MTHFR) gene. Because 5,10-MTHFR 677TT homozygosity and tobacco smoking are associated with low folate status, we tested the hypothesis that smoking in mothers with 5,10-MTHFR C677T or A1298C polymorphisms would be independently associated with lower birth weight among their offspring.Intracellular folate hemostasis depends on the 5,10-methylenetetrahydrofolate reductase  were extracted from recruitments that took place during the period from February 2003 to March 2006. Maternal serum folate were assayed by chemiluminescent immunoassay, and genotyping of 5,10-MTHFR 677CT genotype was independently associated with an increase of 36.40 g  in mean infant birth weight and an increase of 90.70 g  among male infants of nonsmokers. Female infants of 677TT homozygous passive smokers were 99.00 g  lighter. The birth weight of the offspring of smokers with 5,10-MTHFR 1298AA homozygosity was lower by 107.00 g .The prevalence of folate deficiency (<6.8 nmol/L) was 0.3%. The 5,10-MTHFR C677T polymorphism, but not the 5,10-MTHFR A1298C variant, is independently associated with improvement in infant birth weight, especially among nonsmokers. However, 5,10-MTHFR 1298AA might be associated with folate impairment and could interact with tobacco smoke to further decrease birth weight.The results suggest that, in this population, maternal 5,10- MTHFR) gene, which is located at position 36 on the short arm of chromosome 1. This gene codes for the enzyme MTHFR, which catalyses the irreversible conversion of 5,10-MTHFR to 5-metyltetrahydrofolate, a substrate for methylation of homocysteine to methionine. Thus far, 14 rare mutations in MTHFR have been described, but the 2 most common single nucleotide polymorphisms (SNPs) are 5,10-MTHFR C677T (dbSNP ID: rs1801133)—a missense mutation in exon 4, characterized by an alanine to valine substitution on codon 222—and 5,10-MTHFR A1298C (dbSNP ID: rs1801131)—a point mutation in exon 7 characterized by a glutamate to alanine substitution on codon 429.1 5,10-MTHFR C677T is located in the catalytic N-terminal domain of the enzyme, while 5,10-MTHFR A1298C is located in the regulatory domain of the enzyme.2Intracellular folate hemostasis depends on the 5,10-methylenetetrahydrofolate reductase , low birth weight (LBW), small for gestational age (SGA), neurodevelopmental delays, and other congenital anomalies.3–15 However, several other investigators have found no such associations.16–25 This confusion might be explained by the fact that phenotypic expression of this genetic trait depends on folate status and other environmental factors that vary by geographic region and race.Biochemically, the 5,10-MTHFR A1298C variant are not well known, it is a risk factor for neural tube defects,26,27 and compound heterozygosity  has been reported to have a biochemical profile similar to that of 677TT homozygosity.1,28Although the functional consequences of the 5,10-29 More recently, smoking has been associated with nutritional deficiencies, including folate deficiency.30–32 LBW secondary to IUGR or PTD remains a public health concern because it increases the risk of morbidity and mortality throughout life.Maternal smoking during pregnancy is an established risk factor for intrauterine growth retardation (IUGR), SGA, PTD, and other adverse pregnancy outcomes.Helicobacter pylori infection, and periodontal diseases. However, only a few such studies have investigated obstetric events, and none has considered infant birth size.3,6,17,33–35 We therefore tested the hypothesis that maternal smoking in the presence of the 5,10-MTHFR C677T or A1298C polymorphisms independently reduces birth weight.In Japan, there have been genetic association studies of the relation between folate and cardiovascular pathologies, cancers, 36 Briefly, all indigenous Japanese women who reserved antenatal care at any of 37 participating hospitals within Hokkaido during their first trimester of pregnancy were considered eligible. Health care personnel introduced the study, after which each potential participant was given an invitation that included a consent form, baseline questionnaire, and self-addressed envelopes for return of the signed consent forms and completed questionnaires. The participants were recruited between February 2003 and March 2006. Only participants with linked and integrated data  were included in the baseline population of this study. The response rate for each variable was at least 70.0% from various sources. Based on the population allele frequencies of the 5,10-MTHFR C677T37 and A1298C38 polymorphic variants specific to Japanese and the prevalence of tobacco smoking during pregnancy,39 minimum sample sizes were calculated by using genetic software.40 We randomly selected 1805 extracted genomic DNAs, attempted to discriminate the alleles of 5,10-MTHFR polymorphisms, and successfully genotyped 1784, which were ultimately used in the data analysis , and gynecologic and obstetric histories. Infant birth records from hospitals had information about birth weight, gestational age at delivery, sex, obstetric events during index pregnancy, and congenital anomalies, among other information. Postpartum questionnaires collected information on infant anthropometric parameters, active or passive tobacco smoking during the index pregnancy, and whether the index pregnancy was eventful. Each variable in the dataset had a response rate of at least 70.0%, although the item on smoking status decreased from 99.0% at baseline to 70.0% after pregnancy. Whole-blood specimens were collected during the first trimester for serum folate assays; subsequent collections of whole blood specimens were stored at −80°C for genetic analyses.41 The specific assay method for this study was the ADVIA Centaur technique, which has a coefficient of variation between 4.0% to 4.3%.42 Analyses were conducted in batches, which were scheduled with regard to recruitment period and laboratory procedure.Serum folate was assayed by a commercial laboratory  using an automated competitive protein binding (CPB) chemiluminescent enzyme immunoassay (CLEIA) technique according to the manufacturer’s protocols. This type of assay has an intra- and inter-assay imprecision of 10.0% or less and has become common in in vitro studies, as it is less costly, faster, and convenient. In addition, the need for smaller samples is advantageous in large scale epidemiologic studies.37 and 19.0%,38 respectively, among Japanese. Genomic DNAs were extracted using a Maxwell 16 Instrument . DNA amplifications were performed in batches on 96-well micro-amp reaction plates using validated TaqMan probes for MTHFR C677T and A1298C (assay IDs: C_1202883_20 and C_850486_20), respectively, on a Gene Amp 9700 thermal cycler  with an end-point allelic discrimination (AD) assay on a 7300/7500 Real-time PCR System43 . We randomly selected 95 samples (5.0% of the successfully genotyped samples) and repeated genotyping to check for genotyping quality. The results were 100% concordant.We chose the 2 most common SNPs of this gene, namely C677T and A1298C, which have minor allele frequencies (MAF) of 35.2%Overall smoking status was classified into 3 categories using both self-reported active smoking and passive exposure to environmental tobacco smoke (ETS) at home. Nonsmokers had no history of active smoking or exposure to ETS at home. Nonsmokers and quitters with ETS exposure were classified as the passive smoking group, while smokers consisted of active smokers irrespective of ETS exposure status. Quitters with no ETS exposure during the first trimester had mean infant birth weights similar to those of nonsmokers; hence, they were added to the nonsmoking group. Mothers who quit during the second or third trimesters were added to the active smoking group.MTHFR C677T and A1298C genotypes were categorized as dominant homozygous, heterozygous, and recessive homozygous genotypes .The 5,10-MTHFR C677T and A1298C polymorphisms and smoking on serum folate levels, while ANCOVA was used to investigate the interactive association between smoking and 5,10-MTHFR C677T and A1298C polymorphisms in relation to folate status and infant birth weight. Serum folate was log-transformed before the analyses and back-transformed after the analyses. Known major predictors of infant birth weight  were adjusted for in the multivariate regression analyses. Use of a folic acid supplement, which was highly correlated with serum folate levels, was also included as a covariate. Smoking status was adjusted for when we assessed the predictive power of each SNP on birth weight. Categorical covariates were dichotomized to fit the regression equation. Subgroups with few participants  were excluded from the regression analyses. We used the codominant genetic model and per-allele approach. Our preliminary analyses revealed that mean serum folate was highest for the MTHFR 1298AC genotype. Because adequate folate status is an integral part of our hypothesis, we decided that it was biologically plausible to set 1298AC as the reference category in the regression analyses. Predictors were entered simultaneously into the equation. Assessment of the 5,10-MTHFR C677T and A1298C genotypes for deviation from the Hardy-Weinberg equilibrium, and other evaluations of data quality, were conducted using Haploview version 4.2 software.44 All other analyses were performed using SPSS version 16.00 for Windows . The level of statistical significance was set at less than 0.05.Univariate ANOVA with multiple comparison tests was performed to assess the main effects of maternal 5,10-MTHFR C677T and A1298C genotypes did not deviate from the Hardy-Weinberg equilibrium . The frequencies of MTHFR 677CC, 677CT, and 677TT were 37.3%, 46.7%, and 16.0%, respectively, while those of MTHFR 1298AA, 1298AC, and 1298CC were 62.7%, 33.1%, and 4.2%, respectively. A strong LD (D′ = 0.943) between MTHFR C677T and A1298C was also observed, and the minor allele frequencies were 0.392 and 0.205, respectively  was observed in the 677TT homozygous group. Tukey’s honestly significant differences (HSD) of 1.0 nmol/L (P = 0.008) and 3.8 nmol/L (P < 0.001) were observed between 677CC versus 677CT and between 677CC versus 677TT, respectively. In contrast, carrying the 1298C allele was associated with higher mean serum folate levels. A Tukey’s HSD of 1.5 nmol/L (P < 0.001) was observed between 1298AA versus 1298AC . With regard to 5,10-MTHFR A1298C genotypes, the lowest mean folate level was observed among smokers with the 1298AA homozygous genotype,  level was 16.4 (1.5) nmol/L. The prevalence of folate deficiency (<6.8 nmol/L) was 0.3%; most (73.0%) mothers had adequate folate status (≥13.6 nmol/L). The prevalence of folic acid supplementation was 10.0%. Mean infant birth weight (SD) was 3040 (374) g. The prevalence of active smoking during pregnancy was 15.9%, while that of passive smoking was 53.0%. The distributions of the 5,10-P = 0.045) reduction in mean birth weight of infants in the normal birth weight group (data not shown).We initially explored the role of maternal serum folate during the first trimester as a predictor of infant birth weight and found no significant linear association. After stratification by folate status, low folate status (<13.6 nmol/L) was associated with a nonsignificant reduction in birth weight. After stratification by birth weight status, low folate status was associated with a 34.00 g  for 5,10-MTHFR C677T. The 677CT genotype was associated with a 36.40 g increase in mean infant birth weight . Carrying the 677T allele was associated with a marginally significant 27.00 g increase in infant birth weight .To investigate whether the maternal 5,10-MTHFR A1298C or carrying the 1298C allele was not significantly independently associated with birth weight, although the adjusted mean infant birth weight was highest (3048 g) in the 1298AC heterozygous group. The adjusted mean birth weight of infants of active tobacco smokers was lowest (2978 g) and was 85.00 g  less than that of children born to nonsmokers  heavier than the infants in the reference category, which was marginally statistically significant, while among passive smokers, female infants of 677TT homozygous mothers were 99.00 g  lighter than reference. None of the minor-allele genotypes among smokers showed any significant effect on infant birth weight as compared with those in the major-allele genotypes (data not shown). Per-allele analyses revealed that carrying the 677T allele was associated with a 68.00 g  reduction in mean birth weight among infants of smokers and an 89.00 g  reduction among female infants  reduction in mean birth weight. Males were lighter by 79.00 g , while females were lighter by 107.00 g ; male newborns were 90.70 g  heavier than the male infants of nonsmoking 677CC mothers,  decrease in mean infant birth weight among smokers. Stratification by infant sex did not yield obvious differences in birth weight, Pinteraction = 0.040; Table MTHFR 1298AA genotype was associated with a 107.00-g  decrease in mean infant birth weight in smokers; the effect was more obvious in male infants .In cross-classification interactive analyses, infants born to nonsmokers with 5,10-h weight 092 g; maMTHFR 677CT heterozygosity with higher infant birth weight, while 5,10-MTHFR 677TT homozygosity was associated with lower birth weight among female infants of passive tobacco smokers. In addition, among smokers, 5,10-MTHFR 1298AA homozygosity was associated with low folate status and lower birth weight. To our knowledge, this is the first study to report such findings for a Japanese population.Among nonsmokers, we found an association of maternal 5,10-MTHFR 677T allele and low folate status, which agrees with the findings of earlier reports.6,46 677TT homozygosity was associated with low folate status, and values were much lower among active and passive smokers, which suggests independent and combined effects of tobacco smoke and 5,10-MTHFR C677T polymorphism on folate status.Our results showed an association between the 5,10-MTHFR 1298C allele was associated with higher serum folate levels, while the 1298AA genotype was associated with lower folate levels. Although the metabolic and clinical functions of this SNP have not been fully characterized, it is currently being studied by a number of investigators. In a recent study of Koreans, mean plasma homocysteine was higher among 1298AA homozygotes as compared with those carrying the 1298C allele.48 Because serum folate is inversely correlated with plasma homocysteine, we inferred that our study population might have a similar plasma homocysteine distribution across genotypes. A report from Portugal noted that 1298AC heterozygosity was associated with a high plasma folate level and that the level was lowest among 1298CC homozygotes,49 which is similar to the findings of the present study. Our findings contradict those of a study of a Dutch population, in which MTHFR A1298C alone was not associated with any biochemical abnormalities except when in combination with MTHFR C677T, specifically compound heterozygosity 5,10-MTHFR 677CT/1298AC.28 The fact that our findings were similar to those from a report on a Korean population is genetically plausible because racial, geographic, and nutritional disparities might account for differences in the functional characteristics of 5,10-MTHFR SNPs.50,51In contrast, the 5,10-MTHFR 1298AA genotype in reducing the birth weight of infants delivered by tobacco smokers might be due to low folate status associated with the 1298AA genotype. Perhaps some essential folate-dependent cellular processes were compromised. Cells that lack folate have been observed to accumulate in the S-phase of the cell cycle. Such cells have higher uracil misincorporation and DNA damage,52 which might have a role in the impairment of fetal growth. Moreover, chronic deficits in extracellular and intracellular folate due to the effects of tobacco smoke might have been severe enough to inflict nutritional stress. In our study, we could not examine the role of the 1298CC genotype among smokers because of its low frequency. However, previous reports observed that the maternal 1298CC genotype was associated with a greater reduction in the risk of low birth weight as compared with the 1298AA genotype.21 The 1298CC homozygous genotype was also reported to be protective against IUGR in Canadians.53 Hyperhomocysteinemia might have increased the risk of placental vasculopathy via oxidative stress, endothelial cell dysfunction, and/or coagulopathies leading to feto-placental hypoperfusion.5Smokers carrying 1298A alleles delivered infants with lower mean birth weights, especially female infants; however, these results must be interpreted with caution because alleles do not act in isolation. The effect of the maternal 5,10-12 status has been shown to decrease total plasma homocysteine levels in Japanese.54 However, among smokers, the possible coexistence of nutritional deficiencies, including vitamin B12 deficiency, might have compromised the methylation of homocysteine to methionine, resulting in impaired fetal growth. The folate level was probably not adequate to silence the phenotypic expression of 1298AA among smokers. Higher exogenous folate may be needed to correct deficits and maintain ideal levels for optimal fetal growth.Adequate serum vitamin BMTHFR gene structure, the A1298C variant is located on the regulatory C-terminal domain, which contains protein retention signals that prevent delivery of proteins to the secretory pathway. It is possible that allosteric inhibitory interplay in the s-adenosyl methionine (SAM) and s-adenosyl homocysteine (SAH) cycle is involved in the functional behavior of this SNP in relation to folate status and mediation of fetal growth. Recently, the 5,10-MTHFR A1298C polymorphism was found to be associated with increased folate levels in red blood cells, in an inverse relationship with 5,10-MTHFR C677T polymorphism,55 which suggests that both SNPs have different functional characteristics with regard to phenotypic expression.With regard to the 5,10-MTHFR A1298C polymorphism, especially among Japanese, further research is needed to verify this observation and elucidate the biological mechanisms associated with this SNP.Due to limited evidence on the functional role of the 5,10-MTHFR 677T allele is associated with low folate and high homocysteine levels. In this study, the 677T allele was associated with lower birth weight in offspring of active smokers. The 677CT genotype was protective against low birth weight, especially among male offspring, but only in the absence of active or passive tobacco smoke. This might be due to the presence of higher mean serum folate levels among nonsmokers. In a Korean population, the 677T allele had a weak protective association with lung carcinoma.56 The protective effect of adequate folate status might be mediated by the stabilization of flavin-adenine-dinucleotide (FAD) binding at the catalytic domain.2 The birth weight of female infants of 677TT homozygous passive smokers was significantly lower than that of female infants born to passive smokers of similar genotypes. Male fetuses were favored, probably because pregnant mothers carrying male fetuses have a higher nutritional intake than those with female fetuses.57 Poorly understood phenomena on fetal sex-specific signals have been implicated in fetal growth, especially in response to glucocorticoid activity that might modify the fetal response to stress.58The 5,10-59 Therefore, the findings of the present study are likely to be reliable. Nevertheless, this does not diminish the importance of using biomarkers like cotinine. The findings might have been confounded by other B vitamins, which were not studied, or by other unidentified sources common to cohort study designs. Finally, this study was hospital-based; therefore, our findings should not be generalized. Further studies of other factors in the folate-homocysteine pathway should prove interesting.The participants were indigenous Japanese; hence, we overcame issues of population stratification in genetic association studies. Overall, this study might be limited by selection bias, as we utilized integrated data from only 61% of the total recruitment during the study period. However, because we randomly selected the final study population based on known pooled population frequencies of the genetic factors and tobacco smoking among pregnant women, we believe that this study does not substantially differ from one with a higher participation rate . The sample size of the study was adequate to detect gene-environment interactions; however, multiple comparisons and small sub-group sample sizes might have affected the study power. There could be misclassification bias from self-reported tobacco smoking; however, a previous study reported very low misclassification bias among Japanese women.MTHFR A1298C polymorphism shows that the frequency of the 1298AA genotype is greater than 60.0% in the Japanese population.38 Its association with low folate status is thus a considerable public health concern. With the recent increasing prevalence of smoking among young Japanese women, particularly in Hokkaido,60 maternofetal morbidity and mortality might also increase. Smoking cessation and targeted use of folic acid supplements could therefore prove to be very important public health tools in this population.Analysis of the 5,10-MTHFR C677T polymorphism is independently associated with higher infant birth weight, especially among nonsmokers, while the 5,10-MTHFR A1298C variant is not independently associated with birth weight. In addition, the 5,10-MTHFR 1298AA polymorphism might be associated with folate impairment and could interact with tobacco smoke to further decrease birth weight.Our findings suggest that the maternal 5,10-


Elevated total plasma homocysteine (tHcy) in humans is associated with cardiovascular disease but prevention trials have failed to confirm causality. Reported reasons for this association have been that homocysteine and its major genetic determinant methylenetetrahydrofolate reductase (MTHFR) may have an effect on HDL and Apolipoprotein (Apo) A1 levels. We wanted to study if tHcy and its major determinants were correlated with Apo A1 levels in a large population without folate fortification.This study was a prospective incident nested case-referent study within the Northern Sweden Health and Disease Study Cohort (NSHDSC), including 545 cases with first myocardial infarction and 1054 matched referents, median age at inclusion was 59 years. Univariate and multiple regression analyzes was used to study the associations between apolipoproteins Apo A1 and B, tHcy, folate and vitamin B12 in plasma as well as MTHFR polymorphisms 677C>T and 1298A>C.Apo A1 and Apo B were strongly associated with the risk of a first myocardial infarction. tHcy was not associated with Apo A1 levels. Instead, folate had an independent positive association with Apo A1 levels in univariate and multiple regression models. The associations were seen in all men and women, among referents but not among cases. MTHFR polymorphisms had no clear effect on Apo A1 levels.Analyzing over 1500 subjects we found an independent positive association between plasma folate (major dietary determinant of tHcy) and Apo A1 levels among those who later did not develop a first myocardial infarction. No association was seen between tHcy and Apo A1. Hyperhomocysteinemia has been claimed to be a risk factor for cardiovascular disease. Evidence for this has come from meta-analysis based on several epidemiological studies and also from prospective studies -7. The rSeveral different mechanisms how hyperhomocysteinemia can lead to atherosclerosis are described, such as inflammation, oxidative stress and enhanced lipogenesis ,12. ApolOnly a few reports have been published regarding the relationship between folate and Apo A1. In a Norwegian case–control study with 107 patients with acute myocardial infarction (MI), whole blood folate correlated positively and tHcy negatively with Apo A1 and HDL . In a laThe aim of this study was to investigate if tHcy and its major determinants folate, vitamin B12 and MTHFR polymorphism are correlated with Apo A1 levels in over 1500 subjects, without folate fortification, including both men and women and to see if this correlation was present among those who did and did not develop a first myocardial infarction. Using multiple regression models, an independent positive association was seen between plasma folate and Apo A1 levels among those who later did not develop a first myocardial infarction.The baseline characteristics including the apolipoprotein levels and the MTHFR 677 C>T and 1298 A>C distribution, are presented in Table The correlations of clinical data, analytes and MTHFR genotypes with Apo A1 in univariate linear regression analysis for men and women separately are seen in Table Among all men and male referents Vitamin B12 and folate levels were associated with Apo A1.Univariate analyses were performed in subgroups based on fasting status. Only 3.6% (n=41) of the men had fasted for less than 4 hours. An association was seen between homocysteine and Apo A1 in this non-fasting group. But among the male fasting subjects no significant associations were seen for tHcy with Apo A1. In contrast, folate remained significantly associated with Apo A1 among men that had fasted more than 8 hours. Among women no significant association was seen for tHcy and Apo A1 in the subgroups. For folate and Apo A1 a significant association was seen in the fasting group of 4–8 hours.Regarding MTHFR genotypes, no associations with apolipoproteins were seen in all men and women respectively, but among male referents, a negative association was found between Apo A1 and MTHFR 677 in a recessive model.2 of the total model was 0.144, i.e. it explains 14.4% of the variation in Apo A1 levels. Clinical data and plasma variables had R2 values 0.085 and 0.074 respectively. Among all women, corresponding R2 values were 0.106, 0.066 and 0.065.Multiple regression analysis for the association with ApoA1 is presented in Table The correlations of clinical data, analytes and MTHFR genotypes with Apo B in univariate linear regression analysis for men and women separately are seen in Table 2 values were 0.058, 0.045 and 0.018 in the total model, for clinical data and for plasma variables respectively and the R2 values for women were 0.191, 0.170 and 0.022. Adding homocysteine to the model did not change the major findings, but in female referents the correlation between Apo B and diabetes was no longer significant and in male cases a significant correlation between Apo B and creatinine was seen.Regarding MTHFR genotypes, no associations with apolipoproteins were seen in all men and women respectively, but in male referents between Apo B and MTHFR 1298A>C and with 1298 in a recessive model. Multiple regression analysis for the association with Apo B is presented in Table This study on the relationship between apolipoproteins, B-vitamins, homocysteine and MTHFR is to our knowledge the largest, including both men and women. We have previously reported that homocysteine levels were positively associated and folate concentrations in plasma were inversely associated with first myocardial infarction in this cohort . The assAs expected, Apo A1 and Apo B showed strong associations with risk for first myocardial infarction, except the association with Apo A1 in women where the odds ratio increased after adjustments and was no longer significant. Adjusting Apo A1 for folate did not change the significant findings, which is in line with our previously reported study [Analyzing over 1500 subjects, from a population without folate fortification, we found no clear relationship between plasma tHcy and Apo A1 levels in multiple regression. In contrast folate was positively associated with Apo A1 levels in multiple analyses in all men and women, as well as among male and female referents. This association was not seen among cases which could be due to lower statistical power as the beta-values for male cases and referents are similar -deficient mice suggested that cysteinemia rather than homocysteinemia is negatively correlated with Apo A1 . AnotherFolate, in the form of 5-methyl tetrahydrofolate, is the main source of methyl groups used for methylation, for example by DNA methyl transferases. Thus, one-carbon metabolism is closely linked to methylation of genes, for example promotor regions. This epigenetic mechanism may either silence or activate a gene. We have previously published data on the effects on CpG island methylator phenotypes . AlteredOther studies in mice and cells including the modulation of PPAR α, supports a negative correlation between tHcy and Apo A1 ,44. AlsoA main strength in our study is the design where we could study subjects that did not have previous MI, stroke or cancer, at inclusion, with both men and women in the cohort. These findings are probably representative for the general population as we only excluded subjects with prior MI, stroke or cancer and we were able to analyze separately those who later did or did not have a first MI. We also excluded all subjects with lipid lowering drugs. Other strengths are that we analyzed the B-vitamins folate and B12, and that the results were not confounded by folate fortification. A limitation of this study is that lifestyle factors can be a confounding factor, as healthy eating and exercise could lead to higher levels of both Apo A1 and folate. The number of cases may be too low to reveal a weak association between folate and Apo A1. Not all subjects were fasting at time of blood sampling. This may have had an effect on the results, as tHcy may be influenced by a high methionine meal. However, the number of subjects that were not fasting was low and excluding those subjects had no effect on the associations. As alcohol is known to have impact on lipoproteins, it is also a limitation that alcohol intake data was only available for a subset and thus could not be corrected for .In conclusion, tHcy was not associated with Apo A1 levels in a large cohort with over 1500 subjects without folate fortification. Instead, plasma folate, the major dietary determinant of tHcy, was found to be independently and positively associated with Apo A1 levels. This association was seen for both men and women, but restricted to referents. Those who later developed MI showed no association with folate. Future studies on the association between tHcy and endpoints or biomarkers should also take into account major determinants of tHcy, e.g. folate.This was a prospective incident nested case-referent study within the Northern Sweden Health and Disease Study Cohort (NSHDSC) . The cohAll participants were invited to complete a standardized questionnaire including life-style factors and subjects were interviewed by a trained nurse for all sorts of medication used. Those participating within the MSP completed a short questionnaire. The standardized questionnaires were used to calculate alcohol intake (g/day). This information was only available for a subset  and thus not included in the analysis.In the study, all cases with fatal and non-fatal first MI and suspected fatal MI , between the ages of 25 and 64 years and from 1 January 1985 to 31 December 1999 were included. The case identification was done in a standardized manner in accordance with WHO and MONICA criteria . Using SDiabetes was defined as fasting plasma glucose ≥ 7.0 mmol/L and/or post-load plasma glucose ≥ 11.0 mmol/L  and/or self-reported diabetes. For classification as non-diabetic, measurement of fasting or post-load glucose was required. Fasting glucose and/or 2-hour oral glucose load tests were available for 444 cases and 855 referents. Patients with lipid-lowering drugs (n=9) were excluded. Hypertension was defined as systolic blood pressure (BP) ≥ 140 mm Hg and/or diastolic blood pressure ≥ 90 mm Hg and/or use of medication to lower blood pressure. Current smoking was defined as daily smoking, excluding non-, previous-, and non-daily smokers. Information about pre- or postmenopausal status among women was not available in this study. Postmenopausal woman was therefore defined as ≥ 56 years of age at inclusion (n=311), the age when 95% are estimated to be in menopausal status in a Swedish material . AccordiAll participants have given written consent. The Research Ethics Committee of Umeå University, Umeå and the National Computer Data Inspection Board have approved the data handling procedures.Peripheral venous blood samples were collected in evacuated test tubes containing heparin, in MONICA and VIP, generally in the morning after at least four hours fasting. In MSP blood samples were collected throughout the day. In the study, 66.9% (n=762) of the men had fasted for more than 8 hours and 15.7% (n=179) had fasted between 4–8 hours, while 3.6% (n=41) had fasted for less than 4 hours. Information about fasting status was missing for 13.8% (n=157) of the men. Corresponding fasting status for women was, 36.5% (n=168), 14.8% (n=68) and 43.0% (n=198). Information about fasting status was missing for 5.7% (n=26) of the women.Plasma was aliquoted after centrifugation at 1500g for 15 minutes. Until analysis they were stored frozen at −80˚C. Plasma specimens were analyzed in triplets of one case and two referents. To avoid systemic bias and interassay variability the position for the case was varied in random and the laboratory staff were blinded to case and referent status.Apo A1 and Apo B were analyzed with immune turbidimetry on a Hitachi 911 multianalyzer with reagents from DAKO . Homocysteine was analyzed in batch with fluorescence polarization immunoassay on an IMx® unit . P-B12 and P-folate were analyzed in batch using DPC® Solid Phase No Boil Dualcount radio assay . Creatinine, albumin and cystatin C were analyzed with a Hitachi 911 multianalyzer, creatinine  with kits from Roche/Boehringer . Albumin was analyzed with immune turbidimetry and Cystatin C with an immunological method, both with reagents from DAKO . C-reactive protein (CRP) was analyzed with an automated method, IMMULITE 1 . Analyses below were analyzed in batch and thus intra-assay coefficients of variation are presented. For Apo A1 coefficients of variation (CV:s) were 0.96% and 0.61% at concentrations 0,91 and 1,65 g/L, respectively and for Apo B 1.21% and 0.62% at concentrations 0.47 and 0.81 g/L, respectively. Homocysteine had CV:s of 2.7% and 1.9% at concentrations 13.1 μmol/L and 25.7 μmol/L respectively. P-folate had CV:s of 15.8% and 12.1% at concentrations 7.3 mol/L and 30.4 nmol/L, respectively and P-B12 16.5% and 10.4% at the concentrations of 263 pmol/L and 1001 pmol/L. For control samples, for creatinine CV:s were 1.59% and 3.21% at concentrations 84 and 367 μmol/L respectively, for albumin 0.89% and 2.25% at 38.0 g/L and 64.3 g/L and for Cystatin C 2.90% and 1.20% at 1.12 and 4.59 mg/L. For CRP the interassay coefficient of variation was <6%.MTHFR genotyping was performed by the TaqMan allelic discrimination method, using Minor Groove Binder (MGB) probes. TaqMan assays and reagents were from Applied Biosystems . The PCR reactions were performed on GeneAmp PCR system 9700, PCR programs  at the Center for Genome Research, Umeå University and the PCR products were analyzed on the ABI PRISM 7900HT Sequence Detection System.2-test.Calculations were performed using IBM SPSS Statistics version 19.0 . Hardy-Weinberg equilibrium was calculated, based on the χTo compare baseline characteristics and study variables for cases and referents T-test and Chi-square test for independence were used for continues and categorical variables respectively. Plasma apolipoprotein quartile limits, based on referents, were separately calculated for men and women and treated as categorical variables, except when testing for trend, where they were included as continuous linear variables in the regression analyses. Odds ratios (OR) for disease and 95% confidence intervals (CI) were calculated by conditional logistic regression. Adjustment was done only for folate and with two other models both including smoking, systolic blood pressure, BMI and CRP but with and without diabetes. Missing values were treated as missing.To find factors influencing Apo A1, linear regression analysis was performed. Normal distribution could not be applied for CRP in our population and instead transformation was done to logarithmic values. In univariate linear regression MTHFR genotypes were coded as follows: one model using the three outcomes coded 0,1,2; one recessive model (wild type and heterozygote mutant v.s. homozygote mutant ) the other, dominant model (wild type v.s. heterozygote mutant and homozygote mutant).Analytes and genotypes that were non-significantly (p<0.05) associated with apolipoproteins in univariate analysis of variance were excluded in the multiple regression models. An exception was homocysteine were we wanted to test the hypothesis regardless the result in univariate analysis.tHcy: Total plasma homocysteine; Apo A1: Apolipoprotein A1; Apo B: Apolipoprotein B; MTHFR: Methylenetetrahydrofolate reductase; MI: Myocardial infarction.The authors declare that they have no competing interests.JHJ, GH, LW and ME designed the cohorts. The study was designed by JH, ES, ME, JHJ and OJ. JH carried out biochemical and genetic analysis. JH and ES performed the statistical analysis and interpreted the data. Drafting was made by JH and ES and all other authors revised it critically. All gave final approval of the version to be published.


The (pro)renin receptor [(P)RR] has been recognized as a multifunctional receptor. The purpose of this study was to assess the association between plasma soluble (P)RR [s(P)RR] concentration in human cord blood  and small for gestational age (SGA) birth.th percentile for gestational age. Logistic regression analysis was performed to assess the association between cord plasma s(P)RR concentration (quartiles) and incidence of SGA births.Participants were women with a singleton pregnancy who delivered at the National Center for Child Health and Development between January 2010 and December 2011. Inclusion criteria were availability of maternal pre-pregnancy and paternal body mass index, and the absence of structural anomalies in neonates. s(P)RR concentration in cord blood was measured in 621 neonates. The 621 pairs of mothers and neonates were categorized into four groups based on quartiles of s(P)RR concentrations in cord blood. SGA was defined as a birth weight below the 10P-value for trend was also significant (P = 0.020).Among 621 neonates, 55 (8.9%) were diagnosed as SGA (SGA group) and 566 (91.1%) were not (non-SGA group). Average s(P)RR concentration in cord blood was 66.1±12.6 ng/ml (mean±standard deviation). There were 155 pairs in the first plasma s(P)RR concentration quartile (Q1: <58.2 ng/ml), 153 pairs in the second quartile (Q2: 58.2–65.1 ng/ml), 157 pairs in the third quartile (Q3: 65.1–73.1 ng/ml) and 156 pairs in the fourth quartile (Q4: >73.1 ng/ml). The distribution of SGA births was 18 (11.6%) in Q1, 14 (9.2%) in Q2, 16 (10.2%) in Q3 and 7 (4.5%) in Q4, respectively. The odds ratio of SGA births was 0.24  for the fourth quartile compared to the first quartile in multivariate models. The High s(P)RR concentration is associated with a lower SGA birth likelihood. To confirm our hypothesis, we conducted a cross-sectional study to assess the association between plasma s(P)RR concentration in human cord blood  and SGA.th percentile for gestational age was classified as SGA. Birth weight for gestational age was determined using the percentile scale derived from the formula used in Japan This study was approved by the Ethics Committee of the National Center for Child Health and Development (NCCHD). The study included Japanese women who delivered at the NCCHD hospital between January 2010 and December 2011, and their neonates. Inclusion criteria were a singleton pregnancy, availability of maternal pre-pregnancy and paternal body mass index (BMI) data and absence of structural anomalies in the neonates. We enrolled and obtained written informed consent from all participants during pregnancy (before labor pain onset). All blood samples were obtained from the umbilical vein at delivery. We weighed all neonates just after birth using neonatal digital scales . A birth weight below the 10Assays were performed by someone who was blinded to the outcome of the pregnancy. Enzyme-linked immunosorbent assays (ELISAs) for human s(P)RR were performed in duplicate, as previously described, using commercial kits  Differences in baseline characteristics and birth outcomes between SGA and non-SGA groups were compared using the chi-square test or Fisher's exact test for categorical variables, and the t-test or Wilcoxon rank sum test for continuous variables.To examine the association between baseline characteristics, demographics, or birth outcome, and s(P)RR concentration, participants were categorized into four groups based on s(P)RR concentration in cord blood . Associations between s(P)RR quartiles and baseline characteristics or birth outcome were assessed by regression analysis for continuous variables and logistic regression for categorical variables.Logistic regression analysis was used to investigate the association between cord plasma s(P)RR concentration quartile and SGA. All multivariate models were adjusted for variables if p values were below 0.2 in the aforementioned analyses RR quartiles and baseline characteristics or birth outcome). We performed two sub-analyses to assess sensitivity, which excluded participants with 1) complications , and 2) gestational age <39 weeks, as these factors might be associated with s(P)RR levels in vitro fertilization was lower in the SGA group than in the non-SGA group . Gestational weight gain was smaller in the SGA group than in the non-SGA group . Birth weight and placental weight were both lower in the SGA group than in the non-SGA group . Prevalence of hypertensive disorder was higher in the SGA group than in the non-SGA group . Finally, s(P)RR concentration in cord blood was lower in the SGA group than in the non-SGA group .A comparison of baseline characteristics and birth outcomes between SGA and non-SGA groups are shown in P<0.001; Q2 vs. Q4: P = 0.002 and Q3 vs. Q4: P = 0.009). There were no significant differences in other baseline characteristics and birth outcomes among the four quartiles.P = 0.026). The P-value for trend was significant (P = 0.048). This association was also significant in multivariate models. The odds ratio of SGA was 0.24 for Q4 compared to Q1 . The P-value for trend was also significant (P = 0.020). That is, neonates with a higher plasma s(P)RR concentration in cord blood were unlikely to be SGA.In the unadjusted model, the odds ratio of SGA was 0.36 for Q4 compared to Q1 , gestational diabetes mellitus and hypertensive disorders in pregnancy. This revealed an SGA odds ratio of 0.16 (95% CI: 0.043–0.60) for Q4 compared to Q1, which was similar to the point estimate for the whole study population.Second, we performed a logistic regression analysis excluding women who delivered at 39 weeks of gestation or earlier, and found an SGA odds ratio of 0.27 (95% CI: 0.91–1.10) for Q4 compared to Q1, which was also similar to the point estimate for the whole study population.To the best of our knowledge, this is the first study to demonstrate the association between cord plasma s(P)RR concentration and fetal growth in humans. Specifically, neonates in the highest s(P)RR concentration quartile (Q4) were 0.24-fold less likely to be SGA than neonates in the lowest quartile (Q1), indicating that s(P)RR might be associated with appropriate fetal growth.In the present study, s(P)RR concentrations in cord blood  were markedly higher than reported for normal adult volunteers Among the three known functions of (P)RR , we surmise that the relationship between (P)RR and WNT signaling is highly significant, although the role of (P)RR in embryonic and fetal development remains unclear. Recent studies have identified (P)RR as an essential adaptor protein in the WNT signaling pathway Interestingly, a previous study found that higher maternal s(P)RR concentrations were associated with elevated blood pressure and a higher prevalence of preeclampsia Furthermore, as SGA prevalence was significantly lower only in the fourth quartile group and showed no significant differences between the first, second and third quartile groups, we wonder if the association between s(P)RR concentrations in cord blood and SGA births applies only under a certain threshold.Several studies suggest that SGA increases the risk for neurodevelopmental delay Our study has some limitations worth nothing. First, we could not determine whether decreased s(P)RR concentration in fetal blood induced abnormal fetal body development or abnormal fetal development decreased s(P)RR levels because we assessed this association at only one point . Assessment of s(P)RR concentration in fetal blood during pregnancy would clarify the contribution of (P)RR to fetal growth, although such a study may not be feasible. Second, the origin of s(P)RR in cord blood could not be determined, since the mechanism of placental transfer of s(P)RR from maternal circulation remains unclear. However, given that the molecular weight of s(P)RR is 28,000 In conclusion, our study showed that higher s(P)RR concentration was associated with appropriate intrauterine fetal growth . Conversely, neonates with lower s(P)RR concentrations in cord blood may be abnormal in their intrauterine growth and represent follow-up candidates for adult diseases and neurodevelopmental disorders. The association between (P)RR and fetal origin of adult disease is an exciting and interesting theme for future investigation. Large, long-term cohort studies of (P)RR are needed to clarify this association.


Best practice guidelines to promote breastfeeding suggest that (i) mothers hold their babies in naked body contact immediately after birth, (ii) babies remain undisturbed for at least one hour and (iii) breastfeeding assistance be offered during this period. Few studies have closely observed the implementation of these guidelines in practice. We sought to evaluate these practices on suckling achievement within the first hour after birth.Observations of seventy-eight mother-baby dyads recorded newborn feeding behaviours, the help received by mothers and birthing room practices each minute, for sixty minutes.Duration of naked body contact between mothers and their newborn babies varied widely from 1 to 60 minutes, as did commencement of suckling (range = 10 to 60 minutes). Naked maternal-infant body contact immediately after birth, uninterrupted for at least thirty minutes did not predict effective suckling within the first hour of birth. Newborns were four times more likely to sustain deep rhythmical suckling when their chin made contact with their mother’s breast as they approached the nipple  and if their mothers had given birth previously . Infants who had any naso-oropharyngeal suctioning administered at birth were six times less likely to suckle effectively .Effective suckling within the first hour of life was associated with a collection of practices including infants positioned so their chin can instinctively nudge the underside of their mother’s breast as they approach to grasp the nipple and attach to suckle. The best type of assistance provided in the birthing room that enables newborns to sustain an effective latch was paying attention to newborn feeding behaviours and not administering naso-oropharyngeal suction routinely. The Baby Friendly Hospital Initiative (BFHI) recommends that newborn babies be placed in skin-to-skin contact with their mothers immediately following birth for at least one hour, and mothers helped to initiate breast feeding within the first half-hour following birth ,2. ‘Ten The term ‘skin-to-skin contact’ is defined as ‘naked newborn placed on his/her mother’s bare chest’ and is widely used in practice. However some mothers and midwives may misunderstand the term and believe that placing babies cheek to cheek with the mother or resting the baby’s face on their mother’s breast might be ‘skin-to-skin’. We argue that the term ‘naked body contact’ more clearly portrays the concept of naked baby on mother’s bare chest. In this paper, the term ‘skin-to-skin contact’ will be used when reference is made to existing research studies and ‘naked body contact’ will be used for presenting the findings of the present study.The benefits of skin-to-skin contact between mother and newborn during the first hours after birth are well known -15. SkinThe first 2 hours after birth is a sensitive period in which mothers are particularly responsive to their newborns ,23. Rececorrect’ positioning and ‘correct’ attachment [BFHI accreditation standards recommend that mothers receive help to breastfeed within 30 minutes of birth and emphasize ‘tachment ,27. Howetachment -31, midwtachment  in the btachment ,20,33. Wtachment . These fTo our knowledge, no published studies have explored relationships between time newborns are in skin-to-skin contact and effectiveness of suckling. Furthermore, there has been very little research on the extent of help mothers receive in the birthing room. Descriptions of best practice for breastfeeding commencement could inform birthing room protocols. The aim of this study was to identify predictors of newborn suckling behaviour within the first of hour after birth. It was hypothesised that naked newborn infants placed immediately on their mothers’ bare chest after birth and left undisturbed for at least 30 minutes would commence breastfeeding and suckle effectively sooner than infants not receiving this level of contact.Pregnant women over 18 years of age and intending to breastfeed were recruited from the antenatal clinic of the participating hospital. Participants were at least 36 weeks gestation, able to communicate in English, and with no major prenatal complications or underlying medical problems likely to impact on their ability to hold their naked baby immediately after or within 30 minutes of birth. One hundred and three women enrolled in the study. Participating women returned a signed consent form and completed demographic information plus a breastfeeding intention/history questionnaire. In terms of the recommended ratio of variables to subjects ,35 the 7The study was conducted within the Women and Birthing Services at a public metropolitan hospital in Queensland, Australia. The site was preparing for BFHI accreditation. Approval by the Human Research Ethics Committees for Griffith University and Metro South Health Service District was obtained.Personal information such as age, income and education and obstetric data in relation to wellbeing, previous pregnancies and breastfeeding history was sought.The birthing room observation tool was devised using a time line grid with items listed down one side. The list of items was developed in consultation with midwifery colleagues and drew on standard maternity procedures, perinatal data collection forms , and a cThe observation tool was based on four overarching sets of variables related to: 1) Birthing events and placement of babies immediately following birth; 2) Newborn feeding behaviours; 3) Maternal behaviour to initiate breastfeeding; and 4) Assistance mothers received to position and attach their baby to the breast.Birthing events and placement of babies immediately following birth consisted of 65 items. There were 5 sub-categories. i) Birth: items recorded information such as time of birth and immediate observations associated with the birth for example, baby weak/vigorous cry. ii) Baby placed: determined what happened to babies from the first moment of birth and at any time throughout the hour, for example, if babies were put on their mother’s abdomen, resuscitation cot or elsewhere. iii) Maternal response to baby: recorded how mothers responded to their infants, and the point in time mothers held their babies, for example, mother picks newborn up, holds baby on bare skin. iv) Baby held: ascertained by whom babies were held, at what point in time and whether wrapped, covered or clothed, for example, by mother, by father, loosely wrapped, nappy on. v) Resuscitation: verified the point in time any resuscitation event occurred such as mouth/nose suction, nasal oxygen . From th2) Newborn feeding behaviours had 32 items. This was a list of expected, known, and defined feeding behaviours ,37,47,483) Maternal behaviour to initiate breastfeeding had 21 items. This section clarified actions mothers took to position themselves or their baby to offer their babies a breastfeed at any point in time, e.g. mother positions herself, positions baby close etc.4) Assistance mothers received to position and attach their baby to the breast had 19 items such as breastfeeding assistance offered, baby held to breast for mother, and readiness to breastfeed discussed . This deThe tool was pilot tested on two births before commencing data collection. Three new observations were added and several repetitious items deleted in order to refine the tool.Midwives who worked regularly in the birthing suite (n = 23 out of a possible 40 permanent midwifery staff) agreed to the researcher being present and recording observations of consenting mothers in their care. Midwives were requested to not make changes to their normal practice, and behave as if the researcher was not present. Staff at the Unit informed the researcher of a participating woman’s admission. The researcher then arrived at the birthing room, confirmed the consent of the woman and her partner and gained permission to be present from the person managing the birth.Equipment for observations included the observation record sheet on a clip board, a stop-watch, a tape recorder, ear phones, and a pen. When birth was imminent, the researcher entered the room to observe. At the moment of birth the researcher pressed the stop-watch, recorded the time of birth and pressed the play button on the tape recorder . A pre-rObservations were unobtrusive with no interaction with mothers, family or staff ,50. A goData were prepared for analysis in two steps. The first step was data cleaning and data entry in Microsoft Excel prior to importation to the Statistical Package for the Social Sciences (SPSS) . The seci) Continuous variables measured uninterrupted naked body contact time between mother and newborn or time mothers and babies were separated as well as time between, before or after specific events. For example, time infants suckled effectively and longest time mothers’ and newborns’ spent in naked body contact were calculated as continuous variables that could be categorized.ii) Categorical variables were developed from the continuous data as well as from raw data collected such as ‘mode of birth’. Length of time or ‘duration of naked body contact’ was calculated from the first moment baby was placed naked on his/her mother’s bare chest to the time baby was removed. Categorical dichotomous variables were created to measure whether or not naked body contact was interrupted, and the mother, infant or staff performed various behaviours. Four categories of ‘continuous uninterrupted’ and ‘interrupted naked body contact’ were ‘0 = naked body contact immediately after birth and continued undisturbed for at least 30 to 60 minutes’, ‘1 = naked body contact interrupted within 1–5 minutes then commenced or continued for at least 30 minutes’, ‘2 = naked body contact commenced after 5 minutes and interrupted before 30 minutes’, ‘3 = no naked body contact at all or none after the first minute’.iii) The third process used to determine variables for analysis involved an analysis of items observed in the birthing room using Principal Components Analysis (PCA) ,52,53. TNaked body contact  means the baby is naked (with the exception of a nappy) and is lying face down on his/her mothers’ bare chest. As described above, ‘continuous uninterrupted naked body contact’ means no interruption of maternal-infant naked body contact from the moment of birth to at least 30 minutes. ‘Interrupted naked body contact’ refers to maternal-infant naked body commenced between 2–5 minutes after birth or interrupted within the first 5 minutes of birth and resumed for at least 30 minutes. ‘Naked body contact later’ means maternal-infant naked body contact commenced later than 5 minutes after birth. Cases where babies were placed with their mother on top of clothing or bed linen or wrapped were not considered naked body contact until the moment they were actually placed bare on the mother’s bare chest.‘Chin contact breast’ means the baby’s chin nudged mother’s breast as he/she approached to suckle either independently or with assistance. The single item ‘suckled at the breast for the first time’ could be verified without doubt by mother and midwife and significant others involved and were defined as ‘commencement of suckling’ or suckling achievement. ‘Effective suckling’ was defined as deep rhythmical suckles sustained for at least four minutes.‘Mother initiates breastfeeding’ was where mothers acted independently to position themselves or their babies to offer their babies a breastfeed. ‘Positioning and entice to attach’ was constructed from any one of four items, ‘turns baby to face herself’, ‘positions baby close’, ‘expresses colostrum for baby or ‘holds nipple to baby’s mouth’.The term ‘assisted to attach’ was defined by one or more actions occurring . Mothers who received hands on assistance to attach baby to the breast regardless of other help received were deemed to be in the category of ‘assisted to attach’. The IV ‘head held and assist to attach’ meant the back of baby’s head was held when ‘assisted to attach’.Definitions for other categorical dichotomous variables were ‘suction’ defined as any nasal, oral, pharyngeal or gastric suction administered to babies. ‘Analgesia’ refers to either Pethidine injection, epidural or spinal anaesthesia. Parity is the number of live born children a woman has given birth to. Primapara are first time mothers and multipara are women who have given birth to two or more live born children.Data were analysed using International Business Machines (IBM) SPSS version 21 . An alphProposed predictor variables were chosen as described above based on clinical observation and experience ,56, the Of recruited mothers n = 103), 78 births were observed  began suckling within 60 minutes following birth. The average time to begin suckling was 38 minutes . Thirty babies (38.5%) ‘breastfed well’ with deep rhythmical suckles sustained for at least four minutes. Twenty- three (29.5%) achieved ‘a few suckles’ or suckled for one to three minutes, fourteen (61%) of these made ‘repeated attempts’ before attaching to suckle. Of the twenty-five (32%) babies who did not suckle, twelve (48%) were unable to latch despite ‘attempts to grasp the nipple’ while nine (36%) only mouthed the nipple or ‘licked and nuzzled’ and four (16%) made no attempt as if ‘not interested in feeding’.PCA identified four components that explained a total of 46% of the variance of newborn feeding behaviours. ‘Effective suckling’ contributed 18.5% of the variance and consisted of three items ‘grasp nipple for the first time’, ‘suckles at breast for the first time’ and ‘sustained suckle ’. ‘Hand to mouth co-ordination’ comprised eight pre-feeding behaviours that explained 12.6% of the variation. Five items related to newborn ‘head movement’ contributing 7.4% were ‘turns head’, ‘lifts head up’, ‘nudges breast with chin’, ‘wide gape’ and ‘attempt to grasp nipple’. ‘Attempt to attach’ contributed 7.3% with items ‘attempt to grasp nipple’, ‘nudges breast with chin’, ‘wide gape’ and ‘attempt to turn head’.Many inconsistencies in practice of naked body contact between mother and newborn during the first hour of birth were evident. Continuous uninterrupted naked body contact from the moment of birth and for a full sixty minutes was experienced by twenty-six (33%) mothers and their newborn babies. Five (6.4%) mothers and their newborns were undisturbed for 43–56 minutes from the moment of birth. Only mothers who gave birth vaginally held their newborns in naked body contact within the first minute of birth .Naked body contact was interrupted within the first 5 minutes of birth for twenty-two (28%) mothers and their newborns. Eleven (14%) infants began contact immediately but were moved away from their mothers for resuscitation procedures or held by another person and then put back to maintain naked body contact for 37–58 minutes. Eleven (14%) began contact between two and five minutes then remained undisturbed for 34–59 minutes.‘Skin contact later’ was experienced by seventeen (21%) mothers and their newborn babies. One (1.2%) infant who commenced naked body contact immediately after birth but was interrupted during the first minute and not recommenced was categorised with infants who had no naked body contact. Seven (9%) mothers and their infants had no time in naked body contact during the first hour of birth. All mothers who had a caesarean section  were initially separated from their babies for at least 24 minutes.The most common event to separate newborns from their mothers during the first hour after birth was resuscitation intervention  , followed by baby held by other person , warming , routine practices such as wrapping babies, vitamin K injection or weighing , assessment of babies , and mothers’ request to stop holding or not to hold her baby . Resuscitation procedures were carried out on all babies born by caesarean section and on two thirds of babies born vaginally (n = 42 out of 61).Three components of maternal behaviour to initiate breastfeeding identified by PCA were ‘state of nipple pain or trauma’ ‘getting ready’ and ‘position to entice and attach’. Fifty-nine mothers (75.6%) actively positioned themselves  or their babies  to begin breastfeeding. Thirty-seven (47%) held their nipple to their babies’ mouth and five (6.4%) expressed colostrum to entice their baby to latch.Mothers were mostly offered help to breastfeed. Mothers received a variety of assistance. PCA results confirmed the definition for ‘assist to attach’ chosen for the independent variable.Five descriptions for practices observed assisting mothers initiate breastfeeding emerged. Firstly, nine mothers (11.5%) received no assistance at all and only one of these was offered assistance. Secondly, seven mothers (9%) received ‘hands off’ midwifery support, meaning any verbal or nonverbal supportive instruction, encouragement or information without any ‘hands on assistance’. Most mothers  received ‘hands on’ help. Three categories of ‘hands on’ help identified were, 1) ‘minimal assistance’ when helpers touched babies to position them optimally or closer to the breast; 2) ‘moderate assistance’ where helpers touched mothers’ breasts or babies to help babies attach to their mothers’ breast and 3) ‘full assistance’ help to both position and attach babies to mothers’ breasts. See Table The back of babies’ heads were held in 22% of total cases, sixteen of moderate assistance and one of minimal assistance. Fourteen (82%) of these mothers had given birth vaginally, ten (59%) of which experienced continuous uninterrupted naked body contact with their infant (34–60 minutes) and four (23%) interrupted naked body contact (44–59 minutes). Twelve out of fourteen infants achieved suckling, but only four (33%) suckled effectively. One infant sustained suckling for 25 minutes and then was held by the father and others in the room. One infant suckled for 23 minutes after several repeated attempts to grasp the nipple. Two babies suckled effectively for 4 minutes, one toward the end of the hour and the other was unable to reattach when the back of his head was held to assist. The eight infants who did not suckle effectively, took a few suckles over a 2–3 minute period with repeated attempts to attach and suckle. One baby failed to sustain suckling when his mother held the back of his head and was then placed lying on his back so he could not reach the nipple for further attempts.All three (3.8%) of the infants born by caesarean section commenced suckling within the hour. However, the one who had no maternal-infant naked body contact suckled effectively. Of the two in naked body contact with their mothers, one suckled effectively for seven minutes, the other suckled off and on for seven minutes and the mother reported nipple pain toward the end of the hour.Chi-square test for independence (with Yates continuity Correction) indicated ‘suction’, ‘chin contacts breast’ and ‘parity’ had significant associations with ‘effective suckling’  (see Table p = .142).A direct logistic regression analysis was performed to assess the impact of four factors on the likelihood of newborn infants suckling effectively during the first hour of life. Predictor variables entered into the model were parity, suction, chin contacts breast, and duration of naked body contact. The hypothesis that infants placed naked on their mother’s bare chest from the moment of birth and left uninterrupted for at least 30 minutes after birth would breastfeed effectively was not confirmed . Babies were four times more likely to suckle effectively when their chin made contact with their mother’s breast as they approached the nipple . Likewise, infants of multipara mothers were four times more likely to suckle effectively  than first time mothers. Results of the direct logistic regression analysis model are presented in Table The key findings of this study are that effective suckling within the first hour of birth can be predicted by a model of best practice, including positioning babies so they can instinctively nudge their chin into the underside of their mother’s breast as they approach the nipple and attach to suckle.This study has several unique features. First, the study reported detailed observations of what actually happened in the birthing room for the first hour. The use of video recording could miss some activities. Second, the detailed observations of infant feeding behaviour and maternal assistance received provide definitions for both suckling commencing and assistance. Third, the analysis identified predictors of effective suckling in the first hour of life that have not been reported elsewhere.The measure used as the outcome variable for effective suckling was that infants grasped the nipple, commenced and sustained suckling for more than three minutes. Previous researchers have described both ‘reaching the nipple’ and ‘effective suckling’ as outcome variables but the latter has been criticised as subjective ,62. Reseor within five minutes after birth” (p.11) [Naked body contact was interrupted on many occasions for both newborns placed immediately on their mothers’ bare chest and those placed in naked body contact after initial separation. The wide range of inconsistent practices observed may be explained in part by maternal choice at the time. For example, some mothers gave birth in positions where it was not conducive or safe to hold their naked baby immediately (such as standing up leaning over the bed). Others started holding their naked baby but requested another person to hold the baby while they got comfortable or needed brief medical attention. In several cases, mothers wanted to take time to look at and examine their baby and adapt to the fact of having just given birth before holding their naked newborn on their bare chest. Results of this study validate revised BFHI Global criteria accreditation guidelines in Australia that newborns are placed “with their mothers in skin-to-skin contact immediately ” (p.11) .The frequent interruptions of naked body contact observed in this study may have interfered with some infants achieving suckling and suckling effectively within the hour. Righard and Alade reported that on average newborns begin active feeding behaviours at around twenty minutes and this is a crucial time for infants to not be disturbed . On the Given that over half of the babies were placed immediately with their mothers but naked body contact was disturbed for a third of them , it was not possible to determine the importance of continuous uninterrupted naked body contact as a predictor of effective suckling within the hour. Since the study ended after 60 minutes it is not known how many newborns suckled effectively over the next few hours or if they remained in naked body contact. Widstrom et al.  emphasisFindings revealed some important insights about positioning and attachment at the breast. When the baby’s chin is in contact with the breast, the gape reflex is elicited enabling the baby to gape widely and take an adequate amount of breast tissue as observed by Widstrom et al. ,18. The ‘correct’ positioning and ‘correct’ attachment are emphasised in the postnatal period [Observation and working with newborn feeding behaviour is supported by revised BFHI guidelines ,27. Howel period ,74,75. DDescriptions of assistance mothers receive are useful measures for clinical application, education and further research. Results of this study quantify terms commonly used in midwifery practice to describe assistance mothers receive to initiate breastfeeding. To our knowledge no formal definitions of these terms have been published to date.‘correctly’, many of the women actively positioned themselves or their babies to begin breastfeeding without any instruction. To date, BFHI guidelines have not adequately defined newborn readiness to begin feeding or the nature of help to give, yet stipulate that help be given within 30 minutes [Although breastfeeding is perceived to be a learnt skill with mothers requiring help to position and attach baby  minutes . Even th minutes  it may b minutes ,56. DescAmongst mothers and infants who received ‘assistance to attach’, the back of the baby’s head was held for almost half the cases (46%). The greater proportions of these were mothers who gave birth vaginally and who held their babies in naked body contact continuously from the start or with only a short interruption at the beginning. This may explain lack of support for the research hypothesis. Holding the back of the baby’s head at a feeding attempt can be counterproductive and lead to breast refusal . ResultsFurther research is warranted to test differences between birth types. BFHI Global Criteria for ‘Step 4’ advocates that mothers who have a caesarean section without general anaesthesia be offered the same care as mothers who give birth vaginally . While bObservations ceased at 60 minutes so it is not possible to know how many continued on in maternal-infant naked body contact and fed well before being disturbed within a 2–3 hour timeframe. It is known that there should not be a rush to have the baby feed in the first hour . It is pThe adverse effect of suction on newborn feeding ability needs attention in midwifery education programs. In this study, the likelihood of babies achieving suckling within the hour diminished if suctioning intervention was administered and confirms available evidence ,20,21. RResults of the present study need to be considered in light of limitations concerned with sample size, “Hawthorne effect” and practice changes since data collection. Although recruitment allowed for losses, there were more missed births than anticipated, reducing the overall sample size. However, in comparison to small samples (n = 21) in other observational studies e.g. Widstrom et al.,  the presSuckling commencement within the first hour after birth is associated with a collective model of practices including infants held and positioned so their chin can instinctively nudge the underside of their mother’s breast as they approach to grasp the nipple and attach to suckle. It is the type of help given in the birthing room that midwives need to pay attention to. The help provided is best if done in conjunction with, paying attention to newborn feeding behaviour s and readiness of babies to begin suckling, no unnecessary suction and if any hands on assistance to attach is needed it should be gentle with helping babies’ chins to approach the breast first.PCA: Principal Components Analysis; DV: Dependent variable; IV: Independent variable; IVs: Independent variables; BFHI: Baby Friendly Hospital Initiative; IBCLC: International Board Certified Lactation Consultant; SPSS: Statistical Package for the Social Sciences; IBM: International Business Machines.The authors declare that they have no competing interests.There are no financial or non-financial competing interests  to declare in relation to this manuscript.RC was involved in the conception and design of the study, protocol writing, designing and analysis of the observation tool, data collection, analysis and interpretation of the results, and in drafting the manuscript and was the lead author on the manuscript. DC was involved in the conception and design of the study, interpretation of the results, and in drafting the manuscript and critical review of the manuscript for significant intellectual input. MC assisted in conception and study design, and critical review of the manuscript. FD assisted in conception and study design and critically reviewed the manuscript for substantial intellectual input. All authors read and approved the final manuscript.RC is a registered nurse, midwife and International Board Certified Lactation Consultant at Metro South Hospital and Health Service, Queensland Health and was the researcher undertaking this observation study (January to July 2004). DC is Professor at Griffith Heath Institute. MC is Professor of School of Nursing and Midwifery and Griffith Heath Institute. FD is Professor of Maternal and Infant Health and director of MAINN.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2393/14/20/prepub


Understanding the fitness consequences of inbreeding is of major importance for evolutionary and conservation biology. However, there are few studies using pedigree-based estimates of inbreeding or investigating the influence of environment and age variation on inbreeding depression in natural populations. Here we investigated the consequences of variation in inbreeding coefficient for three juvenile traits, birth date, birth weight and first year survival, in a wild population of red deer, considering both calf and mother's inbreeding coefficient. We also tested whether inbreeding depression varied with environmental conditions and maternal age.We detected non-zero inbreeding coefficients for 22% of individuals with both parents and at least one grandparent known (increasing to 42% if the dataset was restricted to those with four known grandparents). Inbreeding depression was evident for birth weight and first year survival but not for birth date: the first year survival of offspring with an inbreeding coefficient of 0.25 was reduced by 77% compared to offspring with an inbreeding coefficient of zero. However, it was independent of measures of environmental variation and maternal age. The effect of inbreeding on birth weight appeared to be driven by highly inbred individuals (F = 0.25). On the other hand first year survival showed strong inbreeding depression that was not solely driven by individuals with the highest inbreeding coefficients, corresponding to an estimate of 4.35 lethal equivalents.These results represent a rare demonstration of inbreeding depression using pedigree-based estimates in a wild mammal population and highlight the potential strength of effects on key components of fitness. Inbreeding depression, the reduction in fitness of offspring resulting from matings between related individuals, is of considerable importance in studies of evolution, ecology and conservation [for reviews see -4]. HoweCervus elaphus) on the Isle of Rum, Scotland.One reason for the lack of studies of inbreeding depression in natural populations is the difficulty in collecting the data required to estimate levels of inbreeding. An individual's inbreeding coefficient F, defined as the probability that two alleles at any randomly-chosen locus are identical by descent , can be Drosophila have demonstrated that the magnitude of inbreeding depression is dependent on the environment experienced  a aDrosophed . In . In Drosed  et al. .F≥0.0625; i.e. a mating between first cousins or closer relatives), a number of inbred individuals also have low but non-zero levels of inbreeding  and the majority of inbred individuals have moderately low levels of inbreeding . Thus the depth of pedigree information available for this population allows us to detect numerous low level inbreeding events. Contrary to results from a highly inbred island populations of song sparrows . In thersus 644 , 573 [49rsus 644  in previrsus 644  [but seersus 644 ].1,1108 = 0.73, p = 0.392). Given that inbreeding depression in birth weight was driven by individuals with the highest inbreeding coefficients, it may be that the effects of such high levels of inbreeding are independent of environment. Inbreeding effects on offspring survival also showed no sign of interactions with environmental variation. The effect of inbreeding on offspring survival was strong and therefore perhaps independent of environmental variation, or it is possible that winter conditions are in general stressful enough that effects of inbreeding are independent of environmental variation. It is also possible that we lack the power to detect such effects, as suggested by the large standard errors on some of the fixed effects . The same pattern was true for first winter survival with the quadratic effect of maternal age being marginally significant when birth weight was removed .Patterns of inbreeding depression differed between the juvenile traits examined here. All effects of inbreeding were due to the offspring inbreeding coefficient, but birth date showed no clear evidence for inbreeding depression. Given that this trait is most likely determined by oestrus date and female condition during gestation , thiCanis lupus baileyi) [Canis lupus) [Although offspring birth weight is an important predictor of offspring first year survival, we found that offspring first year survival suffered strong inbreeding depression independent of the effects of inbreeding on birth weight. The first year survival of offspring with an inbreeding coefficient of 0.25 was reduced by 77% compared to offspring with an inbreeding coefficient of zero Figure . The estbaileyi)  and 5.19s lupus) . Given tAlthough the population of red deer studied here has one of the most complete pedigrees of any wild mammal population currently studied, approximately 40% of calves born to the study population have unknown paternity. Consequently the frequency of inbreeding estimated in this study is likely to be an underestimate  as is the severity of inbreeding depression . RestricThis study presents an investigation of inbreeding effects in a wild mammal population using pedigree-based inbreeding coefficients as opposed to marker-based estimates. Although the results are generally consistent with previous studies using marker-based estimates in this population, differences do exist, highlighting the importance of confirming such associations with a pedigree-based approach. The fact that strong inbreeding depression for first winter survival was still apparent after controlling for birth weight highlights the potential for inbreeding to affect multiple facets of early-life performance in natural populations. Long-term studies of pedigreed natural populations such as this represent an ideal situation in which to generate a fuller understanding of inbreeding depression and particularly its context dependence, or lack of, under ecologically relevant conditions.post mortem or by immobilisation. All sampled individuals are genotyped at up to 15 microsatellite loci [The red deer population resident in the North Block of the Isle of Rum, Inner Hebrides, Scotland  has been the subject of intensive study since 1972 . Individite loci ,75,76.Pedigree reconstruction was achieved using a combination of genetic and behavioural information as detailed in Walling et al. . Brieflyhttp://www-personal.une.edu.au/~bkinghor/pedigree.htm[Wright's inbreeding coefficient (F) was calculated for mothers and offspring from the pedigree using the software package PEDIGREE VIEWER version 6.4 igree.htm,82. F waWe examined the effects of both an offspring's inbreeding coefficient and that of its mother on the juvenile traits birth date, birth weight and first year survival among all offspring caught at birth. Date of birth was scored as the number of days since 1st May in the year of birth, birth weight as capture weight (kg) minus 0.01539 times age at capture (hrs) [2 > 0.94) [In addition to the inbreeding coefficient of the offspring and its mother, a number of other fixed effects were fitted that are known to be important from previous studies. Offspring sex was fitted as a two-level factor in all models, as this is known to be an important predictor of birth weight and first winter survival, with males being heavier at birth but yet less likely to survive ,55,83. P > 0.94) . Populat > 0.94) ,55. Fina > 0.94) ,85,86. I > 0.94) ,85,86.S = S0eBf -where S is survival, 0 Sis the survival of non-inbred individuals, B is the number of lethal equivalents per gamete and F is the inbreeding coefficient under consideration. The parameter B is usually estimated as the regression coefficient from a least-squares linear regression of the natural log of survival against inbreeding coefficient. However, this method runs into problems when the average survival observed in a class of individuals with a particular value of F is zero. Kalinowski & Hedrick [B which avoids the zero-survival problem and Kruuk et al. [B [see [The number of lethal equivalents is a standardised measure of inbreeding depression in a population, defined as a gene or group of genes that would cause death in an individual in a homozygous state . Assumin Hedrick  suggest k et al.  extended [B [see  for detasurv,i Nis the number of survivors in class i, total,i Nis the total number of calves in class i, i Fand the log-likelihood is summed over the different inbreeding classes i. The effect of allowing annual variation in the base-line survival where y = 1980, 1981...2009. The significance of the effect of estimating B gives Where  Hedrick  and Kruu Hedrick .Birth date and birth weight are normally distributed and thus analysed using mixed models with normal errors using ASReml version 3.0  to impleIn addition, the minimum inbreeding coefficient that can be assigned to an individual depends on the depth of the pedigree for that individual and this varies between individuals in this population , median = 3, minimum = 0, maximum = 9). To assess the consequences of this variation for our results we re-ran minimal models on a dataset restricted to individuals with all four grandparents known (sample sizes: Birth date 816 (344 with F>0), birth weight 749 (318 with F>0), first year survival 708 (296 with F>0), summer survival 716 (297 with F>0), winter survival 632 (266 with F>0)).CAW and DHN designed and carried out the statistical analysis. JMP, LEBK and THCB participated in the design of the analysis and are involved in the maintenance and management of the long-term monitoring project on Rum and JMP has led the sampling and genotyping of individuals. AM collected much of the data on which the analysis is based. CAW wrote the initial manuscript and DHN, JMP, LEBK and THCB all provided detailed comments and suggestions. All authors read and approved the final manuscript