• Journal of Veterinary Clinics
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• v.33 no.5
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• pp.266-269
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• 2016

Cell-free fetal RNA is useful to determine fetal sex and detect other inherent genetic disorders. However, non-invasive fetal sex determination methods using fetal RNA from maternal plasma is not yet well established in studies pertaining to bovine animals. Thus, the aim of this study was to systematically evaluate the presence of the male-specific ZRSR2Y gene transcript in maternal plasma using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assays, and to verify its accuracy, sensitivity, and specificity in determining fetal sex between 30 and 100 days of gestation. Overall accuracy, sensitivity, and specificity of the ZRSR2Y gene transcripts in determining fetal sex were 89.1%, 86.3%, and 100%, respectively. The 30 to 100 days of gestation were further classified into five stages of gestation, and each stage had relatively high accurate, sensitive, and specific results. Overall, these results indicate that the expression of the ZRSR2Y gene can be used for fetal sex determination in bovine animals using circulating cell-free RNA in maternal plasma during early pregnancy.

• Journal of Embryo Transfer
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• v.28 no.4
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• pp.343-348
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• 2013

Cell-free fetal RNA has been highlighted as useful tools for the fetal sex determination or other genetic inherent disorder. However, there is no knowledge about the sex determination using cell free fetal RNA in bovine field. Thus, the present study aimed to evaluate the presence of transcripts of DDX3Y, USP9Y and ZRSR2Y genes in maternal plasma of pregnant cows to determine the sex of the fetus using real-time quantitative polymerase chain reaction assay, and verify its accuracy, sensitivity and specificity compared with the molecular testing and the calf sex at birth. Transcripts of USP9Y and DDX3Y genes were expressed in the all plasma of males and females both the control group and the experimental group. However, ZRSR2Y gene was matched up with the molecular testing and the true sex in control group and has an overall accuracy of 82.6%, a sensitivity of 75%, and a specificity of 100% in experimental group. Therefore, these results indicated that real time PCR technique, as a noninvasive and cost-efficient method, is possible to determination fetal sex in the bovine species using circulating cell free RNA in maternal plasma and especially ZRSR2Y gene could be a good candidate for the RNA based sex determination work.

• Clinical and Experimental Reproductive Medicine
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• v.6 no.1_2
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• pp.23-28
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• 1979

To determine whether hormone analysis of amniotic fluid could be used for accurate determination of fetal sex, we measured testosterone(T) and follicle-stimulating hormone in 19 amniotic fluid samples. The mean T in amniotic fluid of 8 women earring male fetuses was 310 pg. per milliliter and of 11 women earring female fetuses was 150 pg. per milliliter (P<0.05${\ast}$). The mean amniotic fluid FSH of 1.16 mI.U. per milliliter for 7 women with male fetuses was over trifold lower than that for subjects with female fetuses. The mean amniotic fluid FSH of female fetuses was 3.85 mI.U. per milliliter (P<0.01${\ast}$) Measurement of T & FSH in amniotic fluid may be an adjunct method for fetal sex determination.

• The Korean Journal of Nuclear Medicine
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• v.16 no.2
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• pp.37-47
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• 1982

To evaluate fetal sex-hormonal status before delivery, testosterone and follicle stimulating normone(FSH) levels were measured in 64 amniotic fluid samples at midgestation by radioimmunoassay method. The mean concentration of testosterone in amniotic fluid of 37 cases carrying male fetus was 90.7 pg/ml and 27 cases carrying female fetus was 62.3 pg/ml. The mean :amniotic fluid FSH concentration of male fetus was 1.15 mIU/ml and of female fetus was 11.98 mIU/ml. The amniotic fluid testoserone and FSH concentrations had statistical difference between male and female fetuses. The ratio of testosterone over FSH in the amniotic fluid was 231.2 in male, 9.8 in female respectively and very significant difference was noticed. The levels of testosterone/FSH greater than 25 were found over 92% of male fetus and lesser than 25 were found over 92% female fetus. Measurement of testosterone and FSH especially testosterone/FSH ratio in amniotic fluid in midgestation may be an adjunct to other method of fetal sex determination.

• Journal of Genetic Medicine
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• v.8 no.1
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• pp.1-16
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• 2011

Owing to the risk of fetal loss associated with prenatal diagnostic procedures (amniocentesis, chorionic villus sampling), noninvasive prenatal diagnosis (NIPD) is ultimate goal of prenatal diagnosis. The discovery of circulating cell-free fetal DNA (cffDNA) in maternal plasma in 1997 has opened up new probabilities for NIPD by Dr. Lo et al. The last decade has seen great development in NIPD. Fetal sex and fetal RhD status determination by cffDNA analysis is already in clinical use in certain countries. For routine use, this test is limited by the amount of cell-free maternal DNA in blood sample, the lack of universal fetal markers, and appropriate reference materials. To improve the accuracy of detection of fetal specific sequences in maternal plasma, internal positive controls to confirm to presence of fetal DNA should be analyzed. We have developed strategies for noninvasive determination of fetal gender, and fetal RhD genotyping using cffDNA in maternal plasma, using real-time quantitative polymerase chain reaction (RT-PCR) including RASSF1A epigenetic fetal DNA marker (gender-independent) as internal positive controls, which is to be first successful study of this kind in Korea. In our study, accurate detection of fetal gender through gestational age, and fetal RhD genotyping in RhD-negative pregnant women was achieved. In this assay, we show that the assay is sensitive, easy, fast, and reliable. These developments improve the reliability of the applications of circulating fetal DNA when used in clinical practice to manage sex-linked disorders (e.g., hemophilia, Duchenne muscular dystrophy), congenital adrenal hyperplasia (CAH), RhD incompatibility, and the other noninvasive pregnant diagnostic tests on the coming soon. The study was the first successful case in Korea using cffDNA in maternal plasma, which has created a new avenue for clinical applications of NIPD.

• Biomedical Science Letters
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• v.20 no.1
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• pp.43-47
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• 2014

To understand the effect of prenatal stress on sex-specific changes in embryonic and placental growth, a synthetic glucocorticoid (dexamethasone) was administered intraperitoneally at a dosage of 1 mg/kg body weight (BW) (Dex1) or 10 mg/kg BW (Dex10) to pregnant ICR mice at the gestational days 7.5, 8.5 and 9.5 post coitum (p.c.). Embryos and placentas were then harvested at days 11.5 and 18.5 p.c., and their body weight and size were measured following the determination of sex through PCR using Sry specific primers in tail tissues. As a result, female embryos presented reduced fetal body weight and size in Dex1- and Dex10-treated groups than those of control group at the embryonic day 11.5 p.c. Interestingly, the growth seems to be recovered at day 18.5 as there was no difference in growth between control and dexamethasone treated groups. In the case of males, Dex1 induced a decrease in fetal weight in day 11.5 and this pattern was maintained until day 18.5, whereas their growth was not affected by Dex10 treatment. Placental growth showed similar patterns to fetal growth in both sexes but the extent of reduction was not statistically significant in most cases. Placental weights in Dex1- and Dex10-treated group were decreased significantly in male only. The results imply that the effect of prenatal stress is largely sex dependent due to different strategies for growth and survival in a stressful environment.

• Journal of Genetic Medicine
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• v.2 no.1
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• pp.35-39
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• 1998

Duchenne/Becker muscular dystrophy are the major neuromuscular disorders with X-linked recessive inheritance. Preimplantation diagnosis of sex determination has been generally used to avoid male pregnancies with these diseases. However, in order to determine if the embryo is normal, carrier or affected regardless of the sex, there is a need for a combined analysis of specific exon on dystrophin gene as well as sex determination of embryo using the same biopsied blastomere. If the exon deletion is not determinable, further diagnosis of carrier or patient can be performed by haplotype analysis. In this study, we applied the primer extension preamplification (PEP) method, which amplifies the whole genome, in 40 cases of single amniocyte and 40 cases of chorionic villus cell. We analysed haplotypes using two (CA)n dinucleotide polymorphic markers located at the end of 5' and 3' region of the dystrophin gene. Exon 46 of dystrophin gene and DYZ3 on chromosome Y were chosen as a target sequence for coamplification PCR. Upon optimizing the conditions, the amplification rates were 91.25% (73/80) for haplotypes (92.5% in amniocyte, 90% in chorionic villus cell) and 88.75% (71/80) for coamplification (85% in amniocyte, 92.5% in chorionic villus cell). The result of the study indicates that haplotypes analysis and coamplification of dystrophin and Y-specific gene using PEP can be applied to prenatal and preimplantation diagnosis in Duchenne/Becker muscular dystrophy making it possible to determine if the fetus is a carrier or an affected one.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.51-56
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• 1997

Recently, through the development of the primer extension preamplification(PEP) method which amplifies the whole genome, simultaneous multiple DNA analysis has become possible. Whole genome from each single cell can be amplified using 15 base oligonucleotide random primer. The greatest advantage of PEP-PCR is the ability to investigate several loci simultaneously and confirm results by analysing multiple aliquots for each locus. This technique led to the development of preimplantation genetic disease diagnosis using blastomere from early embryo, sperm, polar body and oocyte. In this study, we applied PEP-PCR in 20 cases of single amniocyte and 20 cases of single chorionic villus cell for the clinical application of the prenatal and preimplantational genetic diagnosis. We analysed 7 gene loci simultaneously which are 46, 47 exons related to dystrophin gene, two VNTR (variable number tandem repeat) markers using 5'dysIII, 3'CA related to dystrophin gene and DYZ1, DYZ3, DYS14 regions on chromosome Y. In all the tests, 97.5% of PEP-PCR amplifications with single cells were successful. We obtained 38/40 (95%) accuracy in gender determination through chromosome analysis comparison. Therefore, these results have significant implications for a sperm or oocyte analysis and prenatal or preimplantational genetic diagnosis.