OBJECTIVE: To investigate a kallikrein-related peptidase 2 (KLK2) single nucleotide polymorphism (SNP) in relation to male infertility because of its role in semen processing. We investigated the genetic association of the KLK2+255G>A genotype with male infertility. METHODS: We genotyped the SNP site located in intron 1 (+255G>A, rs2664155) of KLK2 from 218 men with male infertility (cases) and 220 fertile males (controls). Pyrosequencing analysis was performed for the genotyping. RESULTS: The SNP of the KLK2 gene had a statistically significant association with male infertility (p<0.05). The odds ratio for the minor allele (+255A) in the pooled sample was 0.47 (95% confidence intervals, 0.26-0.85) for rs2664155. CONCLUSION: The relationship of KLK2 SNP to male infertility is statistically significant, especially within the non-azoospermia group. Further study is needed to understand the mechanisms associated with male infertility.
Alleles ; Genetic Association Studies ; Genotype ; Humans ; Infertility, Male ; Introns ; Male ; Odds Ratio ; Polymorphism, Single Nucleotide ; Semen

PURPOSE: This study aims to establish a new strategy that provides for the rapid establishment of human clonal adipose derived stem cell (hADSC) lines with aspirated adipose tissue and to characterize newly generated hMSC lines for their cell phenotype, differentiation potential, lineage-specific gene expression. METHODS: Human adipose tissue-derived stem cells (hADSCs) were isolated from subcutaneous adipose tissue based on standard protocols. After incubation for 2 h, only the cell culture supernatant was transferred to a new dish. This process was repeated several times with 30 h incubations. RESULTS: We confirmed the difference in growth rate, however, differences were not seen in the differentiation capabilities and stemness of the each cell lines. CONCLUSION: It is necessary to establish cell lines via single cell level for application to disease specific tissue engineering.
Adipose Tissue ; Cell Culture Techniques ; Cell Line ; Humans ; Phenotype ; Stem Cells ; Subcutaneous Fat ; Tissue Engineering

The H1 histone family, member N, testis-specific (H1FNT) is exclusively expressed in the testis, and had its possible role for sperm chromatin formation. The purpose of this study is to investigate any genetic association of H1FNT gene with male infertility, especially at the promoter region. We examined the promoter single nucleotide polymorphisms (SNP) of H1FNT gene which is located within transcription factor binding site for its association with male infertility. The statistical analysis showed that the -1129A>T polymorphism was present at a statistically significance in male infertility (p=0.0059 and 0.0349 for hetero and risk type, respectively). The dual-luciferase promoter assay was performed to examine the polymorphic effect of this promoter SNP by the cloning of promoter region (1700bp fragment) into pGL3-basic vector. In our plasmid based reporter system, there is no big difference between wild and risk type. In conclusion, H1FNT -1129A>T promoter SNP is statistically significant with male infertility, especially with subfertile (non-azoospermia) group. Further analysis of its functional polymorphic effect in vivo may provide the biological significance of testis-specific histone with spermatogenesis.
Binding Sites ; Chromatin ; Clone Cells ; Cloning, Organism ; Histones ; Humans ; Infertility, Male ; Male ; Plasmids ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; Spermatogenesis ; Spermatozoa ; Testis ; Transcription Factors

Human follicular fluid (HFF) includes various biologically active proteins which can affect follicle growth and oocyte fertilization. Thus far, these proteins from mature follicles in human follicular fluid have been poorly characterized. Here, two-dimensional polyacrylamide gel electrophoresis (2-DE) with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was used to identify new proteins in HFF. Mature follicular fluids were obtained from five females after oocyte collection during in vitro fertilization (IVF). We directly rehydrated HFF samples, obtained high-resolution 2-DE maps, and processed them for 2-DE and MALDI-MS. One hundred eighty spots were detected and 10 of these spots were identified. By the 2-DE database, six of them had been reported, as proteins already existing in HFF. Hormone sensitive lipase (HSL), Unnamed protein product 1 (UPP1), Unnamed protein product 2 (UPP2), and apolipoprotein A-IV precursor were newly detected. HSL and apolipoprotein A-IV participate in lipid metabolism. UPP1 has a homology with selenocysteine lyase. We found by RT-PCR that these genes are expressed from human primary granulosa cells. The proteins identified here may emerge as potential candidates for specific functions during folliculogenesis, hormone secretion regulation, or oocyte maturation. Further functional analysis of these proteins is necessitated to determine their biological implications.
Adult ; Electrophoresis, Gel, Two-Dimensional/*methods ; Female ; Follicular Fluid/*chemistry/metabolism ; Gene Expression ; Granulosa Cells/metabolism ; Humans ; Ovarian Follicle/*chemistry/metabolism ; Proteins/*analysis/genetics ; RNA, Messenger/genetics/metabolism ; Research Support, Non-U.S. Gov't ; Reverse Transcriptase Polymerase Chain Reaction ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Complete or partial triplication of human chromosome 21 results in Down syndrome (DS). To analyze differential gene expressions in amniotic fluid (AF) cells of DS, we used a DNA microarray system to analyze 102 genes, which included 24 genes on chromosome 21, 28 genes related to the function of brain and muscle, 36 genes related to apoptosis, 4 genes related to extracellular matrix, 8 genes related to other molecular function and 2 house-keeping genes. AF cells were collected from 12 pregnancies at 16-18 weeks of gestation in DS (n=6) and normal (n=6) subjects. Our DNA microarray experiments showed that the expressions of 11 genes were altered by at least 2-folds in DS, as follows. Ten genes, COL6A1, CASP5, AKT2, JUN, PYGM, BNIP1, OSF-2, PRSS7, COL3A1, and MBLL were down-regulated and GSTT1 was only up-regulated. The differential expressions of GSTT1 and COL3A1 were further confirmed by semi-quantitative RT-PCR for each sample. The gene dosage hypothesis on chromosome 21 may explain the neurological and other symptoms of DS. However, our results showed that only two genes (COL6A1 and PRSS7), among 24 genes on chromosome 21, were down-regulated in the AF cells of DS. Our data may provide the basis for a more systematic identification of biological markers of fetal DS, thus leading to an improved understanding of pathogenesis for fetal DS.
Amniocentesis ; Amniotic Fluid/*cytology/*metabolism ; Apoptosis ; Cells, Cultured ; Chromosomes, Human, Pair 21 ; Collagen Type III/biosynthesis ; DNA, Complementary/metabolism ; Down Syndrome/*genetics/metabolism ; Down-Regulation ; Gene Dosage ; Gene Expression ; *Gene Expression Regulation ; Glutathione Transferase/biosynthesis ; Humans ; Models, Genetic ; *Oligonucleotide Array Sequence Analysis ; Research Support, Non-U.S. Gov't ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; Up-Regulation

To investigate the XIST gene expression and its effect in a Klinefelter''s patient, we used Klinefelter''s syndrome (XXY) patient with azoospermia and also used a normal male (XY) and a normal female (XX) as the control, We were performed cytogenetic analysis, Y chromosomal microdeletion assay (Yq), semi-quantitative RT-PCR, and the Northern blot for Klinefelter''s syndrome (KS) patient, a female and a male control, We extracted total RNA from the KS patient, and from the normal cells of the female and male control subjects using the RNA prep kit (Qiagen), cDNA microarray contained 218 human X chromosome-specific genes was fabricated. Each total RNA was reverse transcribed to the first strand cDNA and was labeled with Cy-3 and Cy-5 fluorescein, The microarray was scanned by ScanArray 4000XL system. XIST transcripts were detected from the Klinefelters patient and the female by RT-PCR and Northern blot analysis, but not from the normal male, In the cDNA microarray experiment, we found 24 genes and 14 genes are highly expressed in KS more than the normal male and females, respectively. We concluded that highly expressed genes in KS may be a resulted of the abnormal X inactivation mechanism.
Azoospermia ; Blotting, Northern ; Cytogenetic Analysis ; DNA, Complementary* ; Female ; Fluorescein ; Gene Expression ; Humans ; Klinefelter Syndrome* ; Male ; Oligonucleotide Array Sequence Analysis* ; RNA ; X Chromosome Inactivation* ; X Chromosome*

OBJECTIVE: To investigate the association of genetic background between MTHFR A1298C genotype and male infertility. MATERIALS AND METHODS: We compared 377 infertile males with 396 healthy fertile males with one or more offspring. Infertile males were classified into four subtypes (281 azoospermia, 26 oligoasthenoteratozoospermia (OAT), 59 severe OAT and 11 remnants) by World Health Organization (WHO). Pyrosequencing analysis for MTHFR (methylenetetrahydrofolatereductase) A1298C variation was performed on polymerase chain reaction (PCR) product of study group. To validate pyrosequencing data of A1298C variation for randomly selected 50 samples, we compared the pyrosequencing result with the PCR-RFLP (Restriction Fragment Length Polymorphism) result of MTHFR A1298C genotype. RESULTS: We studied MTHFR A1298C variation by pyrosequencing. A1298C variation data (1298 AC; p=0.2166 and 1298 CC; p=0.5056) of MTHFR gene was no significant difference in between fertile and infertile males. CONCLUSION: The genetic analysis in MTHFR gene didn't appear genetic difference in Korean fertile and infertile males. We require further study for MTHFR gene in infertile males.
Avena ; Azoospermia ; Genotype ; Humans ; Infertility, Male ; Korea* ; Male* ; Oxidoreductases* ; Polymerase Chain Reaction ; World Health Organization

OBJECTIVES: Methylenetetrahydrofolate reductase (MTHFR) mutation are commonly associated with hyperhomocysteinemia, and through their defects in homocysteine metabolism, they have been implicated as a risk factor for recurrent spontaneous abortion. Recent report describe that 28-bp tandem repeat polymorphism in thymidylate synthase enhancer region (TSER) that influence enzyme activity would affect plasma homocysteine level. We have investigated the relationship between TSER genotype and plasma homocysteine level in 54 patients with recurrent spontaneous abortion. METHODS: Plasma homocysteine level was measured by fluorescent polarizing immunoassay. MTHFR mutation (C677T and A1298C) was identified by PCR-restriction fragment length polymorphism assay and TSER mutation was analyzed by PCR method. The data were analyzed using the program SAS 8.2 for Windows. RESULTS: Total homocysteine level was significantly higher in MTHFR 677TT genotype (9.80+/-3.87 mumol/L) than MTHFR 677CC genotype (8.14+/-1.74 mumol/L) in Korean patients with unexplained recurrent spontaneous abortion (p=0.0143). However, the plasma homocysteine level was not significantly different in the MTHFR 1298AA (8.42+/-2.65 mumol/L) and 1298CC (6.09+/-0.32 mumol/L; p=0.2058) and, TSER 2R2R (8.61+/-1.68 mumol/L) and 3R3R (8.05+/-2.81 mumol/L; p=0.9319) mutant genotypes, respectively. In this study, we found the combination effects of TSER and MTHFR C677T genotypes. Plasma homocysteine levels were the highest (11.47+/-4.66 mumol/L) in individuals with TSER 3R3R (8.05+/-2.81 mumol/L) and MTHFR 677TT (9.80+/-3.87 mumol/L) genotypes. Individuals with a combination of both TSER 2R2R/2R3R and MTHFR 677CC/CT genotypes (7.69+/-1.77 mumol/L) had lower plasma homocysteine levels than TSER 2R2R (8.61+/-1.68 mumol/L) and MTHR 677CC (8.14+/-1.74 mumol/L) genotypes, respectively. The effect of MTHFR polymorphism in the homocysteine metabolism appears to be stronger than that of TSER polymorphism. CONCLUSION: Although statistically not significant, we found the elevated level of plasma homocysteine in combined genotypes with TSER and MTHFR (C677T and A1298C) in Korean patients with unexplained habitual abortion. In this study, we reported the possibility that TSER polymorphism is a genetic determinant of plasma homocysteine levels in the Korean patients as well as MTHFR C677T polymorphism. A large prospective study is needed to verify our findings.
Abortion, Habitual ; Abortion, Spontaneous* ; Female ; Genotype ; Homocysteine* ; Humans ; Hyperhomocysteinemia ; Immunoassay ; Metabolism ; Methylenetetrahydrofolate Reductase (NADPH2) ; Plasma* ; Polymerase Chain Reaction ; Pregnancy ; Risk Factors ; Tandem Repeat Sequences ; Thymidylate Synthase*

OBJECTIVE: Previous studies have suggested that hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR C677T) mutations are associated with increased risk of recurrent spontaneous abortion (RSA). Recently, a second site polymorphism in MTHFR, 1298A-->C, which changes a glutamic acid into an alanine residue, was shown to be associated with a decreased enzyme activity. We tested whether the variant alleles of MTHFR C677T and A1298C are risk factor (biomarker) for RSA. MATERIALS AND METHODS: We analyzed DNA from a case-control study in the Korean DNA was extracted from blood samples of 118 patients with RSA and 123 healthy fertile patients as the controls. MTHFR variant alleles were determined by a PCR-restriction fragment length polymorphism assay. RESULTS: We found no evidence for an association between 677TT genotype and risk of RSA (OR=1.95, 95% CI=0.84~4.50, p=0.12). However, the MTHFR 1298AC (OR=0.36, 95% CI=0.20~ 0.63, p=0.0004) and 1298AC+CC (OR=0.35, 95% CI=0.20~0.61, p=0.0002) genotypes were lower among 118 RSA cases compared with 123 controls, conferring a 2.8-fold decrease in risk of RSA, respectively. Moreover, the combined genotypes of MTHFR 677CC/1298AC (OR=0.30, 95% CI= 0.10~0.88, p=0.029) and 677CT/1298AC (OR=0.77, 95% CI=0.60~0.99, p=0.043) also showed significantly lower risk than those with MTHFR 677CC/1298AA type. CONCLUSION: MTHFR 1298AC, MTHFR 677CC/1298AC and 677CT/1298AC genotypes may represent genetic markers for the protection of RSA at least in Korean women.
Abortion, Spontaneous* ; Alanine ; Alleles ; Case-Control Studies ; DNA ; Female ; Genetic Markers ; Genotype ; Glutamic Acid ; Humans ; Hyperhomocysteinemia ; Methylenetetrahydrofolate Reductase (NADPH2) ; Oxidoreductases* ; Pregnancy ; Risk Factors ; Vascular Diseases

Trinucleotide repeat (TNR) instability can cause a variety of human genetic diseases including myotonic dystrophy and Huntington's disease. Recent genetic data show that instability of the CAG/CTG repeat DNA is dependent on its length and replication origin. In yeast, the RAD27 (human FEN-1 homologue) null mutant has a high expansion frequency at the TNR loci. We demonstrate here that FEN-1 processes the 5'-flap DNA of CTG/CAG repeats, which is dependent on the length in vitro. FEN-1 protein can cleave the 5'-flap DNA containing triplet repeating sequence up to 21 repeats, but the activity decreases with increasing size of flap above 11 repeats. In addition, FEN-1 processing of 5'-flap DNA depends on sequence, which play a role in the replication origin-dependent TNR instability. Interestingly, FEN-1 can cleave the 5'-flap DNA of CTG repeats better than CAG repeats possibly through the flap-structure. Our biochemical data of FEN-1's activity with triplet repeat DNA clearly shows length dependence, and aids our understanding on the mechanism of TNR instability.
Base Sequence ; DNA, Single-Stranded/*metabolism ; Endodeoxyribonucleases/genetics/*metabolism ; Flap Endonucleases ; Gene Expression Regulation ; Genetic Diseases, Inborn/*genetics ; Human ; Nucleic Acid Conformation ; Trinucleotide Repeat Expansion ; *Trinucleotide Repeats