Maternal immunity and pregnancy outcome: focus on preconception and autophagy.

Modulation of the maternal immune system before conception has a major role in determining subsequent pregnancy outcome. However, this has been a neglected area of investigation. There is a correlation between the length of time a woman is exposed to semen from her male partner and the development of regulatory T cells that limit a maternal antifetal immune response. Similarly, the composition of the vaginal microbiota influences the capacity of microorganisms to bypass the cervical barrier and colonize the uterus before pregnancy. The extent that this preconception colonization influences pre- and post-implantation gestational events depends on the types of microbes present, the genetic make-up of the mother and environmental influences on the magnitude and direction of her immune responses. Prepregnancy uterine and placental colonization with commensal bacteria may be beneficial to the fetus and newborn by generating tolerance to organisms that enhance postnatal well-being. Efforts to prevent or stop the progression of premature myometrial contractions have been limited because of an incomplete understanding of the mechanism(s) that trigger this occurrence. Based on recent studies of autophagy during gestation and parturition, inhibition of autophagy in myometrial cells may be the critical factor leading to a sequence of events culminating in induction of myometrial contractions either prematurely or at term.

Risk factors for development of isthmocele following cesarean section.

AIM: The aim of this paper was to analyze the risk factors contributing to the development of isthmocele following cesarean section (CS). METHODS: We retrospectively analyzed a cohort of patients presenting to our outpatient clinic for hysteroscopy. Only premenopausal patients with a history of CS were included. The risk factors investigated were: occurrence of previous vaginal delivery antecedent to CS, patient's age at CS, weeks of gestation at CS, phase of labor, type of suture (single/double layer) employed, and uterine flexion (anteversion/retroversion). RESULTS: The association between the identified risk factors and the occurrence of isthmocele was found statistical significant concerning indication for hysteroscopy (c2), stage of labor (c2), age at CS (Mann-Withney U test), according to the univariate analysis. This association was then confirmed in the multivariate logistic regression model pooling all the variables together. CONCLUSION: The significance of stage of labor to the development of isthmocele confirms previous observations and may be part of the information needed to the clinical decision of the CS time setting decision for the clinicians. Nevertheless, further prospective studies employing larger cohorts are warranted to further clarify the aforementioned risk factors before suggesting any modifications of clinical practice.

Vulvar melanoma: a 33 years single Italian center experience.

AIM: Vulvar melanoma is a rare disease with a poor prognosis. The purpose of this study was to report our experience on vulvar melanoma. METHODS: This is a retrospective study on patients with primary melanoma of the vulva admitted to our hospital during the last 33 years. Clinical characteristics, surgical therapy and follow-up are reported. Patients were classified following the 2009 edition of the melanoma staging system. RESULTS: The predominant symptom was pain; five patients reported ulceration and one patient presented bleeding from the vulvar lesions. The average age at diagnosis was 61.4 years. Surgical treatment was performed: radical vulvectomy in five cases, emivulvectomy in three cases, large regional excision in one case. Average time to follow-up was 50.2 months. In four cases (44.4%), regional recurrence occurred and the patients died as a result of the tumor; one patient died of other causes; four patients were still alive at the time of the study. CONCLUSION: Current treatment protocols have moved towards less aggressive treatment in view of the current available evidence. Sentinel lymph node biopsy and adjuvant therapy are still under debate. Our study confirms the overall poor prognosis for vulvar melanoma.

Altered autophagy induction by sera from pregnant women with pre-eclampsia: a case-control study.

OBJECTIVE: Mechanisms leading to pre-eclampsia remain incompletely defined. Autophagy is a conserved process necessary for cell survival under adverse conditions. We hypothesised that sera from women with healthy pregnancies and women with pre-eclampsia differed in autophagy induction. DESIGN: A case-control study. SETTING: Weill Cornell Medical College. POPULATION: Twenty-four normotensive pregnant women and 20 women with pre-eclampsia. METHODS: Sera were incubated with peripheral blood mononuclear cells (PBMCs) from female donors. After 48 hours the PBMCs were lysed and the intracellular concentration of p62 was determined by enzyme-linked immunosorbent assay (ELISA). Its concentration is inversely proportional to the extent of autophagy induction. Serum endoglin, interleukin 13 (IL-13), insulin-like growth factor 1 (IGF-1), and transforming growth factor ß1 (TGF-ß1) levels were quantitated by ELISA. MAIN OUTCOME MEASURES: Differences in autophagy induction and serum mediator levels in the two groups. RESULTS: Autophagy induction increased with gestational age in sera from normotensive women (P = 0.0045), but not in women with pre-eclampsia. In the presence of an autophagy inducer, the capacity for autophagy induction decreased with gestational age in sera from women with pre-eclampsia (P = 0.0235), but not from controls. Endoglin concentrations were positively associated with the extent of autophagy induction in controls only (P = 0.0141). There was no association between autophagy and serum IL-13, IGF-1, or TGF-ß1 levels. CONCLUSIONS: Sera from women with pre-eclampsia differ from normotensive women by their inability to induce autophagy as a function of gestational age.

Oxidative stress in pathological pregnancies.

Oxidative stress (OS) plays a role in pregnancy at risk of pre-eclampsia, diabetes and premature labour. We measured three markers of OS: total antioxidant capacity (TAC), thiolyte capacity and pro-oxidant capacity in 45 women: 15 normal pregnancies, 17 pathological pregnancies (pre-eclampsia and pregestational diabetes) and 13 delivered pre-term. Plasma TAC (µmol/ml) values in patients with pathological pregnancies (235.67 ± 70.08) (p(1) = 0.0086) and pre-term labour (243.51 ± 50.52) (p(2) = 0.0479) were significantly reduced as compared with the controls (306.78 ± 70.08). Thiolyte capacity (µmol/ml) in the pathological pregnancies (326.03 ± 78.24) (p(3) = 0.0029) and in pre-term labour (335.94 ± 76.63) (p(4) = 0.0084) groups were significantly reduced compared with the control group (417.48 ± 39.76) (p < 0.05). Pro-oxidant capacity (mg/100 ml) in the pathological pregnancies (94.11 ± 26.13) (p(5) = 0.00034) and in pre-term labour (87.18 ± 20.28) (p(6) = 0.00044) groups were significantly higher compared with the controls (60.27 ± 6.33). Elevated OS values were seen in pathological pregnancies. This supports the important role of OS in diseases in pregnancy, particularly pre-eclampsia, diabetes and pre-term birth.

Genomewide search for type 2 diabetes mellitus susceptibility loci in Finnish families: the Botnia study.

Type 2 diabetes mellitus is a heterogeneous inherited disorder characterized by chronic hyperglycemia resulting from pancreatic beta-cell dysfunction and insulin resistance. Although the pathogenic mechanisms are not fully understood, manifestation of the disease most likely requires interaction between both environmental and genetic factors. In the search for such susceptibility genes, we have performed a genomewide scan in 58 multiplex families (comprising 440 individuals, 229 of whom were affected) from the Botnia region in Finland. Initially, linkage between chromosome 12q24 and impaired insulin secretion had been reported, by Mahtani et al., in a subsample of 26 families. In the present study, we extend the initial genomewide scan to include 32 additional families, update the affectation status, and fine map regions of interest, and we try to replicate the initial stratification analysis. In our analysis of all 58 families, we identified suggestive linkage to one region, chromosome 9p13-q21 (nonparametric linkage [NPL] score 3.9; P<.0002). Regions with nominal P values <.05 include chromosomes 2p11 (NPL score 2.0 [P<.03]), 3p24-p22 (NPL score 2.2 [P<.02]), 4q32-q33 (NPL score 2.5 [P<.01]), 12q24 (NPL score 2.1 [P<.03]), 16p12-11 (NPL score 1.7 [P<.05]), and 17p12-p11 (NPL score 1.9 [P<.03]). When chromosome 12q24 was analyzed in only the 32 additional families, a nominal P value <.04 was observed. Together with data from other published genomewide scans, these findings lend support to the hypothesis that regions on chromosome 9p13-q21 and 12q24 may harbor susceptibility genes for type 2 diabetes.

Heritability of albumin excretion rate in families of patients with Type II diabetes.

AIMS/HYPOTHESIS: To study whether albumin excretion rate is an inherited trait in families of patients with Type II (non-insulin-dependent) diabetes mellitus. METHODS: We used three different approaches. Heritability of albumin excretion rate was studied in 267 nuclear families from the Botnia Study in Western Finland using parent-offspring regression. Albumin excretion rate was also measured in 206 non-diabetic offspring of 119 Type II diabetic parents with or without albuminuria (albumin excretion rate > 20 microg/min). Finally, albumin excretion rate was measured in altogether 652 siblings of 74 microalbuminuric and 320 normoalbuminuric probands. To study the potential confounding effect of blood pressure, the heritability of blood pressure was estimated in 718 nuclear families. RESULTS: Using parent-offspring regression, the heritability of albumin excretion rate was about 30 %, being the strongest from mothers to sons (35-39 % resemblance). The heritability for systolic blood pressure ranged from 10 to 20 % and for diastolic blood pressure from 10 to 27 %. Offspring of albuminuric Type II diabetic parents had higher albumin excretion rates (median 5.4 [range 1.0-195] vs 4.0 [1.0-23] microg/min, p = 0. 0001) and a higher frequency of microalbuminuria (11 vs 2 %, p = 0. 012) than offspring of normoalbuminuric parents. Further, siblings of microalbuminuric probands had higher albumin excretion rates than siblings of normoalbuminuric probands (4.1 [0.6-14.5] vs 3.6 [0.2-14. 4] microg/min, p < 0.01). CONCLUSION/INTERPRETATION: The data suggest that albumin excretion rate is an inherited trait in families of patients with Type II diabetes. [Diabetologia (1999) 42: 1359-1366]

A paired-sibling analysis of the XbaI polymorphism in the muscle glycogen synthase gene.

AIMS/HYPOTHESIS: We have previously shown an association between a XbaI polymorphism in the muscle glycogen synthase gene (GYS1) and both Type II (non-insulin-dependent) diabetes mellitus and hypertension. Association studies are, however, hampered by the selection of the control group. To circumvent these problems we addressed the same question using a novel genotype discordant paired-sibling approach. METHODS: We identified 122 sex-matched sib-pairs discordant for the Xba1 polymorphism among a new set of 743 Finnish subjects from 227 families with Type II diabetes and paired analyses were done by McNemar test of symmetry and by permutation tests. RESULTS: Paired analysis showed that siblings with the A2 variant had more hypertension (p = 0.0067), obesity (p = 0.033) and microalbuminuria (p = 0.031) but not significantly more Type II diabetes (p = 0.27) than siblings with the A1 variant. Siblings with the A2 variant were more often treated by insulin (p = 0.050) or anti-hypertensive medication (p = 0.0060) or both. Diabetic A2 variant carriers had higher triglyceride (p = 0.023) and lower HDL cholesterol (p = 0.0059) concentrations and an earlier age at onset of diabetes (p = 0.022) than diabetic siblings with the A1 variant. In non-diabetic sib-pairs the presence of the A2 variant was associated with higher diastolic (p = 0.0014) blood pressure. Finally, the allele frequency of the XbaI polymorphism differed between 216 randomly chosen unrelated Type II diabetic patients and 115 unrelated healthy control spouses without a family history of Type II diabetes (12.7 vs. 6.5 %, p = 0.013). CONCLUSION/INTERPRETATION: The A2 allele of the XbaI polymorphism in the GYS1 confers an increased susceptibility to different features of the metabolic syndrome and Type II diabetes.

Insulin-regulated mitochondrial gene expression is associated with glucose flux in human skeletal muscle.

To identify abnormally expressed genes contributing to muscle insulin resistance in type 2 diabetes, we screened the mRNA populations from normal and diabetic human skeletal muscle using cDNA differential display and isolated abnormally expressed cDNA clones of mitochondrial-encoded NADH dehydrogenase 1 (ND1), cytochrome oxidase 1, tRNA(leu), and displacement loop. We then measured mRNA expression of these mitochondrial genes using a relative quantitative polymerase chain reaction method in biopsies taken before and after an insulin clamp in 12 monozygotic twin pairs discordant for type 2 diabetes and 12 matched control subjects and in muscle biopsies taken after an insulin clamp from 13 subjects with type 2 diabetes, 15 subjects with impaired glucose tolerance, and 14 subjects with normal glucose tolerance. Insulin infusion increased mRNA expression of ND1 from 1.02 +/- 0.04 to 2.55 +/- 0.30 relative units (P < 0.001) and of cytochrome oxidase 1 from 0.80 +/- 0.01 to 1.24 +/- 0.10 relative units (P < 0.001). The ND1 response to insulin correlated with glucose uptake (r = 0.46, P = 0.002). Although the rate of insulin-mediated glucose uptake was decreased in the diabetic versus the nondiabetic twins (5.2 +/- 0.7 vs. 8.5 +/- 0.8 mg x kg(-1) fat-free mass x min(-1), P < 0.01), insulin-stimulated ND1 expression was not significantly different between them (2.4 +/- 0.5 vs. 2.7 +/- 0.5 relative units). Neither was there any significant intrapair correlation of ND1 expression between the monozygotic twins (r = -0.15, NS). We conclude that insulin upregulates mitochondrial-encoded gene expression in skeletal muscle. Given the positive correlation between ND1 expression and glucose uptake and the lack of intrapair correlation between monozygotic twins, mitochondrial gene expression may represent an adaptation to intracellular glucose flux rather than an inherited trait.

Familial hypercholesterolemia in the Finnish north Karelia. A molecular, clinical, and genealogical study.

A specific mutation termed FH-North Karelia [FH-NK] accounts for almost 90% of familial hypercholesterolemia [FH] cases in the Finnish North Karelia, with a population of about 180,000. Extensive search for its presence in the entire North Karelia province revealed 340 carriers of this mutation. Other mutations of the LDL receptor [LDLR] gene accounted for 67 cases of heterozygous FH. This gives a minimum FH prevalence of 1 in 441 inhabitants in North Karelia, with the highest density of patients in the Polvijärvi commune (1 in 143 inhabitants). Old parish records, confirmation records, and tax records were used to track a common ancestor for most of the present-day North Karelian FH-NK patients in the village of Puso, located within an area where the FH prevalence today is the highest. DNA analysis indicated that 2% of the subjects aged 1 to 25 years would have been diagnosed as false-negative and 7% as false-positive FH patients on the basis of LDL cholesterol [LDL-C] determinations alone. Common genetic variations of apolipoprotein E [apoE], XbaI, polymorphism of apolipoprotein B [apoB], and PvuII polymorphism of the intact LDLR allele contributed little to serum lipid variation in established carriers of the FH-NK allele, although apoE2/4 genotype and the presence of the PvuII restriction site tended to be associated with relatively low LDL-C levels. Coronary heart disease (CHD) was present in 65 (30%) out of the 179 FH gene carriers aged > or = 25 years, and 19 individuals had a previous history of acute myocardial infarction (AMI). The average age (mean +/- SD) at onset of CHD was 42 +/- 7 years for males and 48 +/- 11 years for females (P < .05). In stepwise logistic regression analysis carried out in carriers of the FH-NK allele, age, gender, smoking, and apoE allele E2 all emerged as independent determinants of risk of CHD or AMI. It may be concluded that the relatively high prevalence of FH patients in North Karelia province provides a unique founder population in which genetic and nongenetic factors modifying the course of FH can be effectively investigated.

Characterization of the MODY3 phenotype. Early-onset diabetes caused by an insulin secretion defect.

Maturity-onset diabetes of the young (MODY) type 3 is a dominantly inherited form of diabetes, which is often misdiagnosed as non-insulin-dependent diabetes mellitus (NIDDM) or insulin-dependent diabetes mellitus (IDDM). Phenotypic analysis of members from four large Finnish MODY3 kindreds (linked to chromosome 12q with a maximum lod score of 15) revealed a severe impairment in insulin secretion, which was present also in those normoglycemic family members who had inherited the MODY3 gene. In contrast to patients with NIDDM, MODY3 patients did not show any features of the insulin resistance syndrome. They could be discriminated from patients with IDDM by lack of glutamic acid decarboxylase antibodies (GAD-Ab). Taken together with our recent findings of linkage between this region on chromosome 12 and an insulin-deficient form of NIDDM (NIDDM2), the data suggest that mutations at the MODY3/NIDDM2 gene(s) result in a reduced insulin secretory response, that subsequently progresses to diabetes and underlines the importance of subphenotypic classification in studies of diabetes.

Mapping of a gene for type 2 diabetes associated with an insulin secretion defect by a genome scan in Finnish families.

Non-insulin dependent diabetes mellitus (NIDDM) affects more than 100 million people worldwide and is associated with severe metabolic defects, including peripheral insulin resistance, elevated hepatic glucose production, and inappropriate insulin secretion. Family studies point to a major genetic component, but specific susceptibility genes have not yet been identified-except for rare early-onset forms with monogenic or mitochondrial inheritance. We have screened over 4,000 individuals from a population isolate in western Finland, identified 26 families (comprising 217 individuals) enriched for NIDDM and performed a genome-wide scan using non-parametric linkage analysis. We found no significant evidence for linkage when the families were analysed together, but strong evidence for linkage when families were classified according to mean insulin levels in affecteds (in oral glucose tolerance tests). Specifically, families with the lowest insulin levels showed linkage (P = 2 x 10(-6)) to chromosome 12 near D12S1349. Interestingly, this region contains the gene causing the rare, dominant, early-onset form of diabetes MODY3. Unlike MODY3 families, the Finnish families with low insulin have an age-of-onset typical for NIDDM (mean = 58 years). We infer the existence of a gene NIDDM2 causing NIDDM associated with low insulin secretion, and suggest that NIDDM2 and MODY3 may represent different alleles of the same gene.

Polymorphism at the rad gene is not associated with NIDDM in Finns.

Recently, a trinucleotide repeat polymorphism at the rad (ras associated with diabetes) locus (RAD1) on chromosome 16q was described in association with NIDDM in white Americans. In an attempt to replicate this finding, we screened RAD1 and another microsatellite marker at the D16S265 loci, which is located near the rad locus, with a radioactive polymerase chain reaction method in 290 unrelated Finnish NIDDM patients and 270 control subjects and related the findings to measures of insulin sensitivity. Both groups were randomly selected from the western (189 NIDDM patients and 184 control subjects) and southern (101 NIDDM and 86 control subjects) parts of Finland. The allele frequency distributions of RAD1 and D16S265 did not differ between NIDDM patients and control subjects in the studied population groups. The genotype distribution was also analyzed by structural classes of the RAD1 polymorphism, and no difference was detected between the NIDDM and control groups. In addition, carriers of allele classes I, II, and IV (reported to be preferentially associated with NIDDM in white Americans) did not differ from the class III homozygotes with respect to age at onset of NIDDM, BMI, or rates of insulin-stimulated glucose disposal. In conclusion, we found no association between the rad locus and NIDDM or insulin resistance in Finnish NIDDM patients.

Association of a polymorphism in the beta 3-adrenergic-receptor gene with features of the insulin resistance syndrome in Finns.

BACKGROUND: Because visceral obesity predicts insulin resistance, we studied whether alterations in the gene encoding for the beta 3-adrenergic receptor in visceral fat are associated with insulin resistance. METHODS: We studied the frequency of a cytosine-to-thymidine mutation that results in the replacement of tryptophan by arginine at position 64 (Trp64Arg) of the beta 3-adrenergic receptor by restriction-enzyme digestion with BstOl in 335 subjects from western Finland, 207 of whom were nondiabetic and 128 of whom had non-insulin-dependent diabetes mellitus (NIDDM). We also determined the frequency of the mutation in 156 subjects from southern Finland. Sensitivity to insulin was measured by the hyperinsulinemic-euglycemic clamp technique in 66 randomly selected nondiabetic subjects. RESULTS: In the subjects from western Finland, the frequency of the mutated allele was similar in the nondiabetic subjects and the subjects with NIDDM (12 vs. 11 percent). The mean age of the subjects at the onset of diabetes was lower among those with the mutation than those without it (56 vs. 61 years, P = 0.04). Among the nondiabetic subjects, those with the mutation had a higher ratio of waist to hip circumference (P = 0.02), a greater increase in the serum insulin response after the oral administration of glucose (P = 0.05), a higher diastolic blood pressure (82 vs. 78 mm Hg, P = 0.01), and a lower rate of glucose disposal during the clamp study (5.3 vs. 6.5 mg [29 vs. 36 mumol] per kilogram of body weight per minute; P = 0.04) than the subjects without the mutated allele. In an analysis of sibling pairs, the siblings with the mutation generally had higher waist:hip ratios (P = 0.05) and higher responses of blood glucose and serum insulin after the oral administration of glucose than their siblings without the mutation (P = 0.02 and P = 0.005, respectively). CONCLUSIONS: The Trp64Arg allele of the beta 3-adrenergic receptor is associated with abdominal obesity and resistance to insulin and may contribute to the early onset of NIDDM:

Determination of mannose and fructose in human plasma using deuterium labelling and gas chromatography/mass spectrometry.

We used gas chromatography/mass spectrometry to measure mannose and fructose in human blood plasma. The plasma samples were treated with sodium borodeuteride. Characteristic ion fragments for (1-d)mannitol, (2-d)mannitol and (1-d,2-13C)mannitol were selected for use in sugar fragmentography and to build an algorithm for calculation of the sugar concentrations. The analytical recovery of mannose and fructose and the precision of the mannose assay were satisfactory. The measurement of fructose was marked by poor precision, which was accounted for, at least in part, by the low fructose content of the plasma samples. The method may prove useful in profiling monosaccharides in biological fluids. The method leans on the measurement of polyols but it can also provide auxiliary information on the occurrence of aldoses and ketoses.