Natal features (e.g. Julian birth date and birth mass) often have fitness consequences and can be influenced by endogenous responses by the mother to seasonal fluctuations in nutritional quality and photoperiodic cues. We sought to further understand the biological and environmental factors that influence the natal features of a polytocous species in an environment with constant nutritional resources and limited seasonal variation. During a 36-year study we assessed the influence of biological factors  and environmental factors  on Julian birth date and birth mass using linear-mixed effects models. Linear and quadratic functions of maternal age influenced both natal features with earliest Julian birth dates and heaviest birth masses occurring at prime-age and older individuals, which ranged from 5–9 years of age. Litter type influenced Julian birth date and birth mass. Interestingly, environmental factors affected Julian birth date and birth mass even though mothers were continuously allowed access to a high-quality diet. Random effects revealed considerable variation among mothers and years. This study demonstrates that, in long-lived polytocous species, environmental factors may have a greater influence on natal features than previously supposed and the influence from biological factors is also complex. The documented responses to environmental influences provide unique insights into how mammalian seasonal reproductive dynamics may respond to current changes in climate. The amount of body development that an individual achieves in the first year of life is fundamental to survival in long-lived species –4. The aMuch of the current theory about seasonal breeding is contingent on maternal condition and photoperiod ,18–20. MTiming of reproduction in mammals has evolved as a response to seasonal availability of resources at high latitudes, where there is a strong correlation between photoperiod and plant growth. Because of this correlation, it is possible to use photoperiod as a predictive cue to time late gestation and parturition for when nutritional resources are most available. The mammalian neuro-endocrine pathways use photoperiodic and metabolic information from the individual to stimulate the reproductive processes in preparation for breeding . HoweverWhile a substantial amount of research has focused on understanding factors that affect parturition and survival of neonates in regard to environmental settings and maternal condition –7, most Our study provides a unique opportunity to understand the factors that influence natal features of long-lived species. For 36 years, a known-age population of captive white-tailed deer was fed a high-quality diet at a latitude with benign winter conditions. Within this population of captive white-tailed deer, measurements of parturition date (Julian birth date) and birth mass were recorded for all live births. Further, the study site was located, latitudinally, at the transitional zone suggested by Bronson  in whichPrior to the initiation of the study, all animal research was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the United States Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS). Statutes of the Animal Welfare Act were followed by Kerr Wildlife Management Area personnel and registered with the USDA APHIS Animal Care permit# 74-R-0146.Marrubium vulgare) and cowpen daisy (Verbesina encelioides), as well as scattered live oak (Quercus virginiana) trees, which had been browsed out of reach of white-tailed deer since 1974 [The study was conducted at the Donnie E. Harmel White-tailed Deer Research Facility (hereafter pens) located on the Kerr Wildlife Management Area in Kerr County, Texas, USA . The nunce 1974 . Each peFrom 1974–2012, several types of breeding programs occurred at the pens; however, much of the study program was the same throughout this time span. At ≥1.5 years of age, female deer were separated into one of five to seven mating pens. Each October, one male  was placed into each mating pen and allowed to mate with all available females until the end of December, when the male was removed. Within one day of parturition, neonates were captured, individually marked, and weighed to the nearest 0.1 kg. After weaning, fawns were placed into rearing pens where they spent the next year of development, until the female portion was reintroduced into the mating pens at 1.5 years of age. Deer were initially collected from several ecological regions in Texas, establishing the original stock in 1974. Five sires collected from the Edwards Plateau ecological region were added to the closed research herd in 2007. Because all individuals were uniquely marked, ages were known for all females. To reduce the number of categorical variables describing the heterogeneity of breeding programs, all programs were fitted into three main program types. In the first program (study program 1), no animals experienced any nutritive stress and were fed a continuous 16% crude protein diet throughout their lifetime. The second program (study program 2) was a selective breeding program in which sires were selected as breeders if their first set of antlers possessed spike antler characteristics. The last program (study program 3) consisted of sires that encountered nutritive stress (8% crude protein) from 0.5–1.5 years of age and were given the ad libitum high-quality diet thereafter.SE = 38.5 mm, CV = 0.30) with most precipitation occurring from May–June (average = 195.0 mm) and September–October , which was located approximately 38 km from the pens. Precipitation variables consisted of monthly total precipitation during periods deemed important to conception and parturition of neonates (October–December and April–June). We included two additional predictor variables by summing the monthly total precipitation from October–June (prior to conception and throughout gestation) and April–June (encompassing the months surrounding the last trimester of gestation). Months encompassing the last trimester of gestation were deemed important because most fetal growth occurs during this period ,40. AverDuring this study, females had the opportunity to breed several times throughout their lifetime. Because of this, mixed-effects models were used in all analyses with unique identifiers for both maternal identity and birth year included as random effects. Since <5% of births during this study consisted of triplets, only births of singletons and twins were included in analyses. Also, since there were only two births by females 14 years or older, these births were removed from all analyses. A Kolmogorov-Smirnov test was first conducted to test for normality in Julian birth dates . Linear Biological factors included in the analyses for both response variables were maternal age, the quadratic term for maternal age, and litter type as well as the nuisance variable for study program. The quadratic term for maternal age was included to assess the possibility of a reproductive threshold or senescence in older deer –44. To dF-ratios (P < 0.05) in the previous analysis  with environmental variables  that were present during the period when dams could conceive, and encompassing the last trimester of gestation. A total of 19 regressions were built that assessed environmental influences from October–December (influence on conception), April–June (influence encompassing the last trimester of gestation), and October–June (influence throughout gestation). The first regression included only the biological factors that significantly influenced the two response variables in the previous analyses. All further models considered these biological influences with additional environmental influences added as covariates. The next set of six regressions assessed the influence of precipitation on the two response variables by incorporating individual months into each regression . We then considered three more regressions by summing total precipitation during the possible length of conception (October–December), the period encompassing the last trimester of gestation (April–June), and the entire length of gestation (October–June). The next set of nine regressions followed the same design as noted previously, but included mean maximum temperatures instead of total precipitation as a predictor variable.We then evaluated linear mixed-effects models for both response variables that assessed the added influence of environmental factors. In each model, we included the biological variables with statistically significant c, [c units different. Maximum likelihood estimation was used to calculate parameters during the model-selection process. Parameter estimates and 95% CI for the selected model were then reported with restricted maximum likelihood estimation [R2) and by fixed and random factors . Model c, , and wastimation . Coefficnal R2, ). In anaSE = 0.2). Average maternal age was 4 years (SE = 0.05), with a range of 2–13 years. The mean date of parturition during our study was 13 June , with the earliest birth occurring on 13 April (Julian date = 103) and the latest on 10 September (Julian date = 253). A Kolmogorov-Smirnov 1-sample test revealed that dates of Julian birth were normally distributed during this study .During the 36-year study of white-tailed deer, 2,290 neonates were born to 510 individual mothers for a total of 520 singletons  and 885 twin litters . Number of births per individual female varied during the study, with an average of 4.5 young born during the lifetime of an individual mother , thus, there was a threshold effect. On average, mothers in the youngest age class (2 years of age) gave birth on the latest dates  and the oldest mothers (13 years of age) gave birth to individuals around the same time as a 6-year old mother . Predicted values of Julian birth date for each litter type indicate that mixed-sex litters were born earlier than all other litter types . Julian birth dates for all other litter types  were not significantly different from each other (CV = 0.51). Variance components for the random effects in the Julian birth date analysis could not be derived from the model-averaged analysis, thus, we reported values derived from the model with the lowest AICc . Variance components for this model were dam id (SD = 11.26), birth year (SD = 4.85), and residual error (SD = 15.22). The marginal R2 for this model was 0.09 and the conditional R2 was 0.45. Inclusion of the random effects (dam id and birth year) and fixed effects explained more variation in birth mass than fixed effects alone.All biological factors tested  influenced Julian birth date, as well as the nuisance variable , thus, there was a threshold effect. The lightest birth masses occurred at 2 years of age and heaviest at 5 years of age and older. Predicted values of birth mass for each litter type demonstrated that birth mass varied significantly among litter types. Females from mixed litter types had the lowest birth mass  followed by females of twin litters , males of mixed litters , males of twin litters , females of singleton litters , and the heaviest birth masses were males of singleton litters . Total birth mass for singleton and twin litters varied with total birth mass of singleton litters weighing less than twin litters . Variance components for the random effects in the birth mass analysis were dam id (SD = 0.28), birth year (SD = 0.14), and residual error (SD = 0.41). The marginal R2 was 0.12 and the conditional R2 was 0.44. Inclusion of the random effects (dam id and birth year) along with the fixed effects explained more variation in birth mass than fixed effects alone.Results for the birth mass analysis demonstrated that all biological factors influenced birth mass . InclusiThis study assessed the influences of biological and environmental factors on natal features (Julian birth date and birth mass) of a long-lived, polytocous species. The first objective was to understand the biological influences of litter type and maternal age on Julian birth date and birth mass. As expected, the biological factors of maternal age and litter type affected both of these natal features. The second objective was to assess the influence of environmental factors on Julian birth date and birth mass. Interestingly, we found that environmental conditions influenced these natal features even while holding nutritional resources constant and accounting for maternal traits. Total precipitation during the months that encompassed the last trimester of gestation (April–June) influenced Julian birth date, and December temperatures influenced birth mass.Natal features are commonly dependent on biological factors related to age, social rank, nutritional condition, and previous breeding experience of mothers. We found that maternal age influenced both natal features until 5–9 years of age, which is considered prime to late-prime age for this species . There wa priori expectation for study program and found an influence among study programs on Julian birth date and birth mass. Factors within study programs that could influence natal features might include genetic characteristics and age of the sire (study program 2) or reduction of nutritional quality to sires at 0.5–1.5 years of age (study program 3). Because of study-design constraints in the current study, it was not feasible to utilize sire traits as informative variables. The inclusion of sire as a random effect could have been beneficial to explain some of the variation in natal features. However, each sire was, generally, only bred during a single year and, thus, it was more appropriate to allow the variation among sires to be accounted for within the study program nuisance variable.Based on the differences in study program during the 36 years, study program was considered a confounding variable that could influence natal features. We had no In many environments, precipitation and temperature are drivers of primary productivity  that dirBecause of the constant high level of nutritional quality in this study, we anticipated that environmental influences would not be important predictors of Julian birth date or birth mass. However, mothers still used precipitation during the period encompassing the last trimester of gestation as a cue for favorable environmental conditions in which to birth their young, and birth mass was influenced by temperature during the period closer to conception. Interestingly, these two natal features are influenced at different periods of gestation rather than being connected to each other . These findings suggest that the presumably well-conditioned mothers were exhibiting risk-sensitive reproductive allocation in both Julian birth date and birth mass –63. MothIn a large-scale study on reproductive seasonality in captive ruminants in zoological parks, Zerbe et al.  reportedAlthough environmental and biological factors were influential predictors of Julian birth date and birth mass, only 9% of the variation in Julian birth date and 12% of the variation in birth mass was explained by those factors. With the inclusion of the random effects for dam id and birth year, the explanation of variation increased to 45% and 44%, respectively. This result indicates that it is necessary to account for subject-specific effects, because of individual variation in longitudinal studies. Recently, much emphasis has been placed on the role of individual variation in maternal care ,25. ThosUnderstanding factors that influence natal features in long-lived species is often difficult because nutritional state of mothers is affected by recruitment of young from previous reproductive events and climatic heterogeneity ,28,63. BS1 Table(XLSX)Click here for additional data file.


The experimental parameters have been investigated and the dynamic concentration range of the sensor has been assessed by the selection of two apomorphine surface enhanced Raman scattering (SERS) peaks. The sensor behavior used to detect apomorphine in unfiltered human blood plasma is presented and discussed.Artificially roughened gold surfaces with controlled nanostructure produced by pulsed laser deposition have been investigated as sensors for apomorphine detection aiming at clinical application. The use of such gold surfaces has been optimized using aqueous solutions of apomorphine in the concentration range between 3.3 × 10 In recent years, the analytical applications of Raman spectroscopy and its enhanced variant employing plasmonic media, the surface enhanced Raman scattering (SERS) effect, have significantly grown –7. TheseThe various SERS substrates reported in the literature and the associated experimental approaches to detect the spectroscopic signal attest the significant interest for SERS as a powerful, molecule sensitive, analytical technique. For instance, gold colloids synthesized by laser ablation in liquids, suitably functionalized and tagged with Raman reporters, have effectively revealed specific biomolecules, even in chemically complex environments such as cells –12. On tIn this work, we report on the use of gold nanoparticle (NP) arrays deposited on standard glass substrates to quantitatively detect apomorphine (APO) aiming at the realization of a biomedical sensor. APO has been long used in the treatment of patients with complicated Parkinson’s disease (PD). Indeed one of the challenging problems in the management of PD is the development of severe, long-term motor complications that oral medication cannot adequately control at tolerable doses. Presently, APO delivered via continuous subcutaneous infusion is one of the viable alternatives to oral medication in such patients. Adjusting the APO concentration as it fluctuates with time is critical and is monitored via standardized clinical observations and tests of motor condition . Standar−4 Pa. A KrF excimer laser  was focused onto a pure gold target (99.99%) mounted on a rotating holder. The substrates were deposited on pieces of Corning Glass 5049, or Si(100), placed at a distance of 35 mm from the target and held at room temperature. The target holder was rotated to avoid target surface cratering under repetitive ablation. The ablation was performed in an Ar atmosphere at a pressure of 100 Pa. The number of laser pulses was fixed at 10,000. The laser fluence was kept constant at 2.0 J/cm2. The sample morphology was observed by scanning electron microscopy (SEM) using a Zeiss Supra 40 field ion microscope.The substrates were prepared in a vacuum chamber at a base pressure lower than 10Mw = 303.78332 g/mol) as the active ingredient, corresponding to a molar concentration of 3.3 × 10−2 M of R-apomorphine, plus sodium metabisulfite and hydrochloric acid as excipients.For the preparation of APO solutions at different concentrations, the commercially available Apofin solution for subcutaneous injection  was used. The commercial drug (3 mL) contains 30 mg of apomorphine hydrochloride (−4 M to 3.3 × 10−7 M). The pH values of all solutions were in the range between 5.5 and 6.5, as determined by indicator paper.The APO solutions were obtained by suitable dilution of the commercial drug with sterile water for injection (B. Braun) to reach the final concentrations of 100, 10, 1 and 0.1 µg/mL  for a fixed time and air-dried before acquiring SERS spectra. Three different dipping times were considered, namely 2, 5 and 10 min, in order to evaluate the best contact time for the solution and the substrate.−1 grating and a Peltier-cooled CCD detector. Notch filters for 785 nm were used to suppress the Rayleigh scattering. The laser was focused on the sample through the same 50× Olympus objective (NA = 0.75), with a nominal laser power irradiation at the samples of 1 mW, over a spot size of about 1 µm, and an exposure time of 2 to 30 s (average of 2 accumulations). The spectra were recorded in the region between 900 and 1800 cm−1, where the most intense Raman features of apomorphine lie [The gold substrates subjected to the dipping and drying procedure were observed with an Olympus BX41 microscope using a 50× Olympus objective (NA = 0.75) coupled to the spectrometer, and different points for each sample were chosen for SERS measurements. All SERS spectra were collected by a Jobin-Yvon LabRAM HR800 Raman spectrometer with a solid-state laser  operating at 785 nm, equipped with a 600 grooves mm−1 and 1515 cm−1, were selected as intense, stable and reproducible markers for APO detection in aqueous solutions. The corrected areas under each peak were evaluated by manual selection of the baseline using Omnic 8 software . From repeated measurements at different spots, a mean value and standard deviation from the mean were calculated and studied as a function of drug concentration in order to assess the dynamic range of concentration of the sensor. Different fits were applied to the data  to describe the concentration dependence in the range between 1 and 100 µg/mL.Two SERS peaks, namely at 1353 cmThe SERS peak areas were normalized with respect to the peak area for the highest concentration studied , and plotted as a function of the logarithm of the APO concentration, in order to compare experimental data at different dipping times. A linear regression was applied to the data sets.In order to prove the feasibility of the method for the detection of APO in real samples, some initial trials were carried out on unfiltered blood plasma from a healthy volunteer . The samples of plasma were supplemented with apofin to produce the desired concentrations of 200, 20, and 2 µg/mL of APO in plasma.−1 spectral range with exposure times of 5, 10, 30 and 60 s (average of 2 accumulations).Starting from the lowest APO concentration (2 µg/mL), one SERS sensor was dipped for 5 min in 1 mL of plasma and dried in air before acquisition of the spectra. The experimental procedure was repeated, dipping the same sensor in plasma containing APO at 20 µg/mL for 2 min. The sensor was finally dipped for 2 min in the most concentrated sample (200 µg/mL), air-dried and measured. All spectra were collected in the 900–1800 cm3 nm2, as shown in 7 [own in 7 .The SERS spectra of 100 µg/mL APO collected with different exposure times, ranging from 2 to 30 s, on different areas of the gold substrate are shown in By comparing the spectra, no meaningful changes in the spectral profile are observed during the measurements for the different exposure times. The selected experimental conditions  are suitable to detect the analyte without producing any chemical degradation of the drug molecule during the measurements by interacting with the gold surface. The rapid acquisition time, together with a relatively low laser power, represent promising conditions for a rapid method for the measurement of the concentration of drugs in biological fluids. A spot size of about 1 µm ensures the collection of an average SERS signal from the selected area, given that inter-island channels of the nanostructured Au substrate are, on average, in the 10 nm range see .−1, in agreement with previous experimental and theoretical investigations [The main SERS active modes of APO are found at 1009, 1035, 1091, 1179, 1199, 1223, 1353, 1392, 1515, 1579 and 1618 cmigations .The closeness of the molecule to the nanostructured metal surface is an inherent condition for SERS enhancement. Therefore, the assignment of experimental peaks to specific vibrational modes of the analyte is important to understand the kind of interaction between the molecule and the substrate. The assignment of the peaks also has a more practical meaning: it facilitates the selection and definition of appropriate spectral features that can be employed to recognize, and in some cases, to quantify, the analyte in progressively more complex matrices. The effort to resolve the analyte signals with respect to the overall spectrum, including contributions from interfering species, is a critical step once dealing with real samples.−1 , and the one centered at 1515 cm−1 , in order to elucidate any dependence of the SERS signal on the experimental and instrumental parameters. Both wavenumbers fall in spectral regions where the SERS spectrum of blood plasma does not display any characteristic band. Using the near-infrared excitation at 785 nm, the spectra are dominated by protein bands with broad amide bands at 1650 cm−1 (amide I) and 1250 cm−1 (amide III), and sharp bands due to aromatic amino acids at 1004 cm−1 (Phe), 1127 cm−1 (Tyr) and CH2/CH3 deformations at 1450 cm−1 [Among the strongest peaks of the APO SERS spectrum, two signals were selected, specifically the peak centered at 1353 cm450 cm−1 .A manual baseline correction was applied to the raw data, and average values were plotted versus the acquisition time, as shown in To be considered a useful SERS-based method for drug detection, the procedure, based on the use of gold substrates pulsed laser deposition, should be rapid and quantitative. An acquisition time of 5 s, which corresponds to a fast measurement with an adequate signal-to-noise ratio see , was cho−1 is found. Different acquisition times, up to 30 s, did not result in an evident improvement in signal quality.Starting from the concentration of 1 µg/mL, the intensity of APO peaks increases with APO concentration, while the position of all characteristic peaks remains unchanged. Dilution with water produces a change in the measured pH values of APO solutions from 5.5 for the most concentrated solution to 6.5 for the diluted solutions; such variations have no effect on the spectral features.If the peak area is plotted against the concentration of the APO solutions, a characteristic trend is found, where a steep growth for low concentrations  and the onset of saturation near the highest tested concentration  was studied.The SERS peak areas were normalized with respect to the peak area for the highest studied concentration , and plotted on a semi-logarithmic scale . This waA linear regression was applied to the data sets according toI(C) is the Raman intensity of a selected APO peak, C is APO molar concentration and Cmax = 3.3 × 10−4 M in our experiments; α and β are the fitting coefficients.where 1515 = 0.5 ± 0.2 for the 1515 cm−1 peak, and α1353 = 0.486 ± 0.002 for the 1353 cm−1 peak; when the dipping time is 5 min, these become α1515 = 0.34 ± 0.07 and α1353 = 0.40 ± 0.04, respectively. Hence, the dipping time has a moderate role in the sensitivity of the sensor, and it could be conveniently optimized in order to reach the lowest detectable concentration for any chosen nanostructured, gold substrate. On the other hand, for a given dipping time, the α-parameter is constant with respect to the Raman mode. This is expected because the relative Raman intensity of two peaks reflects a molecular property that does not depend on concentration .When the dipping time is 2 min, the slopes are αBased on these findings, an additional increase of the dipping time, while avoiding sensor saturation, could be expected to cause at least a slight sensitivity increase towards lower analyte concentrations. A third set of experiments at different APO concentrations was therefore carried out with a dipping time of 10 min. We observed the growth of APO crystals of a few micrometers on the surface of the sensor, irrespective of the concentration. More scattered data correspond to this observation . This liTo prove the applicability of the proposed method to the detection and quantification of APO in blood samples, a few experiments were performed on unfiltered blood plasma with different APO concentrations. These challenging conditions were chosen to minimize sample preparation, while still demonstrating the feasibility of the proposed technique.−1 (amide I), 1254 cm−1 (amide III), 1034 cm−1 and 1004 cm−1 (Phe).The SERS spectra are shown in −1, selected for APO detection in aqueous solution, are clearly visible and found at 1520 and 1348 cm−1, respectively. Such differences in peak position can be possibly ascribed to the complexity of interactions that the drug molecule experiences in the biological matrix as compared to the aqueous solution. To reach a comparable SERS intensity with respect to 100 µg/mL APO in water, an acquisition time of 10 s was applied, while 60 s was the acquisition time required for a reasonable signal-to-noise ratio of the SERS spectrum of 20 µg/mL APO in blood plasma, as shown in In The kinetics of APO adsorption onto the gold nanostructured surface are expected to be strongly affected by matrix effects. In particular, the presence of proteins, such as human serum albumin, an abundant plasma protein that binds a wide range of drugs including APO , has to Furthermore, once dealing with APO detection in clinical samples, it is possible to introduce minimized sample preparation  to enhance the APO signal intensity.Gold NP arrays pulsed laser deposited on glass proved to be suitable sensors for the quantitative detection of APO in water by means of SERS. The dynamic range of concentrations of the sensor was assessed by the selection of two SERS peaks. Minimal sample preparation and the capability to operate in an aqueous environment make the detection of APO by means of this technique rapid, with good signal-to-noise ratio, and characterized by adequate spatial reproducibility. The method is promising for transfer to APO detection in biological fluids. Experiments performed on unfiltered blood plasma with different APO concentrations proved the applicability of the proposed method to APO detection for samples of clinical origin.


There has been no large nationwide population-based study to examine the effects of maternal smoking status during pregnancy on birth weight that simultaneously controlled for clinical information, socioeconomic status, and maternal weight. Thus, this study aimed to determine the association between maternal smoking status during pregnancy and birth weight, while taking these confounding factors into consideration.This study examined the first-year fixed dataset from a large nationwide birth cohort study that commenced in 2011. The dataset consisted of information on 9369 singleton infants born before December 31, 2011. Children were divided into 4 groups for statistical analysis: those born to mothers who did not smoke (NS), who quit smoking before pregnancy, who quit smoking during early pregnancy, and who smoked (SM). Multiple linear regression models were conducted for each sex to examine the association between maternal smoking status during early pregnancy and fetal growth. Birth weight was estimated using the least-squares method after controlling for covariates.P < 0.001; female infants, 3018.2 g [NS] vs 2893.7 g [SM], P < 0.001).After controlling for potential confounding factors, maternal smoking status during pregnancy was significantly associated with birth weight. There was a significant difference in birth weight between NS and SM for both male and female infants (male infants, 3096.2 g [NS] vs 2959.8 g [SM], Using data from a large nationwide birth cohort study in Japan, we have shown that maternal smoking during pregnancy may reduce birth weight by 125–136 g. Some perinatal outcomes, such as low birth weight (LBW) and intrauterine growth restriction, are considered indicators of inappropriate fetal growth.In recent years, the “Developmental Origins of Health and Diseases” hypothesis, in addition to the established “fetal programming” and “Barker’s hypothesis”, has been suggested to clarify the mechanisms of childhood growth and development.6 Thus, because inappropriate fetal growth might be a risk factor for poor health later in life, it is important to improve fetal growth levels in Japan.In Japan, the incidence of LBW infants has increased. For example, 5.5% of female infants born in 1975 weighed less than 2500 g, a prevalence that increased to 10.7% in 2013.7–10 Moreover, low socioeconomic status (SES),11–13 pregnancy-induced hypertension (PIH),14 pre-gestational maternal body mass index (BMI),15 and insufficient maternal weight gain during pregnancy16,17 are considered risk factors for LBW. Conversely, it has been suggested that expectant mothers with diabetes mellitus (DM) and gestational diabetes mellitus (GDM) are more likely to have macrosomic babies than those without these disorders.18,19Maternal smoking during pregnancy is one of the major causes of LBW and intrauterine growth restriction.12 Limited data in relation to confounding factors can be a major limitation in the analysis of large sets of population-based nationally representative data.With regards to SES as a risk factor for LBW, Fujiwara et al suggested that a high level of income inequality is associated with inappropriate fetal growth. This finding was based on nationally representative data; however, they did not control for the effects of maternal smoking during pregnancy, as this information was not available.20,21 However, there were some limitations to these studies. First, as they were based in a specific area of Japan, it is difficult to apply the results to the general population. Second, because these studies were based on population-based surveys, clinical information, such as weight gain and complications during pregnancy, was not available. Thus, it was difficult to adjust for these factors, particularly for complications during pregnancy, such as PIH, DM, and GDM.We previously examined the association between maternal smoking status during pregnancy and fetal growth in rural Japan.22 Because this report was based on a hospital-based survey, the participants might not be representative of Japanese pregnant women. Thus, the present study aimed to describe the association between maternal smoking status during pregnancy and birth weight while taking these confounding factors into consideration. Our analysis utilized the first-year fixed dataset from the Japan Environment and Children’s Study (JECS), a large population-based nationwide birth cohort study that commenced in 2011.23To the best of our knowledge, there have been no large nationwide population-based epidemiological studies examining the effects of maternal smoking status during pregnancy on birth weight that simultaneously controlled for clinical information, SES, pre-gestational BMI, and maternal weight gain during pregnancy, although there was a nationwide report that utilized the Japan Perinatal Registry of the Japan Society of Obstetrics and Gynecology.23 The response rate of JECS was about 79%.24 The present study is based on the jecs-ag-ai-20131008 dataset, which was released in October 2013. Details of this dataset were published previously.25 This dataset consisted of information on 9369 singleton infants born before December 31, 2011. The mean age (standard deviation [SD]) of participants in this dataset was 31.0 (5.0) years. The mean (SD) gestational duration at their pregnancy registration was 108.6 (42.7) days. The self-administered questionnaire consisted of questions regarding maternal smoking during the second trimester of their pregnancy and other covariates. Of the mothers who participated, 7734 (82.5%) completed the questionnaire.For JECS, pregnant women were recruited between January 31, 2011, and March 31, 2014. Eligibility criteria for participants (expectant mothers) were as follows: 1) residing in the study area at the time of recruitment; 2) expected delivery date after August 1, 2011; and 3) capable of comprehending the Japanese language and completing the self-administered questionnaire. Individuals who resided outside the study area but attended cooperating health care providers within the study area were excluded from the study. Details of the JECS project have been described in a previous article.The JECS protocol was approved by the Institutional Review Board on epidemiological studies of the Ministry of the Environment and the Ethics Committees of all participating institutions: the National Institute for Environmental Studies, which leads the JECS; the National Center for Child Health and Development; Hokkaido University; Sapporo Medical University; Asahikawa Medical College; Japanese Red Cross Hokkaido College of Nursing; Tohoku University; Fukushima Medical University; Chiba University; Yokohama City University; University of Yamanashi; Shinshu University; University of Toyama; Nagoya City University; Kyoto University; Doshisha University; Osaka University; Osaka Medical Center and Research Institute for Maternal and Child Health; Hyogo College of Medicine; Tottori University; Kochi University; University of Occupational and Environmental Health; Kyushu University; Kumamoto University; University of Miyazaki; and University of the Ryukyus. The JECS was conducted in accordance with the Helsinki Declaration and other nationally valid regulations and guidelines.2).Information on SES and the smoking habits of mothers and their partners during the mothers’ pregnancy was collected by questionnaire during the second trimester of pregnancy. This questionnaire also included questions about lifestyle factors  and physical environment . Maternal anthropometric data before pregnancy and data on maternal weight gain during pregnancy; complications before and during pregnancy, including PIH, DM and GDM; history of previous pregnancy; sex of infants; birth order; and perinatal outcomes, such as birth weight and gestational duration, were also collected from medical records, which were provided by subjects’ obstetricians. BMI was used to evaluate maternal weight status and was calculated according to World Health Organization standards (body weight [kg]/height [m]2), maternal weight gain during pregnancy (kg), prevalence of PIH, prevalence of DM/GDM, gestational duration (days), birth order (1 or more than 1), and partners’ smoking status during pregnancy .The primary outcome was birth weight. Maternal smoking status during pregnancy was assessed by the questionnaire item, “Please choose an item that best describes your current smoking status”, and was grouped as follows: “smoked”, “quit smoking during early pregnancy”, “quit smoking before pregnancy”, or “never smoked (during their lifetime)”. The following were also assessed as covariates: annual household income , maternal age at delivery , maternal weight before pregnancy , who quit smoking before pregnancy (QSB), who quit smoking during early pregnancy (QSD), and who smoked (SM). Analysis of variance and chi-square tests were conducted to compare covariates between each group. Multiple linear regression models were constructed to examine the association between maternal smoking status during early pregnancy and fetal growth for both sexes.Birth weight was estimated using the least-squares method, after controlling for the above covariates. The adjusted birth weights of the QSB, QSD, and SM groups were compared with that of the reference group (NS) using the Dunnett method.In addition, because it has been suggested that maternal smoking during pregnancy, gestational duration, and birth weight could be associated with each other, we suspected it might be difficult to control for each effect simultaneously. Therefore, as a sensitivity analysis to examine the association between maternal smoking during pregnancy and birth weight, adjusted birth weights of each group were calculated without preterm birth.All analyses were conducted using SAS version 9.3 .Overall, 3925 (50.8%) infants were male and 2927 (37.9%) were first babies. Moreover, the number of preterm births was 424 (5.5%). The mean (SD) birth weights in male and female infants were 3054.5 421.6) g and 2964.8 (425.1) g, respectively. In addition, minimum and maximum birth weights were 200 g and 4704 g, respectively. Table .6 g and P < 0.001 for both sexes; Table After controlling for partners’ smoking status, annual household income, birth order of children, PIH, DM/GDM, maternal weight before pregnancy, maternal weight gain during pregnancy, maternal age at delivery, and gestational duration, maternal smoking status during pregnancy was significantly associated with birth weight  were close to those seen our previous study.21 Moreover, Vardavas et al reported that smoking during pregnancy was associated with a 120- to 150-g reduction in birth weight.29We confirmed a significant effect of maternal smoking during pregnancy on birth weight. In fact, birth weights in the SM group for male and female infants were 136 g and 125 g lower, respectively, than in the NS group. In addition, after controlling for the effect of preterm birth, the effect of maternal smoking on birth weight was similar to the result of the main analysis. For female infants, birth weight in the QSD group was 40 g lower than the SM group. These results are consistent with the findings of previous studies, although the differences here are smaller.30,31 However, as the sample sizes of previous studies were small, it would be difficult to detect differences in birth weight of less than 100 g. For example, Prabhu et al reported that infants born to mothers who quit smoking during early pregnancy were approximately 50 g lighter at birth than infants of non-smoking mothers, although this difference was not significant.30 In addition, Fasting et al reported a 20 g difference.31 Therefore, the effects of a 100-g difference in birth weight on subsequent growth and development should be examined. Moreover, it is essential to determine the difference in birth weight that confers a clinically significant effect.Mean birth weight in the QSB group was lower than that in the NS group. Particularly in female infants, birth weight in the NS group appeared to be higher than in the QSD group, although this difference was not significant. These results are inconsistent with previous studies.32 a reduction in the diameter of the chorionic villi, vasoconstriction, suppression of active amino acid uptake,33 and increased apoptosis.34,35 Maternal smoking during pregnancy can also result in reduced placental size and atrophic placental villi,36 significantly higher umbilical artery resistance, and reduced estimated fetal weight.37 Fetal growth might be affected by these placental changes.There are some biological explanations for the association between maternal smoking during pregnancy and fetal growth. It has been suggested that smoking in expectant mothers can lead to effects in the placenta, such as calcification,38 In addition, because information on partners’ smoking status was also obtained from pregnant women using the questionnaire, there might be bias. Furthermore, information of household income might be inaccurate for similar reasons. However, other data on other variables are likely to be relatively accurate and objective, as they were collected from medical records. Finally, although the previous reports suggested regional differences of smoking habits,25 it was difficult to examine the regional differences because the numbers of participants in each unit center of JECS were relatively small.There are certain limitations to this study. First, we used a questionnaire to measure maternal smoking status instead of using objective measurements. Moreover, although the response rate was relatively high, there might be selection bias of participants. These might lead to an underestimation of maternal smoking rates, particularly due to the questionnaire-based study design.Despite these limitations, there has been no other nationwide community-based study examining the effect of smoking status during pregnancy on fetal growth while simultaneously controlling for confounding factors that may have influenced the results of previous studies, such as clinical information, SES, pre-gestational BMI, and maternal weight gain during pregnancy. Therefore, this study provides valuable evidence to support the importance of cessation of maternal smoking before and during pregnancy.In conclusion, we have clarified the association of maternal smoking during pregnancy on birth weight using preliminary data from a large nationwide birth cohort study in Japan. Our results suggest that maternal smoking during pregnancy might reduce birth weight by 125–136 g. Further studies describing the effects of maternal smoking during pregnancy on fetal growth in more detail are necessary; we plan to conduct such studies using the complete JECS dataset, which will include data on approximately 100 000 infants and their parents. In addition, it is essential that the effects of maternal smoking during pregnancy on childhood growth are examined.Abstract in Japanese.


Exposure to ambient air pollution has been associated with reduced size of newborns; however, the modifying effect of maternal ethnicity remains little explored among South Asians.We investigated ethnic differences in the association between ambient air pollution and newborn’s size.10; ≤ 2.5 μm, PM2.5), PM2.5 absorbance, and nitrogen oxides  were estimated using land-use regressions models. Using multivariate linear regression models, we evaluated effect modification by maternal ethnicity  on the associations of air pollution and birth weight, head circumference, and triceps and subscapular skinfold thickness.Pregnant women were recruited between 2007 and 2010 for the Born in Bradford cohort study, in England. Exposures to particulate matter , but not in children of Pakistani origin  (pint = 0.03 and < 0.001). In contrast, PM2.5 was associated with significantly larger triceps and subscapular skinfold thicknesses in children of Pakistani origin , but not in white British children  (pint = 0.06 and 0.11). Patterns of associations for PM10 and PM2.5 absorbance according to ethnicity were similar to those for PM2.5, but associations of the outcomes with NO2 and NOx were mostly nonsignificant in both ethnic groups.A 5-μg/mOur results suggest that associations of ambient PM exposures with newborn size and adiposity differ between white British and Pakistani origin infants.http://dx.doi.org/10.1289/ehp.1408675Schembari A, de Hoogh K, Pedersen M, Dadvand P, Martinez D, Hoek G, Petherick ES, Wright J, Nieuwenhuijsen MJ. 2015. Ambient air pollution and newborn size and adiposity at birth: differences by maternal ethnicity (the Born in Bradford study cohort). Environ Health Perspect 123:1208–1215;  Evidenc tissues . Materna tissues .In newborns, measurements of upper body skinfold thickness may provide an estimate of the total body fat of the infant . PostnatInfants of South Asian origin have a higher prevalence of low birth weight than White Europeans , and theDifferences in newborn body composition and the subsequent increased risk of adverse cardiometabolic outcomes has been linked to both newborn’s size and prenatal exposure to ambient air pollution .In the present study, we examined the association of prenatal exposure to ambient air pollution with mean birth weight and head circumference in an ethnically mixed birth cohort population from Bradford, England. Effect modification by maternal ethnicity was examined by comparison of the associations between the two main ethnic groups: white British and Pakistani-origin populations. We also report here the associations between prenatal exposure to ambient air pollution and neonatal adiposity using triceps and subscapular skinfold thickness. Finally, we examined the influence of adjusting for neonatal skinfold thickness on associations between air pollutants and birth weight.Study population. Born in Bradford (BiB) is a longitudinal multi-ethnic birth cohort  were white British and 4,878 (54%) were of Pakistani origin.The participants gave informed consent for the data collection and ethical approval for the data collection was granted by Bradford Research Ethics Committee (Ref 07/H1302/112).Outcome measurement. Birth weight (grams) recorded immediately following the delivery  was obtained from the medical records. Head circumference , triceps skinfold thickness , and subscapular skinfold thickness  of the offspring were measured by trained hospital personal within the first 24–72 hr after delivery  and oxides of nitrogen  were estimated using land-use regression (LUR) models and measurements developed for the European Study of Cohorts for Air Pollution Effects (ESCAPE), as described in detail elsewhere  in 41 monitoring sites at a combination of traffic, urban, and rural background locations across Bradford. The sites were selected to represent spatial variation of air pollution in the residential areas of the participants. PM10, PM2.5, and PM2.5 absorbance were measured in 20 sites across the Thames Valley (southeast England). A number of geographic information system variables on traffic characteristics, land use, population density, and topography were used to model the measured air pollutants, thus to predict the spatial distribution of exposure to mean levels during 2010. To address the temporal variations related to pregnancies occurring during 2006–2011, we adjusted the LUR spatial annual exposure estimates combining the available daily data of NO2, PM10, and PM2.5 from the background routine monitors located in the cities of Bradford and Leeds. Leeds is at 10 km east of Bradford. Then, the daily spatiotemporal estimates were calculated as the spatial exposure estimation multiplied by the ratio between the daily NO2, PM10, or PM2.5 concentrations at the background routine monitoring station and its annual 2010 average, as described in detail by 2 daily temporal adjustment to NOx and PM2.5 absorbance spatial estimates because of their high correlation , second (from day 93 to day 184), and third trimester (from day 185 to the end of pregnancy), and the full pregnancy (from day 1 to the end of pregnancy). Self-reports of home addresses reported by the mothers at the first interview (week 26–28) were used to estimate the exposure at spatial level. Information on changing residence during pregnancy was not taken into account.Covariates. Self-reported maternal ethnicity was used to define offspring ethnicity and included in all adjusted models for the full population analyses; it was also used to define the subgroups of stratified analyses. Adjustment variables and coding were selected a priori  and birth weight, head circumference at birth, and triceps and subscapular skinfolds thicknesses at birth (as outcomes one at a time). We reported the associations for each 10-μg/m3 increase in PM10, 5-μg/m3 increase in PM2.5, 1-10–5/m increase in PM2.5 absorbance, 10-μg/m3 increase in NO2, and 20-μg/m3 increase in NOx. We evaluated the potential effect modification by maternal ethnicity (white British vs. Pakistani) with the inclusion of an interaction term between air pollutants and ethnicity in the models and with model stratification. We used separate regression models to estimate associations with mean exposures during different time windows of pregnancy . The covariates adjusted for in these models were listed above. We further adjusted birth weight models for subscapular skinfolds thickness to test whether subscapular skinfolds thickness acted as a mediator on these associations. We generated standardized z-scores specific for sex and gestational age  for the full cohort for all the outcomes. We chose to follow the complete case analysis approach.a) exclusion of preterm births (< 37 completed weeks of gestation), n = 481 (5%); b) exclusion of women who had changed their residence address during pregnancy ; c) restriction of birth weight and head circumference analyses to children with triceps skinfold data (to test for selection bias), n = 6,188 (68%); d) stratification by maternal working status , unemployed, n = 4,436 (58%) vs. employed n = 3,201 (42%); e) stratification by pregnancy maternal smoking status ; and f) the exclusion of season of conception from the fully adjusted models . We performed all the statistical analyses using Stata/S.E. version 12.1 (StataCorp) and defined statistical significance as an alpha level of 5% (two tails).Sensitivity analyses entailed Study population. Air pollution exposures and birth weight were available for 4,189 (46%) white British and 4,878 (54%) Pakistani-origin mother–child pairs. White British mothers were younger, were more likely to have a BMI > 25 kg/m2 (data not shown), be nulliparous (48% vs. 32%), and have a lower educational level  . Pearson correlations between trimester-specific exposures were moderate to low . The potential collinearity between PM and NOx or NO2 full-pregnancy mean exposures, according to Pearson correlations, was moderate to low .Pakistani-origin participants were exposed to higher mean concentrations of air pollution compared with white British (r = 0.73) and did not differ between the two ethnic groups (data not shown). The correlation between birth weight and skinfold thickness was modest amongst both the white British (r = 0.50) and Pakistani-origin children (r = 0.55). The correlation between head circumference and skinfold thickness was low in both groups (r = 0.35 in white British and r = 0.30 in children of Pakistani origin). All the correlations above were statistically significant. Girls of both ethnicities had on average lower birth weights (≈ 180 g), smaller head circumferences (≈ 0.5 cm), and higher skinfold thicknesses (≈ 0.2 mm) than boys; these sex differences were greater among Pakistani-origin children than white British children (data not shown).For the full population, the correlation between birth weight and head circumference was high  between maternal ethnicity and exposures to air pollution : –76, –10] in white British infants versus a nonsignificant increment of 9 g  in Pakistani-origin infants. For head circumference we also found statistically significant interactions (p ≤ 0.05). In the stratified analyses, the point estimates of the inverse associations with PM were almost double in magnitude in white British compared with Pakistani-origin children. For example a 5-μg/m3 increase in PM2.5 exposure during the third trimester was associated with a reduction of 0.28 cm  in white British infants and a reduction of 0.08 cm  in Pakistani-origin infants.p ≤ 0.1) between PM2.5, NOx, and ethnicity. A 5-μg/m3 increase in PM2.5 during the third trimester was not associated with these outcomes [–0.02 mm  for triceps skinfold thickness and 0.06 mm  for subscapular skinfold thickness] in the white British population, whereas it was associated with an increment of 0.17 mm  in triceps and of 0.21 mm  in subscapular thickness in Pakistani-origin population. On the contrary, a 20-μg/m3 increase in exposure to NOx during the full pregnancy was associated with a reduction of 0.20 mm  in triceps skinfolds in white British babies, but not in Pakistani-origin babies .For triceps and subscapular skinfold thickness, we found statistically significant interactions   compared with the difference observed in the main analyses, and all the interactions became non-statistically significant  , model 2–51, –6) , model 1nificant .Sensitivity analyses. The results were similar when we analysed sex- and age-standardized z-score values to those reported  (results not shown), suggesting that season of conception was a mediator in the association of exposure to air pollution and birth weight in our study.reported . The repreported . ResultsIn the present study, exposure to PM was associated with child size at birth: We found a reduction in mean birth weight in white British infants, but not in those of Pakistani origin. Ethnic differences were also found in the associations between air pollution and head circumference and triceps and subscapular skinfold thickness. Despite the association between exposure to PM and subscapular skinfold being clinically negligible, its potential mechanistic role was strengthened by the assessment of the potential mediating role of this body fat indicator in the associations between prenatal exposure to airborne PM and birth weight in Pakistani-origin infants . PM was Birth weight is recognized and used in epidemiological studies as indicator of intrauterine growth. Birth weight reflects both the skeletal mass and the mass growth composed by the lean mass and the adipose tissues. Skeletal growth can also be measured at birth as the length of the newborn or estimated through ultrasound scan measures as femoral length , which h3 increase in the exposure to PM2.5 during the full pregnancy was associated with a reduction of 11 g ]; however, the point estimates are consistent with those found in a larger study by 3 increase in the exposure to PM2.5 during the full pregnancy was associated with a reduction of 19 cm  in head circumference, whereas 2 and NOx, which was not the case in the larger ESCAPE sample in which associations for head circumference were evident for all pollutants  . However, the results from this analysis should be interpreted with caution because of the large proportion of missing values in this covariate and because in our study population the employed mothers were predominantly white British; furthermore, we cannot exclude exposure misclassification because employed status can be associated with other factors such as maternal or familial diseases and poverty. In our study population, 6% of women had changed residence during pregnancy, and excluding them from the analyses did not substantially change the results .The differences in effect estimates by ethnicity are complex and could be further related to differences in air pollution composition or housing quality or differences in other risk factors for the outcomes studied such as socioeconomic disadvantages, younger age, poor nutritional status, poor prenatal health care, co-exposure to other pollutants, stress, and instability in residential settings . Howeverin utero exposure to PM may influence the size of newborns in a differential manner between white British and Pakistani-origin babies. The associations between ambient airborne PM and skinfold measures in newborns deserve further study and require replication in different settings and confirmation that associations persist as the cohort increases in age.Our findings from a large multi-ethnic British birth cohort suggest that Further assessment of airborne toxicants such as polycyclic aromatic hydrocarbons and the elemental composition of PM, as well as genetic and lifestyle  factors could help further our understanding of mechanisms that may explain the differences in associations between birth size and adiposity between ethnic groups observed in our study.(381 KB) PDFClick here for additional data file.


Long-term, high dosage protamine zinc insulin (PZI) treatments produce adverse reactions. The trace element selenium (Se) is a candidate for the prevention of diabetes due to anti-oxidative stress activity and the regulation of glycometabolism. In this study, we aimed to investigate the anti-diabetic effects of a combination of PZI and Se on type 2 diabetes. Diabetic KKAy mice were randomized into the following groups: model group and groups that were subcutaneously injected with PZI, Se, high or low dose PZI + Se for 6 weeks. PZI combined with Se decreased the body weight and fasting blood glucose levels. Moreover, this treatment also improved insulin tolerance, as determined by the reduced values from the oral glucose tolerance test and insulin tolerance test, and increased insulin levels and insulin sensitivity index. PZI combined with Se ameliorated skeletal muscle and β-cell damage and the impaired mitochondrial morphology. Oxidative stress was also reduced. Furthermore, PZI combined with Se upregulated phosphatidylinositol 3-kinase (PI3K) and downregulated protein tyrosine phosphatase 1B (PTP1B). Importantly, the low dosage combination produced effects similar to PZI alone. In conclusion, PZI combined with Se improved glycometabolism and ameliorated the tissue and mitochondrial damage, which might be associated with the PI3K and PTP1B pathways. Diabetes mellitus is a complex chronic metabolic disease resulting from abnormal insulin secretion, and type 2 diabetes (T2DM) accounts for more than 90% of cases156Insulin is an effective therapy to decrease the blood glucose levels in type 1 diabetes patients and is a selective therapy in type 2 diabetes patients812Pathways mediated by phosphatidylinositol 3-kinases (PI3Ks) are the major signaling pathways involved in the development of diabetes. In addition, dysfunction of protein tyrosine phosphatase (PTP1B) is also strongly related to insulin secretion and signaling15In this study, yellow KK mice, carrying the yellow obese gene (Ay), were used as a spontaneous diabetic animal model17We have shown that a combination of PZI and Se displayed better performance in STZ-induced diabetic rats see the . As showThe baseline fasting blooding glucose (FBG) levels in the KKAy mice were significantly higher than those in the C57 mice, indicating that diabetes had been established. However, PZI, Se, or PZI combined with Se decreased the FBG levels compared with the model group . FurtherInsulin plays an essential role in glucose regulation. In our study, the insulin levels in the PZI combined with Se group were significantly increased compared with the diabetic mice. However, there is no difference between control and model groups, which may implicate the end stage of T2DM in KKAy mice . ConsistOral glucose tolerance test (OGTT) and insulin tolerance test (ITT) are widely used to evaluate glucose and insulin tolerance. In the OGTT, the KKAy mice manifested a higher glucose load at baseline and a strongly elevated load following glucose administration compared with the control group. The glucose load in the treatment groups, including the PZI, Se and PZI combined with Se groups, decreased much more promptly and persisted compared to the model group . The AUC2 area in the PZI, Se, and PZI combined with Se groups were 3.54, 3.78, 6.70 (H) and 4.56 (L) times the values in the diabetic group. Furthermore, the value of the PZI + Se (H) group was 1.90-fold and that of the PZI + Se (L) group was 1.29-fold higher than the value of the PZI alone group. The value of the PZI + Se (H) group was 1.77-fold and that of the PZI + Se (L) group was 1.21-fold higher than the value of the Se alone group , Se group (+15.75-fold), PZI + Se (H) group (+4.25-fold) and PZI + Se (L) group (+18.73-fold) compared with the model group (2 was higher in the control group (+1.75-fold), PZI group (+1.60-fold), Se group (+3.33-fold), PZI + Se (H) group (+1.67-fold) and PZI + Se (L) group (+1.71-fold) than that in the model group /oxidants (MDA and ROS) have been used to detect oxidative stress46Our results showed that the PTP1B mRNA was upregulated in response to diabetes, while the PI3K levels were decreased in the model group . In paraDespite the abundant research regarding the use of insulin in diabetes, the long-term application of insulin is associated with devastating side effects. As a result, physicians prefer to use adjunct methods that are suitable for long-term use. Our study first confirmed that a low dosage of PZI  combined with Se significantly improved the animals’ general characteristics (food and water volume and decrease in BW), decreased the FBG levels, alleviated glucose and insulin resistance, and mitigated skeletal muscle and mitochondrial injury, accompanied by an increase in the PI3K levels and downregulation of PTP1B. Therefore, we highlight a new, safer therapeutics concept that a low dosage of insulin combined with the trace element selenium can be used for long-term diabetes treatment.6) uniform design and 6 × 6 optimal Latin square principles that precisely determines the proportion ratio and largely decreases the number of experimental groupsClinical reports have testified that long-term insulin usage in diabetes patients will lead to adverse reactions21et al.PZI combined with Se decreased the FBG levels and restored the muscle glycogen levels, implying that PZI and Se improved glucose metabolism. The net BW in the treatment group was not decreased compared with the diabetic group, but weight loss was attenuated. Weight loss is associated with hyperglycemia; therefore, attenuated weight loss is due to the presence of normal glucose levels. Sheng et al. found that a high dosage of isophanuminsulinum promoted β cells recess2 area was increased in the treatment groups, further supporting the recovery of β cells function.β cells dysfunction is the main risk factor correlated with T2DMin vivo skeletal muscle oxidative capacity is determined by the intrinsic mitochondrial function and the number of mitochondria. Therefore, diabetes causes skeletal muscle injuryEmerging evidence has indicated that multiple pathophysiological processes are activated by hyperglycemia and ultimately lead to oxidative stress4Both PZI and Se have been found to activate the insulin receptor/PI3K/AKT signaling cascade, as well as the subsequent activation of GLUT4 and inhibition of ROS production1931In conclusion, this is the first study to investigate the protective effects of a combination of PZI and Se on T2DM. Our results indicated that PZI combined with Se at least partially contributed to β cell repair and reduced insulin resistance, skeletal muscle damage and oxidative stress, which may be associated with the upregulation of PI3K and the downregulation of PTP1B. Importantly, a low dosage of PZI combined with Se produced similar effects as PZI alone, and Se might serve as an insulin sensitizer. Therefore, a combination of PZI and Se may be an effective alternative therapeutic treatment for T2DM, when the mechanisms are sufficiently investigated.Spontaneous diabetic KK/Upj-Ay/J (KKAy) mice , an ideal animal model for diabetes research, were developed by crossing the Ay/a mutation onto the inbred KK strain of native Japanese mice1718171835All of the animal experimental procedures were approved by the Ethics Committees of Xi’an Jiaotong University and were reviewed and performed in accordance with the Guideline of Animal Care and Use Committee of Xi’an Jiaotong University. For choosing an appropriate dosage of PZI, we first tried the dosages of 0.5, 1, 4, 10, 50 U/kg/d PZI alone for 3 days, however, they had no significant effect on FBG. The decreasing rates of FBG were lower than 10%. The decreasing rates of FBG of 100 U/kg/d and 200 U/kg/d PZI were about 15% and 30%. In consideration of long-term medication and the aim of the experiment, we chose 100 U/kg/d PZI alone as the positive control. Six male C57BL/6J mice served as the Control group. After a 1 week acclimation, the KKAy mice with fasting blood glucose (FBG) levels > 16.7 mmol/L were randomly assigned into 5 groups (n = 8 per group): (1) Model group, in which the KKAy mice were treated with an equivalent volume of saline; (2) PZI group, in which the KKAy mice were subcutaneously (s.c.) injected with PZI (100 U/kg/d) ; (3) Se group, which was s.c. injected with Se (200 μg/kg/d) ; (4) PZI + Se  group, which was s.c. injected with PZI (100 U/kg/d) and Se (200 μg/kg/d); and (5) PZI + Se  group, which was s.c. injected with PZI (50 U/kg/d) and Se (100 μg/kg/d). The ratio of PZI and Se was 1:2, which was determined through a uniform design using SD rats see the . The conAt the end of treatment and after a 6 h fast, a 2 g/kg glucose solution was orally administered, and then the blood glucose levels in the blood from the tail veins were measured at 0, 30, 60 and 120 min using a glucose meter. The AUC was calculated for the FBG levels observed during the OGTT.After a 6 h fast, the mice were subcutaneously injected with 4 U/kg insulin , and the blood glucose levels were measured at 0, 40 and 90 min after injection using a glucose meter. The AUC was calculated for the FBG levels observed during the ITT.The serum insulin levels were measured with an enzyme-linked immune sorbent assay (ELISA) using a mouse insulin ELISA kit . The homeostasis model assessment of insulin resistance (HOMA-IR) and insulin sensitivity index (ISI) were calculated according to the following formulas: HOMA-IR = FBG (mmol/L) × FINS (IU/ml)/22.5 and ISI = 1/[FBG (mmol/L) × FINS (IU/ml)].Separated sera were used for the estimation of GSP using GSP assay kit . Samples of 100 mg skeletal muscle were homogenized in 2 mL of a cold 0.1 M phosphate buffer with a pH value of 7.4. Tissue homogenates were prepared in a glass tissue homogenizer. The homogenate was centrifuged at 12000 rpm for 15 min and the supernatant was used for the measurement of glycogen, ROS, SOD and MDA using liver/muscle glycogen assay kit , ROS assay kit , total SOD assay kit  and MDA assay kit . GSP, glycogen, SOD and MDA were determined with an ELISA reader  and ROS were measured with fluorescence microplate reader  according to the manufacturer’s instructions .The splenic regions of the pancreas were minced into 1 mm cubes, fixed with 2.5% glutaraldehyde and 2% osmic acid, then dehydrated and embedded in epoxy resin. Ultrathin sections were collected onto 200-mesh copper grids, double stained with uranyl acetate and lead acetate, then observed with a Hitachi H-7650 transmission electron microscope .The skeletal muscle tissue was prefixed in phosphate buffer (pH 7.2) and embedded in paraffin. Semi-thin sections (1 mm × 1 mm) were examined using a transmission electron microscope  at 50,000× magnification, as previously reportedThe total RNA was isolated from the skeletal muscle using TRIzol reagent. The reverse transcription was performed with 4 μg of RNA using Prime Script™ RT Master Mix . Gene expression was analyzed by real-time quantitative PCR. The primers used in this experiment are listed in One hundred milligrams of thigh tissue was homogenized in 1 mL of RIPA lysis buffer and proteinase inhibitors. Sixty micrograms of the proteins were separated on a 10% SDS/PAGE gel and transferred to PVDF membranes. The membranes were then blocked with 5% non-fat milk or 5% BSA and incubated with primary antibodies against PI3K (p85α) , PTP1B , and β-actin  overnight. After an incubation with the secondary antibody, the bands were treated with horseradish peroxidase (HRP)  and then visualized using ECL-Plus reagent . The bands were analyzed with ImageJ 1.42q.P values less than 0.05 were considered statistically significant.The data are shown as the means ± SEM. Normal distribution analysis before ANOVA analysis was made with SPSS 16.0 for windows . Skewness < 1 and Kurtosis < 1 were considered as normal distribution. Differences between groups were analyzed by ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism Version5.01 . How to cite this article: Lu, J. et al. Protamine zinc insulin combined with sodium selenite improves glycometabolism in the diabetic KKAy mice. Sci. Rep.6, 26563; doi: 10.1038/srep26563 (2016).


Mothers’ stress in pregnancy is considered an environmental risk factor in child development. Multiple stressors may combine to increase risk, and maternal personal characteristics may offset the effects of stress. This study aimed to test the effect of 1) multifactorial prenatal stress, integrating objective “stressors” and subjective “distress” and 2) the moderating effects of maternal characteristics  on infant birthweight.Hierarchical regression modeling was used to examine cross-sectional data on 403 birth mothers and their newborns from an adoption study.2 = 0.032, F = 6.782, p = .001). The hierarchical regression model revealed an almost two-fold increase in variance of birthweight predicted by stressors as compared with distress measures  = 5.339, p < .001). Further, maternal characteristics moderated this association  = 3.413, p = .009). Specifically, the expected benefit to birthweight as a function of higher SES was observed only for mothers with lower levels of harm-avoidance and higher levels of perceived social support. Importantly, the results were not better explained by prematurity, pregnancy complications, exposure to drugs, alcohol or environmental toxins.Distress during pregnancy showed a statistically significant association with birthweight (RThe findings support multidimensional theoretical models of prenatal stress. Although both objective stressors and subjectively measured distress predict birthweight, they should be considered distinct and cumulative components of stress. This study further highlights that jointly considering risk factors and protective factors in pregnancy improves the ability to predict birthweight. A core finding of developmental science is that early life experiences predict subsequent child development. Development is most rapid during the prenatal period  and idenStress is often defined as a threat to physiological and/or psychological homeostasis. . AccordiIn utero, maternal personality and stress impact fetal health through a complex set of physiologic reactions that combine to shape the intrauterine environment  to 5 [daily]), and the extent of knowledge about each other  were aggregated and included as a covariate.To examine the potential effect of adoption openness, birth mothers’ and adoptive parents’ reports of openness, the level of contact between the birth and adoptive parents  to estimate the amount of variance in child birthweight explained by stressor and distress variables separately and by the interaction among stressors and maternal characteristics. The Adoption Openness Scales and Pregnancy Risk Index covariate were entered in the first two steps as a control. The distress variables were then entered in a separate step, followed by the block of stressor variables to investigate their effect over and above that of distress scores. That is, we sought in this fourth step to understand whether there were direct effects of stressors that were not mediated by measures of subjective distress. Maternal characteristics were then added. The interaction between characteristics and the stressor variables was entered as a last step. To ascertain the statistical significance of each interaction, each product term was also entered separately in a second regression model before regions of significance analyses were conducted to interpret statistically significant interactions.(n = 3) and smaller than 4.30 lbs (n = 8) were not included in the analyses. The final sample size reflects the number of BMs (n = 403) without missing data on any of the selected variables excluding the identified outliers.Results of the descriptive analyses for all birth mother variables are reported in F = 7.204, p = .008). The Pregnancy Risk Index did not account for any variance in birth weight  = 0.323, p = .570). In the third block, results indicate that distress measures accounted significantly for 3.2% of the variance in birthweight  = 6.782, p = .001). The four stressors were subsequently entered into the regression equation and the significant change in variance accounted for by all of these was equal to 4.9%  = 5.339, p < .001).The final model contained six blocks . First, maternal characteristics and did not account for a signficant change in variance  = 0.270, p = .604). The final step, which included the four product terms between stressors and maternal characteristics, was associated with an additional 3.1% of variance  = 3.413, p = .009). The hierarchical regression model as a whole accounted for 13.1% of the observed variance in birthweight.The fifth step contained only B = 0.12, t(402) = 2.16, p = .031) had a differential impact on birthweight as a function of maternal characteristics. Other interactions were not significant  = 1.97, p = .285, RC: B = -0.10, t(402) = -1.95, p = .052 CFS: B = -0.05, t(402) = -0.88, p = .379). SES was associated with birthweight only for mothers with average  = 3.68, p < .001) and above average maternal characteristics  = 3.95, p < .001) not for mothers with low scores on personal characteristics  = 4.07, p = .003) and the TCI subscale Harm Avoidance  = 2.35, p = .05) drove the interaction with the stressor variables as indices of maternal characteristics.Post-hoc analyses suggested mothers’ perceived social support scores F = 3.08, p = .016).To control for the possible effect of prematurity on birth weight in the present study, the above analysis was repeated with only infants reported by birth mothers as being born at full term (≥37 weeks of gestation) . DespiteWe found small but unique associations of prenatal maternal distress and maternal stressors with fetal development. We also found that maternal characteristics interacted with stressors in predicting birthweight, suggesting the importance of maternal adaptation. Broadly, the results here demonstrated that a multifactorial model of stress with multiple indicators was most accurate at predicting birthweight. Both birthmother distress and stressor variables during pregnancy were found to predict variance in birthweight. Taken together, the observed findings and extant theoretical framework support that a reliable and sensitive operationalization of stress should reflect its multidimensional nature. Limiting measures of stress in pregnancy to mood or life events may underestimate the effect on child development, not capturing overlapping yet distinct components . In lineProtective personal characteristics measured alongside multiple measures of distress and stressor exposure sets the present study apart [9). Specifically, we hypothesized that measures of maternal self-esteem, temperament/character and perception of social support would moderate the impact of stressors on birthweight, as these factors have been found to predict the appraisal of and psychological adaptation to such experiences . In lineincreased levels of education, income, and neighborhood safety were associated with fetal weight gain only for mothers at average or above average levels of such characteristics. It is noteworthy that an average level of SES in the current sample is lower than the general population. However, in addition to operating as a buffer against adversity at lower levels of SES, these chracteristics may also have enhanced mothers’ capacity to gain from favorable circumstances. This result is consistent with well-validated theoretical frameworks of development [Low SES has been construed as a broad risk-marker for low birthweight , 75 and elopment , 77.In summary, the multiple regression analyses demonstrated that distress and stressor variables both overlapped and interacted yet were distinct components of stress , 78, 79.indirectly correlated with birthweight via other health behaviors during pregnancy [However, the present study does not address the causal mechanisms underlying the association between maternal psychological states and child development. For example, particularly in this cohort of birth mothers who decided to place their child for adoption at birth, stress may be regnancy , 28–30. regnancy , 81 and regnancy . Importaregnancy . FurtherAdditionally, the present study cannot fully exclude biases introduced with retrospective reports despite the lack of influence of postnatal parenting experiences and steps taken to assure the accuracy of self-reported data. Furthermore, factors relating specifically to adoption such as the rationale behind placing the child, the timing of the adoption plan, and compensation to the birth mother could not be investigated in the present study. Other potential limitations include the use of SES as a measure of external stress despite the possible contribution of inherent maternal characteristics to this measure.Despite these concerns, the current study provides evidence supporting the use of multiple indicators of distress and stressors to measure the integrated effects of stress in pregnancy. More important, the present findings clarify that both stressors and distress contribute to birthweight and emphasize the importance of considering maternal characteristics as buffers against those adversities in fetal development.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Emergency cervical cerclage is one of the treatment options for the reduction of preterm birth. The aim of this study is to assess neonatal outcome after cerclage with special focus on adverse effects in very low birth weight infants.2max), neonatal complications ). Statistical comparison of perinatal parameters and outcome of neonates <1500 g after cerclage with a birth weight matched control group.Retrospective cohort study. Classification of cerclages in history-indicated , ultrasound-indicated  and emergency/ physical examination-indicated  cerclage. Descriptive analysis of pregnancy and neonatal outcome  and higher rates of neonatal complications . Placental pathologic evaluation revealed a significantly higher rate of chorioamnionitis (CAM) after PEIC. Neonates <1500 g after UIC or HIC show no significant difference in neonatal complications or CAM.Neonates <1500 g after PEIC show significantly impaired outcome, i.e. prolonged respiratory support (total ventilation in days, CPAP, FiOIn our study PEIC is associated with adverse neonatal outcome in infants <1500 g. The high incidence of CAM indicates a potential inflammatory factor in the pathogenesis. Large well-designed RCTs are required to give conclusive answers to the question whether to prolong or to deliver. Preterm birth is a severe pregnancy complication as the impact on the future life of the newborn is dramatic and associated with many severe health problems . AdditioAt the end of pregnancy the cervix progressively softens and shortens, which is mainly caused by a degradation of collagen fibrils and can be monitored by transvaginal ultrasound. Late miscarriage and preterm birth occur when these changes happen too early. A possible treatment option for preterm cervical changes is cervical cerclage . The terIt has to be taken into consideration that infections are well-established factors of preterm cervical shortening, contractions and dilation and therefore “trapping” the infection represents another potential adverse effect. The diagnosis of intrauterine infection or inflammation is difficult as we lack reliable clinical or biochemical indicators . MaternaPerforming a cerclage has been shown to serve as a valid procedure to reduce the incidence of preterm birth without significant reduction in neonatal morbidity . A matteThe goal of this study is to assess neonatal outcome after cerclage with special focus on adverse effects in very low birth weight infants . We focus on preterm babies <1500 grams as they are at increased risk of neonatal morbidity and mortality with high medical relevance . FurtherThis retrospective cohort study includes all patients who were treated by cerclage in the Department of Obstetrics and Gynecology at the University Clinic Heidelberg between 1999 and 2006. The study was approved by the Heidelberg Medical Faculty Ethics Committee. The ethics committee vote 370/2006 allows for anonymous retrospective analysis of patient data for this project. All patient information was anonymized and de-identified prior to analysis.The standard technique at Heidelberg University was Mc Donald cerclage. Patient data of mothers and neonates was collected retrospectively using the medical records and our electronic patient management system . Cases missing relevant information such as gestational age (GA) of treatment, unclear or undefined indication or missing outcome data were not included.Inclusion criteria: The different types of cerclage were defined as: History-indicated cerclage (HIC), in pregnancies with a history of ≥ 1 preterm birth < 34 gestational weeks and/ or second trimester loss; Ultrasound-indicated cerclage (UIC), transvaginal ultrasound cervical length < 25 mm before gestational week 24 during current pregnancy; Physical examination-indicated cerclage (PEIC), cervical dilatation ≥ 2 cm and/ or bulging membranes. Both singleton and multiple pregnancies were included.Exclusion criteria at time of cerclage placement: Patients with preterm rupture of membranes, vaginal bleeding, regular uterine contractions , clinical  or biochemical  signs of infection, including testing for active vaginal infection by swab test, as well as chronic maternal infections  were excluded from this study.control = 373; nPEIC = 20; nUIC = 10; nHIC = 12). Triplet pregnancies were excluded for this analysis. The fraction of twins was equal in all three subgroups and the control group .For statistical analysis of neonatal outcome of infants <1500 g a control group was generated with neonates born in the study period (1999–2006) without a history of maternal cerclage (n = 652). We performed frequency matching on birth weight categories  and VLBW (1000–1500 g)), in order to adjust for birth weight in all three subgroups and the control group  and neonatal complications (intraventricular hemorrhage (IVH); retinopathy of prematurity (ROP)).Description of the variables statistically assessed in the subgroup of neonates <1500 g: Maternal parameters: rate of preterm premature rupture of membranes (PPROM), reasons for preterm delivery, delivery mode. Infection-associated parameters: CRP levels and white blood cell count (WBC) at delivery, placental pathologic evaluation. Prenatal treatment: antibiotic treatment, RDS prophylaxis (corticosteroids), tocolysis . Neonatal parameters: birth weight, gestational age, APGAR scores, cord pH and base excess, clinical risk index for babies (CRIB score), respiratory parameters , maximal fraction of inspired oxygen (FiOhttp://www.R-project.org). Various statistical methods were used, including analysis of variance (ANOVA), Tukey’s test, Chi-squared test, Kruskal-Wallis test, Welch Two Sample t-test, Wilcoxon rank sum test with continuity correction and are referred to in the corresponding results section. Time-to-event analyses were calculated based on the corresponding Kaplan-Meier estimator and compared by log-rank-test. Bonferroni correction was applied to adjust p-values for multiple comparison.Statistical analysis was performed using R (R Core Team (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria, In this study we identified 106 cases of cerclage  that were treated in our department and matched the criteria of inclusion (see above) resulting in 123 live born neonates . PEIC were delivered significantly earlier than UIC and HIC, whereas there was no significant difference for the time of delivery between UIC and HIC see . In seveMaternal morbidity and mortality was overall low. Cervical laceration was observed in five cases due to preterm uterine contractions. No case of preeclampsia or severe maternal complications  were observed during antenatal, delivery or postpartum period.Neonatal outcome was assessed for all preterm neonates , n = 33 of the UIC-group, n = 31 of the PEIC-group). Neonates after PEIC showed impaired outcome with a significant difference for duration of mechanical ventilation, ROP, IVH, duration of stay at NICU and live birth rate. This difference is in line with our expectations as emergency cerclages target a different risk group than HIC or UIC.HIC = 880 [±204], BWUIC = 904 [±290], BWPEIC = 849 [±281], BWcontrol = 870 [±167]; ANOVA, p = 0.72). There was a significant overall difference for GA at delivery  with a significant difference between controls and UIC only (p = 0.042). The three groups of cerclage differed neither for GA nor for BW  between all four groups . The mean respiratory support via CPAP was significantly longer for neonates after PEIC (+12.4 d)  and also 13.9 d greater for UICs . In line with our findings for respiratory support, our data showed a significantly greater maximal fraction of inspired oxygen (FiO2 max) under respiratory support for infants after PEIC as compared to controls , further stressing the overall compromised respiratory outcome after PEIC.Neonatal complications:Deducible from our findings for respiratory outcome, neonates after PEIC also showed a significantly higher rate of ROP . As ROP 1 is not associated with visual impairment we looked at the rate of ROP ≥ 2. Neonates after PEIC showed a higher rate of ROP ≥ 2 of 80% as compared to 40% for controls without cerclage and an odds ratio of 5.76 . Our data does not support an overall significant difference for IVH , however high significance for clinically relevant IVH ≥ II (Chi-squared test: p = 0.0007) with a 30% rate for neonates after PEIC as compared to 5% for controls (OR 6.45 with [95% CI: 2.11–18.23]).Infection or chronic inflammation play a major role in the pathogenesis of preterm birth and are associated with impaired neonatal outcome. We thus screened for infection-associated perinatal differences and circumstances around delivery in our study population.Clinical signs of CAM were causative for preterm delivery in 45% of cases in the PEIC subgroup as compared to UIC (30%) and HIC (33%). Almost all patients received prenatal antibiotic treatment. CRP levels differed significantly . White blood cell counts within 12 hours before delivery were not significantly higher in PEIC pregnancies (p = 0.376).Placental pathological evaluation was performed for almost all cases. The rate of histological CAM was significantly higher in PEIC placentas (68%) as compared to UIC and HIC (both 30%) (p = 0.048).Short-term postpartal neonatal parameters showed a trend to lower 1’, 5’ and 10’ minute APGAR scores in PEIC neonates. Direct respiratory indicators for neonatal hypoxia such as cord pH did not differ between the groups of cerclage, which might be explained by the overall high rate of cesarean section. The CRIB score  as a tool for assessing direct postnatal adaptation with a high positive predictive value for neonatal morbidity and mortality is also higher in PEIC neonates, however not significantly (p = 0.122).Preterm birth is the single most important cause of neonatal morbidity and mortality. Therefore potential treatment options are urgently needed . HoweverHIC and UIC: When considering placement of cerclage in women with previous preterm birth or late miscarriage, the potential management is either to perform HIC or to observe these women closely with serial ultrasound scans and to perform an UIC in case the cervix shortens. Berghella et al. showed no difference for the two strategies and therefore concluded that serial ultrasound scans might be sufficient to monitor these pregnancies safely and additionally avoid unnecessary treatment . The groPEIC: There is limited data on neonatal outcome after emergency cerclage. Althuisius et al. compared emergency cerclage (PEIC) with bed rest versus bed rest alone and found better outcome in cases of cerclage . In our Non-cerclage control group: Stoll et al. published detailed data for outcome of VLBW neonates in a large retrospective cohort analysis . Our conIn a Cochrane review Alfirevic et al. showed a significant reduction of preterm birth before 37, 34 and 28 weeks in women treated with cerclage at risk of recurrent preterm birth [2max) and rate of severe neonatal complications  differ greatly from birth weight matched neonates without maternal cerclage and same GA  was lost (patients delivered in a different clinic) and cases were excluded from the analysis. In small study populations the exclusion due to insufficient data might represent a selection bias.Some women in the PEIC group had previous preterm births and second trimester losses and did not receive HIC see , as theyFurther, the standard prenatal treatment in between the different groups of cerclage did not differ see , besidesThe delivery mode has been described as an independent risk factor for IVH or periventricular leukomalacia (PVL) in neonates ,25, otheDue to the retrospective study design and the small sample size we did not perform adjustments for body-mass-index, calendar year or other possible variables that might affect outcome parameters. This further stresses the need for large multicenter studies in order to be able to control for the numerous confounders .In conclusion, our study indicates that neonates <1500 g after PEIC show impaired outcome. Intrauterine inflammation is an established risk factor for neonatal morbidity and mortality and may play a causal role in our study population. The diagnosis of subclinical intrauterine infection or inflammation is however difficult as valid clinical or biochemical indicators are missing [