Suman Lee

MTHFR C677T polymorphism associates with unexplained infertile male factors

Purpose: To determine whether 5,10-methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) genotype is associated with male infertility.Methods: Analysis of cytogenetic, Y chromosomal microdeletion assay (Yq), and the C677T and A1298C polymorphisms of the MTHFR gene by pyrosequencing and PCR-Restriction Fragment Length Polymorphism (RFLP) method. SAS 8.1 assessed the statistical risk of MTHFR genotype.Results: The homozygous (T/T) C677T polymorphism of the MTHFR gene was present at a statistically high significance in unexplained infertile men with normal karyotype, instead at no significance in explained infertile men with chromosomal abnormality or Y chromosome deletion. There was no statistically significance of A1298C variation in infertile males.Conclusions: The MTHFR 677TT genotype may be a genetic risk factor for male infertility, especially with severe OAT and non-obstructive azoospermia in unexplained infertile males.

Identification of new proteins in follicular fluid from mature human follicles by direct sample rehydration method of two-dimensional polyacrylamide gel electrophoresis.

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.

The Sulfated Polysaccharide Fucoidan Stimulates Osteogenic Differentiation of Human Adipose-Derived Stem Cells

Human adipose-derived stem cells (hADSCs) are an attractive source for cell therapies, because they can be obtained from aspirated adipose tissues with the capacity of proliferation and differentiation into several mesenchymal lineages under certain conditions. Sulfated polysaccharides, including heparin, modulate osteogenic differentiation of stem cells through the regulation of growth factor binding and signaling pathways. Here, we examined the effects of the sulfated polysaccharide fucoidan on osteogenic differentiation of hADSCs. Strikingly, fucoidan treatment resulted in increased alkaline phosphatase (ALP) activity and alizarin red and von Kossa staining. At the molecular level, fucoidan treatment enhanced the expression of osteogenesis-specific marker genes, including ALP, osteopontin, type I collagen, Runt-related transcription factor 2, and osteocalcin. Furthermore, fucoidan also promoted the osteogenic differentiation of another mesenchymal cell lineage, human amniotic fluid stem cells. These findings strongly suggest that fucoidan enhances osteogenic differentiation of hADSCs and possibly other mesenchymal cell lineages, indicating that it may be a potential candidate for promoting bone regeneration.

Gene Expression Analysis of Cultured Amniotic Fluid Cell with Down Syndrome by DNA Microarray

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.

Molecular and cytogenetic characterization of two azoospermic patients with X-autosome translocation

AbstractPurpose: To report two azoospermic patients with reciprocal X–autosome translocations. Methods: Cytogenetic analysis utilizing GTG-banding and Yq microdeletions shown by polymerase chain reaction (PCR) with 12 sequence-tagged site (STS) markers for Y chromosome microdeletions. Results: Cytogenetic analysis showed one man with 46,Y,t(X;19)(q22;q13.3) and the other with 46,Y,t(X;8)(p22;q11). Neither had any Yq microdeletions shown. The patient with 46,Y, t(X;8)(p22;q11) showed a slightly lower than normal testosterone level. By NCBI-Blast search, we found four testis-specific genes, t-complex-associated-testis-expressed 1-like (TCTE1L), Ferritin, heavy polypeptide-like 17 (FTHL17), Testis expressed sequence 13A (TEX13A), and Testis expressed sequence 13B (TEX13B) located near breakpoints on X chromosome. FTHL17, TEX13A, and TEX13B are spermatogonially-expressed, germ-cell-specific genes. Conclusion: This is the first clinical report of azoospermia with reciprocal X–autosome translocations on Xp22 and q22. These translocations on Xp22 and q22 may be direct genetic risk factors for azoospermia.

Functional polymorphism in H2BFWT‐5′UTR is associated with susceptibility to male infertility

H2B histone family, member W, testis‐specific (H2BFWT) gene encodes a testis‐specific histone that becomes incorporated into sperm chromatin. A male infertility‐associated single nucleotide polymorphism (−9C > T) within the 5′ untranslated region (5′UTR) of the H2BFWT gene was identified by direct sequencing. Statistical association studies showed the polymorphism significantly associated with male infertility (n= 442, P= 0.0157), especially in non‐azoospermia (n= 262, P= 0.018). Furthermore, this polymorphism is also associated with sperm parameters, especially sperm count (n= 164, P= 0.0127) and vitality (n= 164, P= 0.0076). We investigated how the genetic variant at 5′UTR confers susceptibility to non‐azoospermia. Western blotting of His‐tag H2BFWT revealed a difference at the translational level between ‐9T and the wild‐type −9C in the absence of change at the transcriptional level. Reporter assays showed that this reducing translational change originated from an upstream open reading frame (uORF) generated by the −9C to −9T change. Finally, in vivo H2BFWT expression in sperm was significantly dependent on the −9C > T genotype from non‐azoospermia (P= 0.0061). Therefore, this polymorphism could affect the translational efficiency of a quantitatively important histone protein by the uORF. Our data implicate H2BFWT as a susceptibility factor for male infertility, possibly with other genetic and environmental factors.

The association of 4a4b polymorphism of endothelial nitric oxide synthase (eNOS) gene with the sperm morphology in Korean infertile men

OBJECTIVE To investigate the association between three polymorphism sites of endothelial nitric oxide synthase (eNOS; -786T>C, 4a4b, and 894G>T) with nonobstructive male infertility. DESIGN Prospective case-control study. SETTING University-based hospital. PATIENT(S) Three hundred seventy-one nonobstructive infertile men in azoospermia (n = 184) or ejaculate semen (n = 187) group were enrolled in this study. Two hundred twenty fertile men who had at least one child without any history of requiring assisted reproductive technology were included as the nationwide control group. INTERVENTION(S) Semen analysis according to the World Health Organization guidelines and cytogenetic and Y chromosome microdeletion assay. MAIN OUTCOME MEASURE(S) Three eNOS polymorphisms were investigated to assess its association with male infertility by pyrosequencing and gel electrophoresis. RESULT(S) The statistical analysis of three eNOS polymorphisms showed no significant association between the polymorphisms of the control and infertile group. We investigated the sperm parameters depending on the genotypes of the ejaculate semen group. The sperm morphology was found to be significantly associated with the 4a4b polymorphism of eNOS. CONCLUSION(S) Endothelial nitric oxide synthase may be important to sperm morphology in infertile men.

Genetic analysis of three polymorphic sites of the luteinizing hormone β-subunit gene in infertile Korean men with nonobstructive azoospermia

OBJECTIVE To investigate the genetic background of nonobstructive male factor infertility. DESIGN Clinical and controlled study. PATIENT(S) Ninety-five nonobstructive male infertile patients (75 with azoospermia, 18 with oligoasthenoteratozoospermia, and two with oligozoospermia) and 200 healthy fertile control men. INTERVENTION(S) Patients were investigated for genetic background including karyotype, Yq chromosome deletion, and three polymorphisms of the LH beta-subunit gene (Trp8Arg, Ile15Thr, and Gly102Ser). MAIN OUTCOME MEASURE(S) To determine three polymorphisms of the LH beta-subunit gene. RESULT(S) An abnormal karyotype was found in 11 of the 75 patients with azoospermia and one of the 18 patients with oligoasthenoteratozoospermia. Eleven (12%) had one or more deleted sites at 13 loci on Yq. The Gly102Ser variant of the LH beta-subunit gene was not detected at all. The frequency of double Trp8Arg and Ile15Thr heterozygotes was similar between the fertile (14.5%, n = 200) and infertile (12.6%, n = 95) groups, with the exception of one homozygous mutation (Arg8 and Thr15) from patient with azoospermia. CONCLUSION(S) Three variants of the LH beta-subunit gene (Trp8Arg, Ile15Thr, and Gly102Ser) may not be associated with male factor infertility. We found one homozygous Arg8 and Thr15 mutation in a patient with azoospermia with normal hormone levels (FSH, LH, PRL, T), a normal karyotype, and no Yq microdeletions.

Comparison of motor-evoked potentials monitoring in response to transcranial electrical stimulation in subjects undergoing neurosurgery with partial vs no neuromuscular block†

BACKGROUND There have been no evidence-based comparisons of motor-evoked potential (MEP) monitoring with no and partial neuromuscular block (NMB). We compared the effects of different levels of NMB including no NMB on MEP parameters. METHODS MEP-monitored 120 patients undergoing neurosurgery were enrolled. The patients were randomly allocated to four groups: Group A was to maintain two train-of-four (TOF) counts; Group B was to maintain a T(1)/Tc of 0.5; Group C was to maintain a T(2)/Tc of 0.5 (T(1,2), first or second twitch height of TOF; Tc, control twitch height); Group D did not maintain NMB. The mean MEP amplitude, coefficient of variation (CV), the incidence of spontaneous respiration or movement, the efficacy of MEP, and haemodynamic parameters were compared. RESULTS The median [inter-quartile range (IQR)] amplitudes of the left leg for Groups A, B, C, and D were 0.23 (0.15-0.57), 0.44 (0.19-0.79), 0.28 (0.15-0.75), and 0.75 (0.39-1.35) mV, respectively. The median (IQR) CVs of the left leg were 71.1 (56.9-88.8), 76.1 (54.2-93.1), 59.8 (48.6-95.6), and 25.2 (17.3-35.0), respectively. The differences between groups of the mean amplitudes of the left arm and both legs were statistically significant (Kruskal-Wallis test, P=0.011 for the left leg). For all limbs, the differences between groups of the CVs were significant (P<0.001, for the left leg). Other parameters were not different. CONCLUSIONS If NMB is used during MEP monitoring, a target T(2)/Tc of 0.5 is recommended. In terms of the MEP amplitude and variability, no NMB was more desirable than any level of partial NMB.

Association between folate metabolism-related gene polymorphisms and methylation of p16INK4A and hMLH1 genes in spontaneously aborted embryos with normal chromosomal integrity

OBJECTIVE To assess prevalent polymorphisms of 5,10-methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase enhancer region (TSER) and methylation patterns of p16(INK4A) and hMLH1 genes in spontaneously aborted embryos (SAEs) with normal chromosomal integrity. DESIGN Retrospectively analyzed, prospectively obtained database. SETTING Bundang CHA General Hospital in South Korea. PATIENT(S) Fifty-nine SAEs (<20 wk of gestational age) with normal chromosomal integrity. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Genotype frequency of MTHFR, TSER polymorphisms, and methylation status of p16(INK4A) and hMLH1 genes in SAEs with normal chromosomal integrity. RESULT(S) The distribution of the MTHFR 677C>T polymorphism differed significantly between SAEs with normal chromosomal integrity and the two control groups. Also, the frequency of combined MTHFR 677 and TSER genotypes was significantly different between the aborted embryos and the adult control group. However, the MTHFR 677C>T and 1298A>C and TSER polymorphisms were not associated with the methylation status of p16(INK4A) and hMLH1 genes in SAEs with normal chromosomal integrity. CONCLUSION(S) Association between the MTHFR 677C>T polymorphism and the risk of SAEs with normal chromosomal integrity in the Korean population.