Metabolic profile of human coelomic fluid.

AIM: Till now there is very limited knowledge on the molecular content of coelomic fluid and cells. This study presents the first attempt to elucidate the metabolic profile of such samples. METHODOLOGY: Samples were collected via coelocentesis from 41 women during the first trimester of gestation. Metabolic content was assessed using four different analytical platforms. For targeted analysis a hydrophilic interaction chromatography ultra high performance LC-MS/MS method was applied. Holistic analysis performed by GC-MS, NMR spectroscopy and ion cyclotron ultra-high resolution MS (FT-ICR-MS) instrumentation. RESULTS & CONCLUSIONS: Our observations suggest coelomic fluid and cells as promising biosamples, rich in metabolites with potential use in mammalian system biology studies.

Exogenous retroelement integration in sperm and embryos affects preimplantation development.

Retroelement transcripts are present in male and female gametes, where they are typically regulated by methylation, noncoding RNAs and transcription factors. Such transcripts are required for occurrence of retrotransposition events, while failure of retrotransposition control may exert negative effects on cellular function and proliferation. In order to investigate the occurrence of retrotransposition events in mouse epididymal spermatozoa and to address the impact of uncontrolled retroelement RNA expression in early preimplantation embryos, we performed in vitro fertilization experiments using spermatozoa preincubated with plasmid vectors containing the human retroelements LINE-1, HERVK-10 or the mouse retroelement VL30, tagged with an enhanced green fluorescence (EGFP) gene-based cassette. Retrotransposition events in mouse spermatozoa and embryos were detected using PCR, FACS analysis and confocal microscopy. Our findings show that: (i) sperm cell incorporates exogenous retroelements and favors retrotransposition events, (ii) the inhibition of spermatozoa reverse transcriptase can decrease the retrotransposition frequency in sperm cells, (iii) spermatozoa can transfer exogenous human or mouse retroelements to the oocyte during fertilization and (iv) retroelement RNA overexpression affects embryo morphology and impairs preimplantation development. These findings suggest that the integration of exogenous retroelements in the sperm genome, as well as their transfer into the mouse oocyte, could give rise to new retrotransposition events and genetic alterations in mouse spermatozoa and embryos.

Synergistic effect of follicle-stimulating hormone receptor and androgen receptor gene variants on semen quality.

Follicle-stimulating hormone (FSH), interacting with its receptor (FSHR), participates in the production of spermatozoa and androgens. Androgens exert their effects on male sex determination, development and sperm production by binding to androgen receptor (AR). In the present study, we sought to explore the potential synergistic effects of FSHR and AR gene variants on sperm quality. 200 oligozoospermic and 250 normozoospermic men were examined. DNA was extracted from spermatozoa, and the FSHR 307 (T/A), FSHR 680 (N/S) and AR (CAG)n polymorphisms were genotyped. Their parallel analysis revealed six combined genotypes. A gradual reduction of sperm motility, from long AR allele-Thr307Thr/Asn680Asn carriers to long AR allele-Ala307Ala/Ser680Ser carriers and from short AR allele-Thr307Thr/Asn680Asn carriers to short AR allele-Ala307Ala/Ser680Ser carriers was revealed in normozoospermic men (P < 0.001). Similar associations were observed in oligozoospermic men (P < 0.001). In our series, the synergism of the long AR alleles with the FSHRThr307/Asn680 allelic variant was associated with increased sperm motility, while the synergism of the short AR alleles with the FSHRAla307/Ser680 allelic variant was associated with decreased motility, supporting the significance of these genes in semen quality.

Semen quality is influenced by androgen receptor and aromatase gene synergism.

STUDY QUESTION: Does synergism between AR(CAG)(n) and CYP19(TTTA)(n) polymorphisms influence the quality of sperm? SUMMARY ANSWER: AR(CAG)(n) and CYP19(TTTA)(n) polymorphisms had a synergistic effect on sperm concentration and motility. WHAT IS KNOWN ALREADY: Androgens exert their action in the testicular tissue by binding to androgen receptor (AR), while their action is mediated by the aromatase P450 enzyme (CYP19). AR(CAG)(n) alleles are associated with sperm motility and CYP19(TTTA)(n) allelic variants have implications for sperm concentration and motility. STUDY DESIGN, SIZE, DURATION: Two hundred oligozoospermic and 250 normozoospermic men who presented for infertility investigation were examined during a period of 2 years. PARTICIPANTS/MATERIALS, SETTING, METHODS: Conventional semen analysis was performed. DNA was extracted from spermatozoa and both polymorphisms were genotyped by polymerase chain reaction. Serum hormone levels were determined. MAIN RESULTS AND THE ROLE OF CHANCE: Six combined genotypes were identified between the 18 AR(CAG)(n) alleles with 12-32 repeats and the 6 CYP19(TTTA)(n) alleles with 7-12 repeats. A gradual reduction in the sperm concentration (10(6)/ml) and motility (%) from long AR allele-non-CYP19(TTTA)(7) allele carriers to long AR allele-CYP19(TTTA)(7) homozygotes and from short AR allele-non-CYP19(TTTA)(7) carriers to short AR allele-CYP19(TTTA)(7) homozygotes was observed in normozoospermic men (means ± SD; concentration: 93 ± 53.1 versus 65 ± 48.6 and 85 ± 60.1 versus 37 ± 17.2l, P < 0.002; motility: 63 ± 10.3 versus 55 ± 14.5 and 52 ± 19.6 versus 41 ± 13.7, P < 0.001, respectively). Similar associations were observed in oligozoospermic men (concentration: 10 ± 4.2 versus 9 ± 5.9 and 10 ± 6.3 versus 6 ± 3.1, P < 0.03; motility: 47 ± 17.1 versus 39 ± 6.2 and 39 ± 22 versus 27 ± 18.3, P < 0.003, respectively). The above associations of the combined genotypes with sperm concentration and motility were confirmed in the total study population (P < 0.006 and P < 0.001, respectively). LIMITATIONS, REASONS FOR CAUTION: Our study population was limited to Greek Caucasian adult males, residents of Northwest Greece. WIDER IMPLICATIONS OF THE FINDINGS: The confirmation of our findings in other populations would verify the significance of AR and CYP19 genes for spermatogenesis. STUDY FUNDING/COMPETING INTERESTS: This study did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. The authors declare no conflicts of interest.

Dramatic reduction in sperm parameters following bariatric surgery: report of two cases.

Severe obesity constitutes the main public health crisis of the industrialised world. Bariatric surgery has been proposed as the most efficient treatment of obesity. In this study, we report the potential effects of bariatric surgery on semen parameters in male partners of couples undergoing assisted reproduction. These patients had been tested in the context of infertility treatment in two consecutive cycles before and after bariatric surgery. A marked reduction in sperm parameters was observed in a period of twelve to eighteen months after surgery. This unfavourable effect had also remarkable effects on the assisted reproduction outcome, necessitating the counselling of patients before bariatric surgery.

The (TAAAA)n polymorphism of the SHBG gene in men with the metabolic syndrome.

OBJECTIVE: Low serum Sex Hormone-Binding Globulin (SHBG) has been proposed as an indicator of the Metabolic Syndrome (MS) and cardiovascular disease in men. On the other hand, the (TAAAA)n repeat polymorphism in the SHBG gene has been shown to affect SHBG levels. The possible role of this polymorphism in the MS was examined in the present study. DESIGN: The study population consisted of 83 men with MS aged 54.9±14.8 years and 166 healthy men of the same age. The diagnosis of MS was based on the criteria proposed by the National Cholesterol Education Program - Third Adult Treatment Panel (NCEP-ATP III). The waist circumference was recorded and blood samples were obtained after overnight fasting for biochemical and hormonal tests. The SHBG(TAAAA)N polymorphism was genotyped in peripheral blood leucocytes. RESULTS: Genotype analysis for the (TAAAA)n polymorphism of the SHBG gene in the patients and controls identified 6 alleles having 6-11 TAAAA repeats. Patients with MS had more frequently short-allele genotypes (with 6/6, 6/7, 6/8, 7/7, 7/8 or 8/8 tandem repeats) compared to controls (53% vs. 39.8%, p=0.047). In the entire study population, men homozygous for the 6 TAAAA repeat allele had lower SHBG levels (p=0.01) and higher waist circumference (p=0.006) than men heterozygous or non-carriers of this allele. CONCLUSION: Short SHBG(TAAAA)N allele genotypes may play a role in the development of the MS. The mechanism of this contribution remains unclear.

Paraoxonase gene polymorphisms and stroke severity.

BACKGROUND: Paraoxonase (PON) is an HDL-associated enzyme that prevents low-density lipoprotein oxidation, playing a major role in the pathogenesis of atherosclerosis. PON genes polymorphisms may affect the corresponding enzyme activity. In this study, we examined the association of ischemic stroke with the three PON genes. METHODS: One hundred and seventy-eight patients hospitalized for ischemic stroke and 181 age- and sex-matched healthy controls were recruited. PON1(Q/R) 192, PON1(M/L) 55, and PON2(S/C) 311 polymorphisms were analyzed. RESULTS: The presence of the PON2 311C allele was significantly increased in patients with severe forms of ischemic stroke according to Modified Rankin Scale (P = 0.02, odds ratio = 2.215). No significant differences in genotype and allele distribution were observed between patients and controls. CONCLUSIONS: The PON2 311C allele was suggested as a possible predisposing factor for severe cases of ischemic stroke. Large-scale multicenter-controlled prospective studies are warranted to further explore the effects of PON polymorphisms on stroke susceptibility and severity.

Estrogen receptor beta gene variants may be associated with more favorable metabolic profile in postmenopausal women undergoing coronary angiography.

AIM: Estrogen action is exerted on the vasculature through estrogen receptors ER alpha and ER beta. We have previously reported significant association of ER alpha gene (ESR1) variants with more severe coronary artery disease (CAD) in postmenopausal women. The influence of ER beta gene (ESR2) variants on the cardiovascular system is not well established. We investigated the association of common ESR2 variants with risk factors for cardiovascular disease and with the severity of CAD in postmenopausal women. METHODS: ESR2 polymorphisms Alu I (1730 G > A) and Rsa I (1082 G > A) were studied in 174 postmenopausal women undergoing coronary angiography (age 45 - 88 yrs). The severity of CAD (0 - 3 vessels with > 50 % stenosis), indices of obesity and other predisposing risk factors for cardiovascular disease, biochemical and hormonal parameters were recorded. RESULTS: 75 women had 0, 39 had one, 37 had two and 23 had three vessels with severe stenosis in the coronary angiography. There was no association between Alu I (allele frequency = 40.2 %) and Rsa I (allele frequency = 2.6 %) variants and CAD severity. Carriers of Alu I had lower BMI (p = 0.044), lower waist perimeter (p = 0.029) and lower total cholesterol (p = 0.033) and LDL levels (p = 0.029). There was no association between Rsa I polymorphism and any metabolic risk factors. CONCLUSIONS: ESR2 Alu I polymorphism may have a favorable influence on risk factors for cardiovascular disease such as obesity indices and cholesterol levels. It does not appear to be associated with the severity of CAD in women.

Evidence for association of sex hormone-binding globulin and androgen receptor genes with semen quality.

The roles of androgen receptor AR(CAG)n gene polymorphisms and sex hormone-binding globulin SHBG(TAAAA)n gene polymorphisms on semen quality were studied. One hundred fourteen men were included in the study: 85 with normal sperm count and 29 oligospermic. The genotype analysis, on DNA extracted from spermatozoa, revealed five SHBG(TAAAA)n alleles with 6-10 repeats and 18 AR(CAG)n alleles with 12-32 repeats. The SHBG allelic distribution showed that in men with normal sperm count and motility, those with short SHBG alleles had higher sperm concentration than men with long SHBG alleles (P = 0.039). As concerns AR(CAG)n polymorphisms, men with short AR alleles had lower sperm motility compared to those with long AR alleles (P < 0.001) in both total study population and normal sperm count men. The synergistic effect analysis of the two polymorphisms revealed an association between sperm motility (P = 0.036), because of the effect of AR(CAG)n polymorphism on sperm motility. In conclusion, long AR alleles were found to be associated with higher sperm motility, while short SHBG alleles were associated with higher sperm concentration, supporting the significance of these genes in spermatogenesis and semen quality.

The role of sex hormone-binding globulin and androgen receptor gene variants in the development of polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) may be programmed in utero by androgen excess. Our aim was to examine the role of the sex hormone-binding globulin (SHBG) and androgen receptor (AR) gene polymorphisms, in the phenotypic expression of PCOS. METHODS: A cohort of 180 women with PCOS and 168 healthy women of reproductive age were investigated. BMI was recorded and the hormonal profile was determined on Day 3-5 of menstrual cycle. DNA was extracted from peripheral blood leucocytes and the SHBG(TAAAA)n and AR(CAG)n polymorphisms were genotyped by PCR. RESULTS: Genotype analysis revealed six SHBG(TAAAA)n alleles with 6-11 repeats and 19 AR(CAG)n alleles with 6-32 repeats, present in both PCOS and control women. Long SHBG(TAAAA)n alleles (>8 repeats) were at greater frequency in PCOS than normal women (P = 0.001), whereas short AR(CAG)n alleles (

Association of estrogen receptor-alpha gene polymorphisms with stroke risk in patients with metabolic syndrome.

OBJECTIVE: The vascular protective effects of estrogens are mediated by their binding to the two known estrogen receptors. In this study, we examine the association of stroke with two common polymorphisms of the ESR1 gene in patients with metabolic syndrome. MATERIALS AND METHODS: DNA from 130 patients hospitalized for ischemic stroke and 240 healthy controls were genotyped for ESR1 PvuII and XbaI polymorphisms. Results - Comparing female and male patients, it was found that CCGG diplotype is more frequent in male patients (P = 0.03). In addition, the AA genotype is associated with the onset of stroke at a younger age in the male patient group (P < 0.05). CONCLUSIONS: These findings suggest that PvuII and XbaI polymorphisms may affect the age at onset of the first stroke and the probability of developing cerebrovascular disease.

Synergistic effects of protein tyrosine kinase inhibitor genistein with camptothecins against three cell lines in vitro.

Genistein, a natural isoflavone product has been shown to induce cellular death and increase the apoptotic cell death induced by several DNA-damaging stimuli. We have explored the combined effect of genistein and camptothecins against three cell lines, HeLa (cervical cancer), OAW-42 (ovarian cancer) and L929 (normal fibroblasts). Combined effect was estimated in 96-well plates using the SRB method and median-effect analysis. Addition of genistein synergistically increased the antiproliferative affect of camptothecins, inhibiting the camptothecin-induced G2/M arrest and increasing the apoptotic cell population. In HeLa cells, genistein inhibited CDK1 phosphorylation after irinotecan treatment. Thus, abrogation of the G2/M checkpoint control by genistein may be a useful maneuver to increase cytotoxicity of agents that damage DNA and inhibit cell-cycle progression in the G2/M boundary.