Comparative proteomic analysis of human amniotic fluid supernatants with Down syndrome using mass spectrometry.

Down syndrome (DS) is an abnormality of the 21st chromosome that commonly occurs in children born to advanced age women. Amniotic fluid (AF) is usually collected from such women for prenatal diagnosis. This study analyzed human AF supernatants (AFS) by mass spectrometric (MS) approach to search for candidate biomarkers of DS pregnancy. AFS were collected from advanced age pregnant women at 16-18th weeks of gestation by amniocentesis for cytogenetic analysis. AFS from pregnancies carrying DS (n=4) or chromosomally normal (n=6) fetuses, as revealed by cytogenetic analysis, were then subjected to global protein profiling by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Affinity chromatography was applied prior to LC-ESI-MS/MS to minimize the masking effect of highly abundant albumin and immunoglobulin and thereby, increased the diversity of identified proteins. Hereby, at least 30 AFS proteins were newly identified and 44 AFS proteins were found to be differentially expressed between DS and normal cases. Six of these proteins were unique to DS cases while 11 proteins were unique to chromosomally normal cases. In addition, 19 AFS proteins were down-regulated and 8 were up-regulated in DS cases with varying fold changes. A western blot analysis confirmed the LC-ESI-MS/MS data that combined detection of Apolipoprotein A-II (apo A-II) and alpha-fetoprotein (AFP) could be a potential tool for diagnosing DS cases.

Acyl-CoA synthetase long-chain family member 6 is associated with premature ovarian failure.

To identify genes that are associated with premature ovarian failure, a linkage disequilibrium-based genome-wide association study with dense single nucleotide polymorphisms as genetic markers was performed. The acyl-coenzyme A synthetase long-chain family member 6 (ACSL6) gene on chromosome 5q31 was associated with premature ovarian failure and identified disease-susceptibility haplotypes.

Parathyroid hormone-responsive B1 gene is associated with premature ovarian failure.

BACKGROUND: Premature ovarian failure (POF) is a complex and heterogeneous disorder that is influenced by multiple genetic components. Here, we performed a two-stage association study to identify POF-associated genes. METHODS: A first stage linkage disequilibrium (LD)-based genome-wide association study was performed using 24 pairs of patients with POF and matched controls and a high-throughput BeadChip assay with 109365 single-nucleotide polymorphisms (SNPs) that are scattered throughout the genome in an exon-centric and evenly spaced manner. A region that was shown to be strongly associated with POF was then tested again for POF association in the second stage by using a larger sample size (101 cases and 87 controls) and additional putative causal SNPs. RESULTS: The first stage analysis revealed that many regions were associated with POF, with part of chromosome 7p14 that contains the parathyroid hormone responsive-B1 (PTHB1) gene showing the strongest association. A POF-susceptible haplotype of PTHB1 (ht1, 'GAAAG', P = 0.00034) and a POF-resistant haplotype (ht2, 'TGTGC') were also identified. The association between POF and two PTHB1 SNPs (rs3884597 and rs6944723) and part of ht1 was confirmed in the second stage analysis. The additional SNP, rs11773504, was considered as a putative causal variant causing an amino acid change, Ala to Thr. CONCLUSIONS: We showed for the first time that PTHB1 is strongly associated with POF, and ht1 confers susceptibility to POF. While causative SNPs were not identified, the polymorphism of the non-synonymous SNP rs11773504 and the repeated association of ht1 with POF suggest that PTHB1 may contribute to POF pathogenesis.

A novel single nucleotide polymorphism of INSR gene for polycystic ovary syndrome.

OBJECTIVE: To investigate several single nucleotide polymorphisms (SNPs) in the insulin receptor (INSR) gene that have significant associations with pathogenesis of polycystic ovary syndrome (PCOS) in a Korean population. DESIGN: Case-control study. SETTING: University-based hospital. PATIENT(S): 134 patients with PCOS and 100 healthy women as controls. INTERVENTION(S): All exons of INSR in DNA samples from 100 healthy women and 134 women with PCOS were sequenced and compared. MAIN OUTCOME MEASURE(S): Frequencies of genotypes for several SNPs in INSR gene that were found as specifically expressed SNPs in a Korean population. RESULT(S): Among nine SNPs analyzed in a large population, the genotypic frequencies of eight SNPs were similar, and they had no statistically significant association with PCOS. However, the frequency of a minor allele for one novel SNP, +176477 C>T, was higher in the control group than the patient group. CONCLUSION(S): Among the analyzed SNPs, +176477 C>T, a novel SNP in the INSR gene, was associated with the pathogenesis of PCOS in a Korean population.

Branched chain alpha-keto acid dehydrogenase, E1-beta subunit gene is associated with premature ovarian failure.

Genetic variants of the human branched chain alpha-keto acid dehydrogenase, E1-beta subunit (BCKDHB) gene were identified and they have been associated with premature ovarian failure (POF). Reconstructed haplotype from these variants was also associated with POF.

Association study of four polymorphisms in three folate-related enzyme genes with non-obstructive male infertility.

BACKGROUND: Three typical folate metabolism enzymes-i.e. methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS) and MS reductase (MTRR) in the folate cycle-play a critical role in DNA synthesis and methylation reactions. We evaluated whether polymorphisms of these three enzymes are associated with non-obstructive male infertility. METHOD: Three hundred and sixty patients with non-obstructive infertility and 325 fertile men without any chromosomal abnormalities were included in this study. The single-nucleotide polymorphism (SNP) analysis was performed by pyrosequencing and PCR-restriction fragment length polymorphism (RFLP) analysis RESULTS: The frequencies of MTHFR 677TT and MTRR 66GG genotypes were higher in non-obstructive infertile men compared with those in fertile men. By classifying 360 infertile patients into 174 azoospermia and 186 oligoasthenoteratozoospermia (OAT) subjects, the MTHFR 677TT and MS 2756GG types were significantly associated with the azoospermia group (P = 0.0227 and 0.0063, respectively). The frequency of MTRR 66GG was significant in the OAT group (P = 0.0014 versus fertile males). CONCLUSIONS: By analysis of a large number of subjects and a more specific patient selection, we showed the first genetic evidence that MTHFR C677T, MS A2756G and MTRR A66G genotypes were independently associated with male infertility. Each SNP of the three enzymes may have a different impact on the folate cycle during spermatogenesis.

Single nucleotide polymorphism in exon 17 of the insulin receptor gene is not associated with polycystic ovary syndrome in a Korean population.

OBJECTIVE: To assess the association between the single nucleotide polymorphism of the insulin receptor (INSR) gene and polycystic ovary syndrome (PCOS) in a Korean population. DESIGN: Case-control study. SETTING: University-based hospital. PATIENT(S): One hundred seventy-four patients with PCOS and 93 healthy women as controls. MAIN OUTCOME MEASURE(S): Frequency of three genotypes for single nucleotide polymorphism found in exon 17 of INSR gene. RESULT(S): The high frequency of the T allele was shown both in patient and control groups. The frequency of C allele, which known as a normal allele, was slightly higher in the patient group than in the control group. CONCLUSION(S): The C/T polymorphism in exon 17 of the INSR gene is not associated with susceptibility of PCOS in a Korean population.

Gene expression profiling of the pre-implantation mouse embryo by microarray analysis: comparison of the two-cell stage and two-cell block.

To improve our understanding of the molecular mechanisms underlying early embryo development, further characterization of gene activity in oocytes and embryos is urgently required. The transition from the two-cell to four-cell stage is particularly important in pre-implantation embryonic development, as it involves transcriptional reprogramming and cellular differentiation. In this study, we used a 7.4 K cDNA microarray to screen mRNA transcript levels in the pre-implantation mouse embryo. Real-time PCR was used to confirm microarray data. We profiled 7,410 genes and identified 4,562 genes that were differentially expressed in the pre-implantation embryo. We selected a total of 248 genes with significant expression changes that are functionally involved in the two-cell and two-cell block embryo. Of these genes, 114 were down-regulated and the remainder (n=134) were up-regulated in the two-cell embryo. This study provides a developmental map of a large number of genes in the pre-implantation mouse embryo with particular emphasis on gene expression in the two-cell embryo and two-cell block embryo. Further investigations based on this data will provide a better understanding of the effects of various external conditions and may facilitate comparative analysis of pre-implantation development in other mammalian species, including human.

Proteomic analysis of recurrent spontaneous abortion: Identification of an inadequately expressed set of proteins in human follicular fluid.

Recurrent spontaneous abortion (RSA), defined as the loss of three or more consecutive pregnancies prior to the 20th week of gestation, affects up to 5% of the child-bearing population. To investigate the proteins associated with RSA, the protein expression in human follicular fluid was analyzed using 2-DE. Follicular fluid contains a variety of biologically important proteins for oocyte fertilization and follicle maturation in the mammalian reproductive process. Therefore, it can be used as a provisional source for identifying proteins involved in RSA. In this study, we identified five aberrantly expressed proteins (complement component C3c chain E, fibrinogen gamma, antithrombin, angiotensinogen, and hemopexin precursor) in follicular fluid from RSA patients with MALDI-TOF-MS and nano-LC MS/MS. Western blot analysis confirmed that the protein expression level of fibrinogen gamma and antithrombin was less in follicular fluid from RSA patients than those from normal controls. Semiquantitative RT-PCR and real-time PCR analyses revealed that mRNA level of these coagulation factors was also decreased significantly in chorionic villi of RSA patients compared with normal samples. Taken all together, it is likely that coagulation factors (fibrinogen gamma and antithrombin) play an important role in maintaining the normal pregnancy.

Intracytoplasmic sperm injection may lead to vertical transmission, expansion, and de novo occurrence of Y-chromosome microdeletions in male fetuses.

Y-chromosome microdeletion in male fetuses conceived by intracytoplasmic sperm injection (ICSI) was screened by polymerase chain reaction for sequence-tagged sites in azoospermia factor (AZF)-b and AZFc regions. Treatment with ICSI may lead to vertical transmission, expansion, and de novo Y-chromosome microdeletion in male fetuses.

Isolation of male germ stem cell-like cells from testicular tissue of non-obstructive azoospermic patients and differentiation into haploid male germ cells in vitro.

BACKGROUND: The purpose of this study was to establish the culture conditions required to isolate, identify and expand male germ stem cell-like cells (GSC-LC) from the testicular tissue of patients with non-obstructive azoospermia (NOA). METHODS AND RESULTS: Testicular tissues obtained from patients (two with maturation arrest (MA, n = 2) and Sertoli cell-only syndrome (SCOS, n = 11) were dissociated and plated into gelatin-coated dishes. After 2-4 weeks, cultures from both MA patients (100%) and four SCOS patients (36.3%) exhibited multicellular colonies, which proliferated successfully until passage 10. GSC-LC in the colonies displayed alkaline phosphatase activity, as well as Oct-4 and integrin b1 expression after every passage. After the fifth passage, GSC-LC were differentiated by encapsulation in calcium alginate and further cultivation. At 2 and 6 weeks, cells expressed c-Kit, Scp3, testis-specific histone protein 2B (TH2B), and transition protein (TP)-1. Fluorescence in situ hybridization additionally disclosed a few tetraploid and haploid cells at 6 weeks. Human oocytes were activated in the absence of artificial activation and cleaved after the injection of presumptive spermatids. CONCLUSIONS: Our novel culture system may be useful for diagnosing the existence of germ cells and facilitating the treatment of NOA patients.

Quantification of mitochondrial DNA using real-time polymerase chain reaction in patients with premature ovarian failure.

OBJECTIVE: To quantify mitochondrial DNA using real-time PCR in women with premature ovarian failure (POF) and a control group. DESIGN: Prospective study. SETTING: Genome Research Center for Reproductive Medicine and Infertility, Korea Ministry of Health & Welfare. PATIENT(S): Thrity patients with POF and 30 control individuals. INTERVENTION(S): The mitochondrial DNA content was quantified using real-time PCR. The effectiveness of the assay was determined by relative quantification using the comparative threshold cycle (CT) method. MAIN OUTCOME MEASURE(S): Relative quantification of mitochondrial DNA content. RESULT(S): The mitochondrial DNA content was significantly lower in the POF group than in the control group (0.58 +/- 0.38 vs. 1.15 +/- 0.67; P < .01). In both groups, there was a significant positive correlation between the mitochondrial DNA/28S rRNA ratio and mitochondrial DNA CT (control group: r = 0.774; P < .001; POF group: r = 0.556; P = .001) and a significant negative correlation between the mitochondrial DNA/28S rRNA ratio and 28S rRNA CT (control group: r = -0.677; P < .001; POF group: r = -0.627; P = .001). CONCLUSION(S): This study has established the clinical feasibility of quantifying amounts of mitochondrial DNA, relative to an internal standard, using real-time PCR. Further studies are warranted to elucidate the roles of apoptosis and mitochondrial function in the pathogenesis of POF.

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.

Expression and functional analyses of mHAUSP regulating apoptosis of cervical adenocarcinoma cells.

p53 tumor suppressor protein is stabilized by the herpes-virus-associated ubiquitin-specific protease (HAUSP), a deubiquitinating enzyme. We previously isolated a mouse orthologue of HAUSP, mHAUSP, encoding 1103 amino acids with a molecular weight of approximately 135 kDa containing highly conserved Cys, Asp (I), His, and Asn/Asp (II) domains. In this study, we investigated the temporal and spatial expression of mHAUSP during the early mouse embryonic development. Northern blot analysis revealed that the expression of mHAUSP was detected throughout the process of embryonic development with the maximal expression between E10.5 and E13.5. In situ hybridization study showed the global expression of mHAUSP in various organs of embryos, including mesencephalon, spinal cord, lung and genital eminence. In addition, we carried out biochemical analysis for 6 conserved amino acids (Cys224, Gln231, Asp296, His457, His465, and Asp482) in Cys box, QQD box, and His box in order to investigate their structural and functional roles of these amino acid residues. The conserved Gln231 was not essential for the catalytic activity of mHAUSP. However, other conserved amino acids were required for deubiquitinating enzyme activity of mHAUSP. Moreover, we observed that the overexpression of mHAUSP induces cell death in HeLa cells.

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.

Oocyte-selective expression of MT transposon-like element, clone MTi7 and its role in oocyte maturation and embryo development.

Previously, we found MT transposon-like element, clone MTi7 (MTi7) is highly expressed in the mouse ovary. Here, we show that the MTi7 is expressed in the oocyte from the primordial to the preovulatory follicles. For RNA interference (RNAi), double stranded RNAs (dsRNAs) were prepared for MTi7 and c-mos, a control gene with known functions. Each dsRNA was microinjected into germinal vesicle (GV) stage oocytes or zygotes with pronuclei (PN), after which developmental changes, mRNA expression, and nuclear and microtubular organization were analyzed. We found a 43.4-53% GV arrest in the microinjected oocytes with a concomitant decrease in targeted mRNA expression. In MTi7 dsRNA-injected early and late PN zygotes, a 92.9% 1-cell arrest and 76.9% 2-cell arrest were observed, respectively. This is the first report of an oocyte-selective expression of MTi7 mRNA, and our results strongly suggest that MTi7 involved in the nuclear membrane breakdown during oocyte maturation and embryo development.

Expression of wee1 and its related cell cycle components in mouse early stage follicles.

Wee1 is a kinase regulator of the M-phase promoting factor (a complex of cdc2 and cyclin B1). The present study was performed to determine the role(s) of wee1 in the early stages of mouse ovarian follicles. Expression of wee1 and the correlated cell cycle components, namely cdc2, cyclin B1, and cdc25C, was evaluated by immunohistochemistry. In addition, expression of Tyr15-phosphorylated cdc2 (cdc2-p) was also examined to determine whether wee1 kinase phosphorylates cdc2. Each component except cdc25C was found in the oocyte cytoplasm at all follicular stages, while cdc25C was not detected in primordial follicles. It was found primarily in ovarian interstitial cells and to a small extent in granulosa cells of the developing secondary follicles. To further confirm the expression of cell cycle components in the primordial follicular oocytes, day 1 ovaries were enzymatically and mechanically dissociated, then oocytes were isolated from somatic cells including pre-granulosa cells, and we confirmed that cdc2-p was expressed in oocytes of primordial follicles. The results of the present study led to the conclusion that wee1, without the counteracting cdc25C, would cause meiotic arrest of oocytes by inhibitory phosphorylation of cdc2. Expression of all these proteins in the granulosa cells of growing follicles may regulate granulosa cell mitosis concurrently with the growth of oocytes and follicles.

Regional differences in age at menopause between Korean-Korean and Korean-Chinese.

OBJECTIVE: To evaluate the difference in age at menopause between Korean-Korean (KK) women and Korean-Chinese (KC) women. DESIGN: A total of 1,972 participants were recruited: 961 postmenopausal women living in Korea, and 1,011 second- or third-generation emigrants to China. A structured questionnaire was used that included current age (at interview), age at menopause, weight, height, duration of education, socioeconomic status (SES), smoking, alcohol intake, number of children and abortions, etc. For every independent variable, the effect of regional difference (ie, living in Korea or China) on age at menopause was analyzed. Student's t test and chi2 test were applied as appropriate, and multiple regression analyses were performed for significant variables. RESULTS: Age at menopause was significantly higher in KK than KC (49.3 +/- 3.5 y vs 48.9 +/- 3.1 y, P = 0.011). Body mass index (BMI) and number of abortions were also higher in KK (23.3 +/- 2.7 vs 23.1 +/- 2.6, P = 0.021; 1.3 +/- 1.7 vs 0.2 +/- 0.7, P = 0.0001). Duration of education was significantly longer in KK. Smokers showed a significantly earlier menopause onset. BMI was significantly and positively correlated with age at menopause, whereas current SES was negatively correlated. When regional differences were assessed in each categorized subgroup, age at menopause showed a significant difference, too. After adjusting for age, SES, BMI, smoking, and number of children, onset of menopause was still earlier in KC (P = 0.0001, R = 0.037). CONCLUSION: KK women were found to have a later onset of menopause than KC women, and our results suggest that, apart from the well-known genetic influence, environmental effects play a significant role in determining age at menopause.

Effects of bone morphogenetic protein-7 (BMP-7) on primordial follicular growth in the mouse ovary.

Previously, bone morphogenetic protein-7 (BMP-7) was suggested as a factor that may act to facilitate the transition of follicles from primordial stage to the pool of developed primary, preantral, and antral follicles (Lee et al. 2001: Biol Reprod 65:994-999.). Thus, aim of the present study was to evaluate effect(s) of BMP-7 on the primordial-primary follicle transition. Neonatal mouse ovaries were cultured in the presence or absence of 100 mIU/ml FSH with various doses of BMP-7 (0, 10, and 100 ng/ml). After 4-day culture period, number of follicles was counted and the expression of transcripts for FSH receptor (FSHR), kit ligand (KL), and c-kit was measured by RT-PCR. BMP-7 alone at 100 ng/ml concentration stimulated follicle development with concurrent increase of mRNA for FSHR. BMP-7 alone down-regulated KL expression however, the ratio between KL1 and KL2 was increased. There was no change in the c-kit mRNA expression. Results of the present study suggest that the BMP-7 is one of the factors involved in primordial-primary follicle transition in the mouse ovary and it may play a role in expression of FSHR for further follicular development.