 195 healthy pregnant women requesting for legal termination of pregnancy (TOP) were recruited in this study. Transcervical cells were collected from them with the use of a cytobrush before TOP. HLA-G+ or CK7+ cells were then isolated by a combination of mucolytic action, fluorescent immunohistochemistry, and micromanipulation. The origin of these cells was subsequently investigated by either fluorescent in situ hybridization (FISH) or allelic profiling by quantitative fluorescent polymerase chain reaction (QF-PCR) based on chromosome 16, chromosome X, amelogenin gene and sex determining region Y (SRY) gene. This study first demonstrated the presence of fetal cells in transcervical samples based on the detection of chromosome Y signal by ordinary PCR. Cells expressing HLA-G and CK7 were also identified among transcervical cells. Immunopositive cells were isolated by micromanipulation under fluorescent microscopy. One isolated cell expressing CK7 was shown to inherit paternal allele at a locus on chromosome 16, suggesting the possible fetal origin of this cell. However, this study was still hampered by a number of technical factors. Further optimization of the protocol is required before transcervical trophoblasts can be retrieved in a reliable manner.
DegreeMaster of Philosophy
SubjectHLA histocompatibility antigens
Fetus - Abnormalities - Diagnosis
Keratin
Prenatal diagnosis
Dept/ProgramObstetrics and Gynaecology
Persistent Identifierhttp://hdl.handle.net/10722/208587

 

DC FieldValueLanguage
dc.contributor.authorWong, Hoi-hei, Vera-
dc.contributor.author王愷曦-
dc.date.accessioned2015-03-13T01:44:03Z-
dc.date.available2015-03-13T01:44:03Z-
dc.date.issued2015-
dc.identifier.citationWong, H. V. [王愷曦]. (2015). Isolation of human leukocyte antigen G/cytokeratin 7 positive fetal cells from transcervical samples for potential use in prenatal genetic diagnosis. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5388026-
dc.identifier.urihttp://hdl.handle.net/10722/208587-
dc.description.abstractThere has been an increase in rates of chromosomal abnormalities in newborns as a result of reproductive aging. For the past decades, a lot of effort has been placed on identifying pregnancies at risk of genetic defects. Conventional prenatal genetic diagnosis is achieved by invasive procedures that have been associated with an increased risk of pregnancy loss. This has led the researchers to explore the use of non-/minimally invasive techniques for prenatal diagnosis. Trophoblasts are known to be shed from regressing chorionic villi into the lower uterine pole of pregnant women during the first trimester. These cells are trapped within cervical mucus, which can be retrieved with a cytobrush. By using human leukocyte antigen G (HLA-G) and cytokeratin-7 (CK7) as trophoblast markers, this study aims to investigate the possibility of isolating individual fetal trophoblast from transcervical samples for genetic diagnosis. 195 healthy pregnant women requesting for legal termination of pregnancy (TOP) were recruited in this study. Transcervical cells were collected from them with the use of a cytobrush before TOP. HLA-G+ or CK7+ cells were then isolated by a combination of mucolytic action, fluorescent immunohistochemistry, and micromanipulation. The origin of these cells was subsequently investigated by either fluorescent in situ hybridization (FISH) or allelic profiling by quantitative fluorescent polymerase chain reaction (QF-PCR) based on chromosome 16, chromosome X, amelogenin gene and sex determining region Y (SRY) gene. This study first demonstrated the presence of fetal cells in transcervical samples based on the detection of chromosome Y signal by ordinary PCR. Cells expressing HLA-G and CK7 were also identified among transcervical cells. Immunopositive cells were isolated by micromanipulation under fluorescent microscopy. One isolated cell expressing CK7 was shown to inherit paternal allele at a locus on chromosome 16, suggesting the possible fetal origin of this cell. However, this study was still hampered by a number of technical factors. Further optimization of the protocol is required before transcervical trophoblasts can be retrieved in a reliable manner.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.subject.lcshHLA histocompatibility antigens-
dc.subject.lcshFetus - Abnormalities - Diagnosis-
dc.subject.lcshKeratin-
dc.subject.lcshPrenatal diagnosis-
dc.titleIsolation of human leukocyte antigen G/cytokeratin 7 positive fetal cells from transcervical samples for potential use in prenatal genetic diagnosis-
dc.typePG_Thesis-
dc.identifier.hkulb5388026-
dc.description.thesisnameMaster of Philosophy-
dc.description.thesislevelMaster-
dc.description.thesisdisciplineObstetrics and Gynaecology-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b5388026-

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