The size of a microsatellite polymorphism of the haem oxygenase 1 gene is associated with idiopathic recurrent miscarriage.

Endothelial damage, impaired microvascularization and immune maladaptation have been described as aetiological factors in recurrent miscarriages. We investigated the relationship between idiopathic recurrent miscarriage (IRM) and a (GT)(n) repeat microsatellite polymorphism of the gene encoding haem oxygenase 1 (HO-1), known to modulate immune functions such as T-helper (TH) cell function and to be associated with cardiovascular disease. We investigated 162 women with IRM and 129 healthy, post-menopausal controls. The length of the HO-1 (GT)(n) microsatellite was assessed by PCR and direct sequencing in all women. Results were correlated with clinical data. The distribution of genotypes was in Hardy-Weinberg equilibrium. The HO-1 (GT)(n) microsatellite repeat numbers ranged from 13 to 37, with (GT)(23) and (GT)(30) being the most common alleles in both groups. We compared alleles consisting of < or =27 GT repeats, termed class S (short) alleles and alleles consisting of >28 GT repeats, termed class L (long) alleles. Seventy per cent of women with IRM had an S allele either in heterozygous (L/S) or homozygous (S/S) form, compared to 56% of controls (P = 0.02; OR 0.54; 95% CI 0.32-0.90). With respect to S allele frequencies, we found no significant difference among women with IRM and controls [P = 0.3; odds ratio (OR) 1.23, 95% confidence interval (CI) 0.86-1.76]. Comparing women with primary and secondary IRM, no difference with respect to the length of the HO-1 (GT)(n) microsatellite was ascertained. In summary, this is the first report on a HO-1 (GT)(n) microsatellite polymorphism among women with IRM, demonstrating that the investigated polymorphism is associated with IRM in a relatively large Caucasian population.

A polymorphism of the interleukin-6 gene promoter and idiopathic recurrent miscarriage.

BACKGROUND: Cytokines have been described to play a major role in the pathogenesis of idiopathic recurrent miscarriage (IRM). We investigated the association between IRM and a polymorphism of the interleukin-6 (IL-6) gene and IL-6 serum levels. METHODS: In a prospective case-control study, we studied 161 women with IRM and 124 healthy controls. Peripheral venous puncture, DNA extraction and PCR were employed to genotype women for the presence of a polymorphism at position -174 in the promoter region of IL-6. Serum IL-6 levels were assessed by a commercially available ELISA. RESULTS: Allele frequencies among women with IRM and controls were 63.4 and 58.1% respectively for allele G (wild type), and 36.6 and 41.9% respectively for allele C (mutant). No association between allele C and the occurrence of IRM was found (odds ratio 0.8; 95% confidence interval = 0.57-1.12; P = NS). IL-6 serum levels were not significantly different between genotypes and between the study and control groups. CONCLUSIONS: This is the first report on an IL-6 polymorphism in IRM. Although known to alter IL-6 expression, the IL-6 polymorphism investigated was not associated with IRM and alterations in IL-6 serum levels in a Middle-European Caucasian population.

The PROGINS progesterone receptor gene polymorphism and idiopathic recurrent miscarriage.

OBJECTIVE: Progesterone inhibits lymphocyte cytotoxicity, natural killer cell degranulation, and release of proinflammatory cytokines and has been shown to protect against spontaneous miscarriage. We investigated the association between idiopathic recurrent miscarriage (IRM) and the PROGINS 306 base pair insertion polymorphism in intron G of the progesterone receptor gene, which is known to segregate with progesterone-dependent neoplasms. METHODS: In a case-control study we investigated 125 women with a history of three or more consecutive pregnancy losses before 20 weeks' gestation and 79 healthy controls with at least two live births and no history of pregnancy loss. Peripheral venous puncture, DNA extraction, and polymerase chain reaction were used to genotype women for the presence of the PROGINS polymorphism. RESULTS: Allele frequencies among women with IRM and controls were 85.2% and 89.2%, respectively, for allele T1 (wild type) and 14.8% and 10.8%, respectively, for allele T2 (mutant). No association between allele T2 and the occurrence of IRM was found (P =.3; odds ratio [OR] 0.69; confidence interval [CI] 0.34, 1.40). Genotype frequencies were not significantly different between the study group (T1/T1 73.6%, T1/T2 23.2%, T2/T2 3.2%) and the control group (T1/T1 79.7%, T1/T2 19%, T2/T2 1.3%) (P =.4). Between women with primary and secondary IRM, there were no statistically significant differences with respect to allele frequencies (82% versus 87%, P =.4 for allele T1 and 12% versus 13%, P =.6 for allele T2). CONCLUSIONS: We found that the PROGINS polymorphism in the progesterone receptor gene was not associated with IRM in white women.

Tryptophan hydroxylase gene polymorphism (A218C) and idiopathic recurrent miscarriage.

OBJECTIVE: To investigate the frequency of a polymorphism in intron 7 of the tryptophan hydroxylase gene among women with idiopathic recurrent miscarriage and healthy controls. METHODS: In a case control study, we studied 125 women with a history of three or more consecutive pregnancy losses before 20 weeks' gestation and 137 healthy controls with at least two live births and no history of pregnancy loss. Peripheral venous puncture, DNA extraction, and polymerase chain reaction followed by restriction fragment length polymorphism analysis were used to genotype women for the presence of the A218C polymorphism in intron 7 of the tryptophan hydroxylase gene. RESULTS: Allele frequencies among women with idiopathic recurrent miscarriage and controls were 32.4% and 38.7%, respectively, for allele A (wild type) and 67.6% and 61.3%, respectively, for allele C (mutant). No association between the presence of allele C and idiopathic recurrent miscarriage was found (P = .3; odds ratio 1.31; 95% confidence interval 0.93, 1.87). Genotype frequencies also were not significantly different between the study group (C/C: 44.8%; A/C: 45.6%; A/A: 9.6%) and the control group (C/C: 37.2%; A/C: 48.2%; A/A: 14.6%; P = .2). Between women with primary and women with secondary idiopathic recurrent miscarriage, no statistically significant differences with respect to allele frequencies were observed (63% vs 62% for allele C and 31% vs 38% for allele A; P = .3). CONCLUSION: The A218C polymorphism in intron 7 of the tryptophan hydroxylase gene is not associated with idiopathic recurrent miscarriage.

Endothelial nitric oxide synthase gene polymorphism in women with idiopathic recurrent miscarriage.

BACKGROUND: Lack of endothelium-derived nitric oxide is associated with vasospasm and vascular infarction. We investigated the relationship between idiopathic recurrent miscarriage and a polymorphism of the gene encoding endothelial nitric oxide synthase (NOS3). METHOD: In a prospective case-control study, 105 women with idiopathic recurrent miscarriage and 91 healthy controls were investigated. We used the polymerase chain reaction to identify the different alleles of a 27 base pair tandem repeat polymorphism in intron 4 of the NOS3 gene. RESULTS: The wild type B allele was identified on 329 out of 392 chromosomes (frequency 0.84). The polymorphic A allele was present on 63 chromosomes (frequency 0.16). The genotype frequencies were as follows: 68% (B/B), 31% (A/B) and.5% (A/A). The distribution of genotype frequencies was significantly different between the study and control groups for allele A/B heterozygotes (NOS3(A/B)) (36.7 versus 23.8%, P = 0.03, OR 1.6, 95% CI 1.1--3.8). Only one individual was homozygous for the A allele (NOS3(A/A)). She was in the study group. Between women with primary and secondary recurrent miscarriages, no statistically significant difference between the distribution of NOS3(A/B) genotypes (28 versus 34%) was observed. CONCLUSIONS: These data support a role for the NOS3 gene as a genetic determinant of the risk of idiopathic recurrent miscarriage.

A polymorphism of the interleukin-1beta gene and idiopathic recurrent miscarriage.

OBJECTIVE: Proinflammatory cytokines have been described to be involved in the pathogenesis of idiopathic recurrent miscarriage (IRM). We investigated the association between IRM and a polymorphism in exon 5 of the interleukin-1beta gene (IL1B) and interleukin-1beta (IL-1beta) serum levels. DESIGN: Case control study. SETTING: Academic research institution. SUBJECTS: One hundred thirty-one women with a history of three or more consecutive pregnancy losses before 20 weeks' gestation and 68 healthy controls with at least two live births and no history of pregnancy loss. INTERVENTIONS: Peripheral venous puncture. MAIN OUTCOME MEASURES: An IL1B exon 5 (position +3953) gene polymorphism was analyzed by PCR amplification followed by restriction fragment length polymorphism analysis. IL-1beta serum levels were analyzed by a commercially available ELISA. RESULTS: Allele frequencies in women with IRM and controls were 77.9% and 80.8%, respectively, for the E1 allele (wild type), and 22.1% and 19.2%, respectively, for the E2 allele (mutant). No association between the E2 allele and the occurrence of IRM was found (P=.57, odds ratio =.83). Genotype frequencies and IL-1beta serum levels were not significantly different between the study group and the control group. CONCLUSIONS: This is the first report on an IL1B polymorphism in IRM. Although known to alter IL-1beta expression, the investigated IL1B polymorphism is not associated with IRM and increased serum levels in a large Caucasian population.

Interleukin 1 receptor antagonist polymorphism in women with idiopathic recurrent miscarriage.

OBJECTIVE: Proinflammatory cytokines have been described as etiologic factors in idiopathic recurrent miscarriage. We investigated the relation between idiopathic recurrent miscarriage and polymorphisms in the gene encoding for the interleukin 1 receptor antagonist, an indigenous modulator of proinflammatory immune response. DESIGN: Prospective case control study. SETTING: Academic research institution. PATIENT(S): One hundred five women with a history of three or more consecutive pregnancy losses before 20 weeks of gestation and 91 healthy, postmenopausal controls with at least two live births and no history of pregnancy loss. INTERVENTION(S): Peripheral venous puncture. MAIN OUTCOME MEASURE(S): Polymerase chain reaction was performed to identify the different alleles of the gene encoding for interleukin 1 receptor antagonist. RESULT(S): Allele frequencies among women with idiopathic recurrent miscarriage and controls were 0.34 and 0.11, respectively, for the polymorphic allele 2 (P=.002; odds ratio: 7.4, confidence interval: 2.9--10.8) and.05 and.05, respectively, for the polymorphic allele 3 (P=.6; odds ratio: 1.3, confidence interval: 0.8--2.3). Allele 2 was present in homozygous form in 9% of women with idiopathic recurrent miscarriage. In contrast, 1% of the control women were homozygous for this allele (P<.001; odds ratio: 13.5, confidence interval: 7.5--21.8). CONCLUSION(S): These data support a role for allele 2 of the gene encoding for interleukin 1 receptor antagonist as genetic determinant of idiopathic recurrent miscarriage.

Flexible versus rigid endoscopes for outpatient hysteroscopy: a prospective randomized clinical trial.

To evaluate patient acceptance, optical properties and the clinical feasibility of flexible compared with rigid hysteroscopes, 142 patients undergoing outpatient hysteroscopy were included in a prospective, randomized clinical trial. The flexible hysteroscope was used in 70 patients, and the rigid instrument in 72. At different stages of the hysteroscopy the level of pain experienced by the women was assessed using a 10 cm visual analogue scale. Optical properties characterized by the parameters intrauterine visibility, hysteroscopic view and diagnostic accuracy were ranked by the surgeons using a 5-point scale (1 = excellent to 5 = insufficient), and duration of the hysteroscopy was measured. Hysteroscopy was successful in 87.5 and 100% of patients in the flexible and rigid groups respectively. With the use of rigid telescopes, discomfort at introduction and during the hysteroscopy was significantly greater (median 1.7 versus 0.7, P = 0.003; 3.1 versus 1.2, P < 0.001 respectively), but optical properties were judged to be far superior (P < 0.001 for all three comparisons) and procedure time was significantly shorter (median 70 versus 120 s, P = 0.003). In conclusion, outpatient hysteroscopy seems to be less painful when using flexible telescopes. However, rigid hysteroscopes provide superior optical qualities and permit a more rapid performance with higher success rates at much lower cost.

Light and electron microscopic study of stress-shielding effects on rat patellar tendon.

In this second part of our study, the histomorphologic changes occurring in the patellar tendon (PT) of rats after sole stress-shielding were evaluated. In seven adult albino rats, both PTs were exposed by straight skin incision and then stress-shielded on one side by a cerclage, while the contralateral PT served as the sham-operated control. One animal died after the operation and was used as a negative control. After 10 weeks of otherwise unrestricted motion, the rats were killed, and the histomorphology of all PT specimen pairs compared by light and transmission electron microscopy. Light microscopy showed mid-portion thickening and irregularity of collagen bundles in the stress-shielded tendons. Intense remodelling was demonstrated by increased cellularity and vascularity, as well as by enrichment in acidic proteoglycans. Ultrastructural evaluation and morphometry revealed a predominance of large diameter (peak between 180 and 260 nm) collagen fibrils in the sham-operated controls, while in the stress-shielded tendons the number of apparently new, small-diameter (peak between 40 and 60 nm) collagen fibrils increased (up to 77% per cross-sectional field of view). The difference in peak diameters was statistically significant (p < 0.0005). This rat model demonstrated that sole stress-shielding not only causes biomechanical alterations, but also intense tissue remodelling and significant morphological changes in the collagen fibrils in the patellar tendon, comparable to so-called 'ligamentization' in experimental and clinical patellar tendon grafts for anterior cruciate ligament reconstruction.

Expression of inducible nitric oxide synthase in human breast cancer depends on tumor grade.

Expression of inducible nitric oxide synthase (iNOS) by tumor cells has been suggested to abrogate metastasis in several tumor models, whereas constitutive NOS expression correlated positively with tumor grade in human breast carcinoma. Whether or not expression of one of the various NOS isoforms could predict the prognosis of breast cancer, however, has not been established. In the present report we investigated the cellular distribution of NOS isoforms in a series of benign and malignant breast tumors and in normal breast tissue. Immunohistochemistry revealed that in samples of benign disease the number of iNOS+ epithelial cells or total epithelial cells was 69+/-16% (n = 50). In samples of grade II invasive ductal breast carcinomas the number of iNOS+ tumor cells or total tumor cells was 62+/-20% (n = 40), compared to 12+/-9% (n = 40) in samples of grade III carcinomas (P<0.0001). iNOS protein was also identifiable in most of the epithelial cells of normal breast tissue (n = 4). In contrast, eNOS protein was restricted to vascular endothelial cells in all of the specimens studied. Since the presence of tumor cell iNOS protein is inversely related to the tumor's metastatic potential, we conclude that endogenous tumor cell mediated iNOS expression might have an inhibitory effect on the metastatic process in breast cancer.

Elevation of inducible nitric oxide synthase activity in human endometrium during menstruation.

Nitric oxide (NO) is a known agonist of programmed cell death (apoptosis). In order to discover its potential role during menstrual shedding, a process associated with extensive apoptosis, we evaluated activity and mRNA levels of the inducible and constitutive isoforms of NO synthase (NOS) in endometrial specimens of the proliferative (n = 11), late-secretory (n = 7), and menstrual (n = 17) phase of the cycle. These levels were compared with the proportion of apoptotic cells by detection of histochemically labeled DNA fragments. Inducible NOS (iNOS) activity during menstruation was six times that of the proliferative or late-secretory phase (p < 0.05), whereas constitutive NOS activity remained unchanged. Competitive reverse transcription-polymerase chain reaction revealed 146% and 77% increases of iNOS mRNA expression in the late-secretory and menstrual phases, respectively, compared to the proliferative phase (p < 0.05), whereas constitutive NOS mRNA expression remained constant. Inducible NOS immunostaining was restricted to epithelial cells, whereas constitutive NOS immunostainig was confined to vascular endothelia. In addition, the proportion of apoptotic cells within the glands of late-secretory or menstrual endometrium was twice that of the proliferative phase (p < 0.05). We conclude that local production of NO is involved in the signal transduction mechanisms leading to endometrial breakdown during menstruation.

Oral administration of methylglyoxal leads to kidney collagen accumulation in the mouse.

Methylglyoxal (MG) is a physiological substrate of the glyoxalase system which is impaired in the diabetic state and implicated in the development of diabetic complications. Like other reactive aldehydes in diabetes mellitus (DM) this carbonyl can bind to and modify proteins which may lead to changes of biochemical and biophysical properties of connective tissue proteins, a hallmark of diabetes mellitus. As previous studies on MG effects were confounded by other aldehydes found in DM, we decided to administer MG to 10 healthy, female OF-1 mice for a period of five months, at a level of 50 mg/kg body weight per day using 10 healthy untreated litter mates as controls. The left kidneys were taken for the determination of total kidney collagen, fluorescence, acid solubility of collagen and the right kidneys were used for the determination of glomerular basement membrane thickness. Total kidney collagen was significantly higher in the MG treated mice compared to control mice. Only about half the amount of collagen could be extracted from kidneys of MG treated animals indicating reduced solubility. Fluorescence in proteins from extracted kidneys of MG treated animals was about twice that of untreated animals. Glomerular basement membrane thickness was significantly higher in MG treated animals. Our findings indicate that MG can increase glomerular basement membrane thickness and the suggested underlying mechanism may be decreased solubility by increased cross linking as reflected by elevated protein fluorescence and decreased acid salt extraction. The involvement of MG in the development of diabetic complications postulated by others is herewith clearly supported by our findings.

[The role of nitric oxide in reproduction]. / Die Rolle des Stickstoffmonoxids bei der Reproduktion.

Nitrix oxide (NO) is a highly reactive and short-lived radical (half-life time: 10-12 s), which is derived from L-arginine by the NO synthases (NOS) in several organ systems. The release of NO by endothelial cells leads to rapid relaxation of vascular smooth muscle cells, whereas release by several neuronal cells causes neurotransmission. When NOS is actively induced in immune cells or certain epithelia it causes cytotoxicity and/or apoptosis of these cells. In the reproductive organs NO is now considered to be an important trigger molecule for several physiological mechanisms. Follicular synthesized NO is involved in rupture of the follicle during ovulation. Moreover, NO participates in the acrosome reaction of spermatozoa during capacitation. Apoptosis and collagenolysis of the functional endometrium may be involved in endometrial shedding during menstruation. Since NO induces both apoptosis and collagenolysis, the newly discovered production of NO in late secretory endometrium could act as a key mechanism in the process of menstrual disintegration of the endometrium. Additionally, NO is necessary to support and maintain the decidualization process and plays a pivotal role in implantation.

Induction of inducible nitric oxide synthase expression in human secretory endometrium.

The endometrial secretory phase is characterized by stromal oedema, a premenstrual increase in stromal macrophages and an increased cytokine production as menstruation approaches. Nitric oxide (NO) is a mediator of vasodilatation and cytotoxicity which is synthesized from L-arginine by NO synthases (NOS). These enzymes are either constitutively expressed or induced by lipopolysaccharides and/or cytokines. The presence and function of the inducible isoform of NOS (iNOS) in normal human endometrium has not been fully elucidated until recently. Frozen tissue sections taken from 22 women who underwent hysterectomy and adnexectomy for benign disease were immunostained with antibodies raised against the different NOS isoforms to investigate the presence of NOS in human endometrium. iNOS stained positive in the glandular epithelial cells of the secretory endometrium. Staining was either weak or absent in the proliferative and inactive endometrium, as well as in the oviduct and the glandular epithelium of the endocervix. The stroma remained uniformly negative. Immunoreactivity for endothelial constitutive NOS (eNOS) was confined exclusively to endothelial cells. Furthermore, epithelial cells from endometrium, oviduct and endocervix and all endothelial cells showed positive staining for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, which is a histochemical marker for NOS activity. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed in order to assess the presence of NOS mRNA. Abundant expression of iNOS mRNA was detected in the secretory phase endometrium only. The strong expression of inducible NO synthase in human secretory phase endometrium suggests that the increased production of NO, probably induced by cytokines, may be relevant to the process of menstruation.