Adverse birth outcomes are major public health problems in developing countries. Data, though scarce in developing countries including Ethiopia, on adverse birth outcomes and the risk factors are important for planning maternal and child health care services. Hence, this study aimed to determine the prevalence and associated factors of adverse birth outcomes among deliveries at Gondar University Hospital, Northwest Ethiopia.Institution based cross-sectional study was conducted in February 2013 at Gondar University Hospital. Data were collected by face-to-face interview of 490 women after verbal informed consent using a pretested and structured questionnaire. Gestational age was determined based on the last normal menstrual period. Birth weight was measured following standards. Multiple logistic regressions were fitted and odds ratios with their 95% confidence interval were computed to identify associated factors.The mean age of women was 26.2 (±5.2 SD) years. HIV infection among laboring women was 4.8%. About 23% of women had adverse birth outcomes . Women having history of either preterm delivery or small baby  were more likely to have preterm births. Similarly, history of delivering preterm or small baby , preterm birth  and hypertension  were associated factors with low birth weight. Ante partum haemorrhage , hypertension , history of perinatal death  and lack of antenatal care follow up  were significantly associated with still birth.Prevalence of adverse birth outcomes  were high and still a major public health problem in the area. Histories of perinatal death, delivering preterm or small baby, ante partum hemorrhage, lack of ante natal care follow up and hypertension were associated factors with adverse birth outcomes. Thus, further enhancements of ante natal and maternal care and early screening for hypertension are recommended. Adverse birth outcomes- such as prematurity, low birth weight and birth defects- represent significant problems in both developing and developed countries. Each year, about 15 million babies in the world, more than one in 10 births, are born too prematurely. More than one million of those babies die shortly after birth; countless others suffer from lifelong physical, neurological, or educational disabilities, often at great cost to families and societies . ComplicFrom a global standpoint, the prevalence rate of preterm birth varies from 47.5 to 137 per 1000 live births. Extreme parity, a previous history of preterm birth or abortion, younger maternal age, inadequacy of prenatal care, reported hypertension, antepartum hemorrhage, premature rupture of fetal membranes and induced labor are significant determinants of preterm birth .Worldwide stillbirth rate has declined by 14%, from 22.1 stillbirths per 1000 births in 1995 to 18.9 stillbirths per 1000 births in 2009. But in the African region, there was only an annual decline of less than 1%. The stillbirth rate for developed countries is estimated between 4.2 and 6.8 per 1000 births, whereas for the developing world, the estimate ranges from 20 to 32 per 1000 births. Two thirds of all stillbirths occur in just two regions: South-East Asia and Africa ,8. In suLow birth weight (LBW) is closely associated with increased fetal and neonatal mortality, morbidity, and impaired growth and cognitive development. It also leads to chronic diseases later in life. Worldwide, more than twenty million infants  are born LBW . SeveralIn Ethiopia, adverse outcome of pregnancy are still major public health problems ,19. The A hospital based cross-sectional study was conducted at the maternity wards of Gondar University hospital in February 2013. This hospital is the only tertiary hospital located in the historical city of Gondar. It serves for over five million people residing in urban and rural parts of northern and northwestern Ethiopia. On average, there are about 20 deliveries everyday in this hospital. The study included 490 laboring women selected consecutively during the data collection period. This study included all women who gave birth throughout the day and night during the one month study period.Low birth weight was defined as a birth weight below 2500 grams (5.5 pounds). If the baby was born before 37 completed weeks of gestation but after 28 weeks of gestation, it was considered as preterm. Stillbirth was defined as the birth of an infant that has died in the womb or during intra partum after 28 weeks of gestation. Gestational age was calculated based on the last normal menstrual period (LNMP). When LNMP-based gestational age was unknown, we relied on ultrasonography measures and nine women were excluded from the analysis since gestational age was not determined using either method. Birth weight was measured for each new born within an hour of birth using a calibrated weight scale.Data were collected using a combination of a structured questionnaire and measurements of weight of the new born by eight midwives who were trained for this purpose. The questionnaire was structured into four logical sections . Data were entered into EPI Info version 3.5.3 and exported to SPSS version 20 for analysis. Descriptive statistics like frequencies and cross tabulations were performed. Multiple logistic regressions were fitted for the three major adverse birth outcomes separately and odds ratio (OR) with their 95% confidence interval (95% CI) were calculated to identify associated factors of adverse birth outcomes. Variables with p-values ≤ 0.2 in bivarate analysis were remained in the model as potential confounders for the next level analysis. The Hosmer -Lemeshow goodness-of-fit statistic was used to check if the necessary assumptions for multiple logistic regressions were fulfilled and the model had p-value >0.05 which proved the model was good.Ethical clearance was obtained from the University of Gondar Institutional Review Board. Permission letter was also obtained from hospital administration office. Data were collected after informed verbal consent was obtained and after the women were stabilized and ready to be discharged. Confidentiality of the information was assured from all the data collectors and investigators sides. The questionnaire was administered anonymously, locked with keys (hard copy) and password protected (soft copy). Those who had adverse birth outcomes were linked for additional services .A total of 481 laboring women were included in this study. The mean age was 26.2 (±5.2 SD) years. Majority were Orthodox Christians (83.8%), and Amhara (97.1%) ethnics. Most (92.7%) were married, and more than half (55.1%) were housewives. About one quarter (24.9%) of the participants attended secondary education. The mean age at first marriage was 19.3 (±3.7SD) years (Table Majority of respondents (86.3%) had ante natal care (ANC) follow and 17.8% had started their follow up during the first trimester of pregnancy. About three fifth (57.8%) of them had at least 4 ANC visits. Nearly three quarters (73.2%) were using modern contraceptives prior to the current pregnancy, 70% injectible methods. Similarly, nearly three quarter (73%) of the respondents had nutritional counseling and about 72% had additional diet during the current pregnancy.Most labors (91.1%) were spontaneously initiated. The mean duration of labor was 9.4 (±5.9 SD) hours. About 81% of current deliveries were spontaneous vaginal deliveries (SVD) and or assisted vaginal deliveries and 13.9% by caesarian section (CS).Historically, 6.7% of women reported pre natal death in the preceding birth. The pregnancies of most (86.3%) women were planned and wanted. More than one fifth 22.5%) of participants had premature rapture of membrane (PROM) in the current delivery  were screened for HIV and about 5% were sero-positive Table .The over prevalence of still birth was 7.1%. As shown in the multivariate analysis model, risk factors like preterm birth, low birth weight, ante partum hemorrhage (APH), hypertension, history of perinatal death, lack of ANC follow up and large family size (>5) were significantly and independently associated with still birth Table .Nearly one in seven births (14.3%) was found to be preterm birth. The mean gestational age was 37.1 (±1.7 SD) weeks. Women who had history of either preterm delivery or low birth weight  were more like to have preterm birth than their counter parts. On the other hand, hypertension was significant  in the bivariate analysis but turned out insignificant in the multivariate analysis.In this study, 11.2% of deliveries were found to be LBW. The mean neonatal birth weight was 2977.7 ±573.5 SD) grams. In multivariate analysis, history of preterm delivery/or small baby (AOR: 8.40 95% CI 2.40- 29.40), preterm delivery (AOR: 5.51 95% CI 2.61- 11.62) and hypertension (AOR: 5.84 95% CI 1.75- 19.55) remained significantly and independently associated with LBW  among deliveries at Gondar University hospital. The prevalence of still birth was 71 per 1,000 total births. This prevalence is higher than would be expected from a community based study since the study center is a tertiary hospital managing referrals from health centers and district hospitals. It is also higher than the previous reports from Nigeria, Zambia and a systemic review for sub-Saharan African studies where the prevalence of still birth ranged from 21-33/1,000 total births ,12,20. MThis study also revealed that nearly one in seven births 14.3%) was preterm. This is lower than a previous finding from Uganda among HIV-positive rural mothers (17.7%) . This va.3% was pThe prevalence of LBW in this study was 11.2%. This is lower than a previous study in the same hospital (17.1%) . This miIn multivariate analysis, women who did not have ANC follow up were more likely to have stillbirth. During ANC follow up women will have access to information related to nutrition and danger signs of pregnancy. Regular ANC follow up will also help a pregnant woman seek early treatment for her potential pregnancy related problems but if failed to showed up for ANC, she will be disadvantaged. Additionally, women who did not have ANC follow up were mostly illiterate (60.3%) and hence may not have good healthcare seeking behaviors. This finding is in line with previous studies in Africa ,20,22. SFurthermore, women having history of perinatal death in the preceding births were at higher risk of having stillbirths. Most poor obstetrics histories are recurrent. LBW was also found to be associated with stillbirth in this study. Similar to other studies ,27, LBW Owing to the fewer number of cases the significant association in the crude analysis between hypertension and preterm birth was insignificant after adjustment. In the adjusted analysis, LBW was more common in women who had hypertension, had previous history of preterm and/or small baby deliveries and among preterm newborns. Hypertension is one of the causes of preterm deliveries and immature newborns are more likely to be LBW . It is pThis study shares the limitations of cross-sectional studies and hence may not be possible to establish temporal relationship between adverse birth outcomes and explanatory variables. Besides, as the study was in a referral hospital, it may not show the real picture of these adverse birth outcomes in the area. Another limitation is possible recall bias while determining the gestational age.Adverse birth outcomes  are still major public health problems in this area.Histories of perinatal death, preterm birth and/or small baby, ante partum hemorrhage, absence of ANC follow up and hypertension were associated with adverse birth outcomes. Hence, further enhancements of antenatal and maternal care as well as early screening for hypertension are important recommendations. We also recommend a more representative community based study.The authors declare that they have no conflict of interests.AA wrote the proposal, participated in data collection, analyzed the data and drafted the paper. BM, TA, LG and BD approved the proposal with some revisions, participated in data collection, analysis and manuscript writing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2393/14/90/prepub


Gastrointestinal duplication cysts are rare congenital anomalies that can be seen in anywhere of the gastrointestinal system from the mouth to the anus. These are prenatally diagnosed through antenatal ultrasonography. However, attention must be paid since these formations might be confused with ovarian or mesenteric cysts. Our patient, who had been diagnosed with ovarian cyst on the ultrasonography performed in another center and with mesenteric cyst based on the abdominal MRI carried out at fifth month of life, presented to our clinic with the only complaint of constipation at 9th month of life. The diagnosis was set through double wall appearance of duplication cyst on the abdominal ultrasonography. The patient's cyst was resected. Gastrointestinal duplication cysts are rare congenital anomalies that can be seen in anywhere of the gastrointestinal system with a prevalence between 1 : 4500 and 1 : 10000. The most common location is ileum. Signs and symptoms differ based on the location, although mostly it manifests as acute abdominal or intestinal obstruction before 2 years of life. More rarely serious complications such as gastrointestinal bleeding or malignant degeneration may also occur. Colonic duplication cysts are further rare. Gastrointestinal duplication cysts are usually seen as two different forms as lumen-communicating and noncommunicating types. The most common form is cystic and noncommunicating type . The treOn the second-level USG ordered at 23rd gestational week of a healthy mother aged 32, a hypoechoic formation with 5 mm and 9 mm in size which was observed at the right side of the bladder in the pelvis was considered and followed up as ovarian cystic formation.The patient was born by normal vaginal route at term, weight of 3000 gr. The patient's Apgar score was found 8 in minute 1 and 9 in minute 5. No pathologic finding was found at the physical examination of the patient. On the contrast enhanced abdominal MRI performed in a different center when the patient was five months old showed simple cyst. It also revealed two lobule-contoured, thick-walled cystic lesion with diameters of 19 mm, 12 mm, and 5 mm in the right lateral part of the abdomen. The mass was diagnosed as mesenteric cyst, and the patient was followed up for this diagnosis.Our patient presented to our clinic at her 9th month of life with only complaint of constipation. On physical examination, biochemical and hematological values were found normal in the patient who had a growth and development of 50 percentile. On her USG performed in our clinic, 19 mm, 12 mm, and 5 mm in size the duplication cyst having double wall appearance was observed in the right lower quadrant. On sonography, we found that the hypoechoic outer wall of the cystic structure was continuous with the hypoechoic outer wall of the adjacent ileal bowel, and the hypoechoic outer wall of the cystic structure was continuous with the hypoechoic outer wall of the adjacent ileal bowel .An exploratory laparotomy was performed. On the exploration, a duplication cyst of 19 mm, 12 mm, and 5 mm in size was found at the ileal level at an approximate distance of 45 cm from the ileocecal valve . About 7Alimentary tract duplications are uncommon congenital anomalies that occur mostly in paediatric patients. The first reported case was made by Calder in 1733 . DuplicaPrimary cystic lesions seen in the fetal and neonatal periods include renal, choledochal ovarian, mesenteric, duplication cysts, or Meckel's diverticulum. Double wall appearance of duplication is monitored on USG as a hyperechoic edge surrounding the mucosal wall inside and a hypoechoic wall surrounding the smooth muscular layer outside. However, the double wall sign can also be a characteristic of Meckel's diverticulum or sonographic artifacts. Furthermore, Meckel's diverticulum can have a spherically shaped cystlike appearance, which is similar to an enteric duplication cyst, on sonography –9. AlthoPresentation of intestinal duplications is variable depending on size, shape, and type of mucosa. They may be asymptomatic and discovered accidentally at surgery. They may be minimally symptomatic and associated with vague abdominal pain, constipation, or failure to thrive . Our patThe enteric duplication cyst can be associated with malrotation. In half of the cases, there are associated malformations, the most frequent of these being esophageal duplications, followed by vertebral abnormalities . No malr Our patient, who had been diagnosed with ovarian cyst on the ultrasonography (USG) performed in another center and with mesenteric cyst based on the abdominal MRI carried out at fifth month of life, presented to our clinic with the only complaint of constipation at 9th month of life. On USG duplication cyst was diagnosed through the double wall appearance which is specific for duplication cyst. The treatment option proposed for duplication cysts is surgery and the surgery of choice is excision because of the potential complications . In our In conclusion, ultrasonography is an inexpensive and practicable imaging modality in evaluation of intra-abdominal cystic lesions. Although it seems asymptomatic and rare, this type of cyst must be surgically excised because of the potential complications and malignant degeneration.


Mortality risk varied significantly across different ages and was higher for males at any age. Maternal reproductive history was associated with large differences in both stillbirth and liveborn mortality risk: first-time mothers had a higher risk of calf loss as did mothers producing another calf soon (<3.7 years) after a previous birth, and when giving birth at older age. Stillbirth (4%) and pre-weaning mortality (25.6%) were considerably lower than those reported for zoo elephants and used in published population viability analyses. A large proportion of deaths were caused by accidents and lack of maternal milk/calf weakness which both might be partly preventable by supplementary feeding of mothers and calves and work reduction of high-risk mothers. Our results on Myanmar timber elephants with an extensive keeping system provide an important comparison to compromised survivorship reported in zoo elephants. They have implications for improving captive working elephant management systems in range countries and for refining population viability analyses with realistic parameter values in order to predict future population size of the Asian elephant.Juvenile mortality is a key factor influencing population growth rate in density-independent, predation-free, well-managed captive populations. Currently at least a quarter of all Asian elephants live in captivity, but both the wild and captive populations are unsustainable with the present fertility and calf mortality rates. Despite the need for detailed data on calf mortality to manage effectively populations and to minimize the need for capture from the wild, very little is known of the causes and correlates of calf mortality in Asian elephants. Here we use the world's largest multigenerational demographic dataset on a semi-captive population of Asian elephants compiled from timber camps in Myanmar to investigate the survival of calves ( Elephas maximus) with a significant proportion of the world's population now living in captivity. Studies on the mortality and fertility rates of such populations contribute to our knowledge of how management practices affect population sustainability. In many species conditions in captivity such as stable food availability, medical interventions, improved hygiene and lack of predation are associated with early age at first reproduction, short inter-birth intervals and increased adult longevity in comparison with wild counterparts  of Amboseli, Kenya The Asian elephant is classified as Endangered on the International Union for Conservation of Nature (IUCN) Red List of threatened species. In parallel with wild populations, many captive populations are also facing rapid decline and extinction because of current low fertility and high calf mortality rates The paucity of information on the determinants of calf mortality in Asian elephants stems from the rarity of long-term demographic datasets. Studies on captive elephant populations in zoological collections use studbook data but both the sample size and data available on causes of calf mortality in such datasets are often limited. Demographic studies on wild Asian elephants mainly use distance sampling techniques to estimate animal population densities, for example via dung counts or ratios of calves to adults, but no longitudinal, individual-based data exists. Limited information is available for captive Asian elephants n>5000) in the world The one exception is Myanmar (Burma), home to the largest captive elephant population (n = 1020) born to captive mothers (n = 391), and mortality causes and correlates. Specifically, given the high risk of stillbirths in many captive elephant populations n = 272) allows for documenting for the first time the range and prevalence of causes for death for captive Asian elephant calves in their range country.Here we use the largest multigenerational demographic dataset on a captive population of Asian elephants, compiled from 260 timber camps in Myanmar over several decades and covering up to four generations of timber elephants. We investigate survival of calves ; date and place of birth; mother's registration number and name; method, year and place of capture (if wild-captured); year or age of taming; dates and identities of all calves born; date of death or last known date alive; and cause of death. The individual elephant log-books are maintained and updated by local veterinarians and regional extraction managers at least bi-monthly in order to check the health condition and ability of each elephant to work. The multiple sources of data recorded by the Myanmar Timber Enterprise  allows effective cross-checking of any apparent errors. Between-individual variation in workload or rest periods is limited by law: all state-owned elephants are subject to the same regulations set by central government for hours of work per week, working days per year, and tonnage to extract per elephant. For example, in 2010 all mature elephants (>17–55) worked 3–5 days a week (depending on weather and forage availability) 5–6 hrs a day (maximum 8 hrs) with a break at noon. All elephants finish their work season by mid-February each year, and work resumes around mid-June depending on the arrival of monsoon. The maximum tonnage of logs allowed to be dragged in a year per elephant was 400 in 2010. The ages of captive-born elephants are exact because precise dates of birth are recorded, and this study concentrates only on the records of captive-born mothers and their offspring in order to have accurate data on maternal age and previous reproductive history, which are incomplete for most wild-captured mothers.In the wild, elephant calves are raised by their mothers with the help of allomothers and other members of their family unit n = 66) from the analyses, given those born in 2000 would have been censored in all analyses under age one. The remaining sample includes records of a total of 1138 calves born to captive-born mothers, of which 45 calves were recorded as stillborn and included in the analyses of factors associated with the risk of stillbirth. Excluding the stillbirths and animals with missing information on sex or dates of birth/death/lost/escapes, 975 liveborn animals born from 1960 to 1999  remained for further analyses from 391 captive-born mothers, themselves born during 1941–1990. These elephants come from 32 timber extraction areas within eight regions  in Myanmar: Ayeyarwaddy, Bago, Chin, Kachin, Magway, Mandalay, Sagaing and Shan. The percentage of elephants from the initial sample that were included in the analyses  is presented in The entire studbook includes 8006 elephants born and/or captured between 1925 and 2000; data from 2000 onwards is not available to us at the time of this study. Because of the lack of data from 2000 onwards, all calves born in 2000 were removed  to investigate the stillbirth probability and a discrete time survival analysis  to investigate the calf mortality from birth to age of five. The discrete time survival analysis allows a detailed analysis of the effects of time-dependent variables on the calf's probability of dying over discrete time intervals, while controlling for repeated terms of the mother identity and area st borns vs. later borns), previous inter-birth interval , birth cohort (decade of birth), mother's age at the birth of the calf as a continuous variable and the repeated measures of the mother and living area . We also included quadratic functions of maternal age in the models. The inter-birth interval preceding the birth of the focal individual was grouped into four classes based on the distribution of interbirth intervals in the population: ‘short’ if the previous birth-interval length was <3.7 years (25% quartile), ‘medium’ if it was 3.7–6.6 years, ‘long’ if it was >6.6 years (75% quartile) and ‘firstborn or missing’ if the calf was first to its mother  or a laterborn with missing information on birth interval length (n = 23). We tested interactions between the fixed variables especially with calf age (discrete time model) to investigate whether any effects change with calf age. The data were restricted to only single-born calves, because of the poor survival prospects and low number of twin births (n = 8). Descriptive statistics and follow-up percentages  with regard to the variables included in the analyses are given in n = 1138, i.e. including 45 stillbirths and animals with missing information on sex or dates of birth/death/lost/escapes that were subsequently excluded from the analysis of livebirths). All analyses were conducted using SAS . Significance levels were set at α = 0.05. All biologically interesting interactions were tested, but omitted if not statistically significant. In all analyses, selection of significant terms retained in the best final models was determined using a backward elimination approach.We investigated the effects of calf age (discrete time model), sex, birth-order  fitted to a binomial error structure with logit link function. The effects of linear and quadratic functions of maternal age, calf sex, birth-order and birth cohort were investigated on stillbirth mortality risk, fitted as fixed terms in the model. Because of the low number of stillbirths, it was not possible to simultaneously estimate the effects of birth intervals and birth-order. Inclusion of only birth-order in our final model was thus chosen, because analyzing both variables separately suggested that birth-order may be more important of the two. Non-independence of stillbirth probabilities of calves from the same mother were accounted by including the mother-id as a repeated term. Mother's area was included as a repeated term to control for similar living conditions of calves from the same area. The analysis was conducted on 1020 calves, of which 45 were stillbirths and 975 live-births.n = 975 calves, n = 378 mothers).We first documented the overall survival pattern  of calves from birth to age of 18 . We then investigated calf mortality from birth  to five years using discrete time survival analysis. This analysis allowed us to estimate the calf's risk of dying in each year from birth while investigating the effects of fixed and repeated terms. For each year from birth to 5 years , the survival of each calf was coded as survived versus died during the observation year (1/0) or missing . Following restrictions (see above), this analysis was carried out on 4121 data points (P = 0.05) using the backward elimination approach. We used GLMM with binomial error structure and logit link function in GENMOD procedure of SAS.The analysis investigated the effects of linear and quadratic terms (non-linear effects) of calf age and mother age, previous birth-interval length, calf sex, birth cohort and birth-order and the repeated terms of mother and area. The assessment of interactions between a fixed variable and calf age provides an indication of whether calves are more likely to die at a given age in relation to the variable and thus, whether the effect changes with calf age. Therefore, all interactions with calf age were first included in the model but removed if they did not reach statistical significance level ; diseases ; mother agalactia (lack of milk after parturition) and related general weakness; heat stroke; taming-related injury; and others including unknown. We then investigated the distribution and generality of the causes of death for calves below age five.21 = 5.55, P = 0.019; maternal age2 χ21 = 0.15, P = 0.70) 21 = 14,61, P = 0.0001). There was no significant difference in stillbirths between males and females  vs. 4.1% , respectively; n male = 517, n female = 503 including 26 stillborn males and 19 females; χ21 = 0.55, P = 0.46). Finally, stillbirth probability appeared slightly higher among calves born in 1990s as compared to the other birth cohorts , 1970: 3.2% , 1980: 3.4% , 1990: 7.1% ) but the differences between birth cohorts were not statistically significant .Overall, 4% (45/1020) of all calves were stillborn. The probability of stillbirth increased significantly with maternal age  times higher mortality risk than did later born calves from the same mothers, and such differences remained from birth until age five  times higher mortality risk than female calves across all ages since birth until age five . As a coThe main cause of death for calves under five was accidents (42.4%) which were distributed across all ages . One in The worldwide populations of both wild and many captive Asian elephants will continue to be in danger of disappearing unless their current fertility and calf mortality rates improve Our finding that the general levels of stillbirth and pre-weaning mortality among Myanmar working elephants were considerably lower than those reported for zoo elephants is of interest. The recent progress in our understanding of physiology One striking difference between zoo populations and working elephants in Myanmar is the stillbirth rate. During the past two decades, the reports on stillbirth rate in zoo-born Asian elephants have ranged between 16% Our results offer possibilities to improve the management and sustainability of the large captive working elephant populations in Asia. Given that in the wild the Asian elephant is listed as endangered and the captive populations form a large proportion of the worldwide remaining Asian elephants It thus appears that maternal health and survival of calves could be improved by systematic supplementation and or along with reduced work-load of females in the risk groups. In order to enhance survival of calves, it might be necessary to ensure that older females and those with short birth-intervals take a long enough rest to replenish their body reserves after producing and suckling an offspring. It may also be advisable to supplement primiparous pregnant females and/or reduce their workload to avoid excess calf mortality. Furthermore, it is likely that social networks with older herd members are particularly important for the breeding success of first-time elephant mothers, given that in the wild, calves are typically raised by their mothers with the help of other related females The largest fractions of deaths were caused by accidents and by maternal agalactia or innate calf weakness. These latter causes might be partly preventable by supplementary feeding and/or work reduction of the high risk first time or ageing mothers. Over 40% of all liveborn calves died in accidents. They were often linked with a calf whose mother did not produce enough milk straying away from the mother in search of additional food, and might thus be avoidable by improving the nutrition of such calves and their mothers. The main causes for calf-weakness related deaths are inability of mothers to produce adequate milk following pregnancy and nutritional stress, as well as parasite infections of calves et al.et al.et al.et al.These results have implications for refining population viability analyses with realistic parameter values in order to understand the sustainability of captive elephant populations. Several attempts have been made recently to estimate the future population of the Asian elephant. For example, Leimgruber


Neorickettsia risticii is the Gram-negative, obligate, and intracellular bacterial pathogen responsible for Potomac horse fever (PHF): an important acute systemic disease of horses. N. risticii surface proteins, critical for immune recognition, have not been thoroughly characterized. In this paper, we identified the 51-kDa antigen (P51) as a major surface-exposed outer membrane protein of older and contemporary strains of N. risticii through mass spectrometry of streptavidin-purified biotinylated surface-labeled proteins. Western blot analysis of sera from naturally-infected horses demonstrated universal and strong recognition of recombinant P51 over other Neorickettsia recombinant proteins. Comparisons of amino acid sequences for predicted secondary structures of P51, as well as Neorickettsia surface proteins 2 (Nsp2) and 3 (Nsp3) among N. risticii strains from horses with PHF during a 26-year period throughout the United States revealed that the majority of variations among strains were concentrated in regions predicted to be external loops of their β-barrel structures. Large insertions or deletions occurred within a tandem-repeat region in Ssa3. These data demonstrate patterns of geographical association for P51 and temporal associations for Nsp2, Nsp3, and Ssa3, indicating evolutionary trends for these Neorickettsia surface antigen genes. This study showed N. risticii surface protein population dynamics, providing groundwork for designing immunodiagnostic targets for PHF. Neorickettsia (formerly Ehrlichia) risticii is an obligate intracellular bacterium and the causative agent of Potomac horse fever (PHF) [N. risticii and develops PHF [N. risticii-infected digenetic trematodes molt and emerge (hatch) from the water as adults [N. risticii [N. risticii-infected cells and by nested polymerase chain reaction (PCR) on blood samples [N. risticii type strain, which demonstrate inadequate efficacy [Discovered in 1984, er (PHF) . The baclops PHF . PHF is s adults ,10. Clins adults . The admristicii -15. Diag samples -22. The efficacy ,24.N. risticii has similar genetic, antigenic, and morphologic characteristics to Neorickettsia helminthoeca [Neorickettsia  sennetsu, was regrouped into the genus Neorickettsia [Stellantchasmus falcatus (SF) agent, isolated from metacercariae in fish from Japan and Oregon [N. risticii also consists of a variety of strains, based on PCR and sequencing of 16S RNA and groEL, Western blot analyses using purified bacteria as antigen, and morphology [It was determined that inthoeca ,26, whicckettsia . In addid Oregon -30 belonrphology ,22,24,31N. risticii surface-exposed proteins, and this missing information is crucial in the understanding of bacterium-host cell interactions. Antigenic and potential surface proteins ranging between 28 and 110-kDa in mass were previously detected by Western blotting, but these proteins were not identified [N. risticii labeled with I125 and N. risticii immune mouse sera revealed potential surface proteins ranging from 25 to 62-kDa in mass, although these proteins were not identified [N. risticii strains were identified as GroEL and the 51-kDa antigen (P51) [Little is known about entified . Immunopentified . Antigenentified . Two higen (P51) , but it en (P51) ,36.Neorickettsia proteins is now achievable with the availability of whole genome sequencing data on both the type strain (Miyayama) of N. sennetsu [N. risticii [N. risticii, 2) horse immune recognition of N. risticii surface proteins, and 3) strain variations in aligned sequences of these major surface proteins with respect to their predicted secondary structures.The identification of sennetsu  and the risticii . In thisN. risticii IllinoisT [1 cells in 75-cm2 flasks containing RPMI 1640  supplemented with 5-10% fetal bovine serum (FBS)  and 4-6 mM L-glutamine  at 37°C under 5% CO2. N. risticii was isolated from highly-infected P388D1 cells as previously described for N. sennetsu MiyayamaT [llinoisT  and a PellinoisT  were culiyayamaT .N. risticii Illinois and PA-1 from twenty-five 75-cm2 flasks using EZ Link Sulfo-NHS-SS-Biotin  and subsequent bacterial lysis and collection of solubilized bacterial proteins were performed as previously described [N. risticii proteins was then performed, followed by SDS-polyacrylamide gel electrophoresis (PAGE) and fixation and GelCode blue (Pierce) staining of the gel [N. risticii Illinois and proteins from four bands or band collections from PA-1 were identified by capillary-liquid chromatography-nanospray tandem mass spectrometry (Nano-LC/MS/MS) as previously described [Biotinylation of purified escribed . Strepta the gel . Proteinescribed .N. risticii Illinois (NRI_0235), and rNsp2 (35 kDa) and rNsp3 (28 kDa), cloned from N. sennetsu Miyayama , were expressed by transformed BL21(DE3) cells using isopropyl-β-D-thiogalactopyranoside induction and His-tag purified as described previously [N. sennetsu Miyayama (NSE_0642), was acquired from stored aliquots [Recombinant P51 , cloned from eviously ,39. Recoaliquots . Fifty μaliquots ,21. The N. risticii in P388D1 cells using the DNeasy Blood and Tissue Kit , according to manufacturer's instructions. PCR amplification was then performed using either Phusion or Taq DNA polymerase  and primers designed for conserved regions through alignment of multiple Neorickettsia spp. and/or N. risticii strains  were analyzed by PHYLIP (v3.66) to obtain bootstrap values for 1000 replicates  and to create dendrograms  [DNA was purified from buffy coats of PHF-positive horses or cultures of rawgram) . Proteinrawgram) ,44.N. risticii strain and Neorickettsia spp. sequences.Predictions for Nsp2 and Nsp3 were based on a combination of the programming algorithm in the PRED-TMBB web server , hydrophN. risticii Illinois  and N. sennetsu Miyayama (YP_746740). Previously-deposited Nsp3 sequences include N. risticii Illinois  and N. sennetsu Miyayama (YP_506742). Ssa3 sequences include N. risticii Illinois  and N. sennetsu Miyayama . The Ssa1 sequence is from N. risticii Illinois , and other Ssas are from 25-D (AAC31427) and 90-12 (AAC31428).GenBank accession numbers of all sequences determined in this study are shown in Table N. risticii Illinois genome (NC_013009) has been sequenced [N. risticii proteins in N. risticii Illinois (1984 isolate) and five N. risticii proteins (with conserved peptide sequences in relation to N. risticii Illinois) in PA-1 (2000 isolate) contained two or more peptide queries identified by Nano-LC/MS/MS , Nsp3, and a conserved hypothetical protein (NRI_0567). The largest protein coverage and the largest number of peptides identified were both from P51. Proteins identified in PA-1 also included P51 and GroEL; the largest number of peptides was from P51. Minor proteins identified in PA-1 strain were DnaK (NRI_0017), ATP synthase F1, alpha subunit , and strain-specific antigen 3 .Given that only the equenced , these dome NC_01009 has bN. risticii Illinois by nano-LC/MS/MS, rNsp2 was included in the Western blotting studies because both Nsp3 and Nsp2 from N. sennetsu Miyayama are significant surface proteins  were unique to all other N. risticii strains. For full-length Nsp2, 081 clustered with SF Oregon, rather than with other N. risticii strains. Additionally, external loop 2  showed similar patterns of clustering as seen in full-length Nsp2 and external loop 4; the exceptions were MN, which was 100% identical to N. risticii Illinois and Herodia, and OH07-4, which had one amino acid difference in comparison to the majority of post-2000 strains in this region (data not shown).Nsp2 sequences of 4 Figure . Full-le4 Figure  and 4c. N. risticii, except for the sequence from N. risticii Illinois have also not been determined. Nsp3 was predicted to have eight transmembrane β-barrel proteins with four external loops. Alignment of a total of 21 Nsp3 proteins and protein fragments demonstrated the highest variation within predicted external loop 2, yet there was less variation in the C-terminal region comprising external loops 3 and 4  [Neorickettsia spp. consisted of anywhere from zero to four repeated 52-aa peptides arranged in tandem followed by a terminal 40-aa peptide similar to the 52-aa repeats . It appears that the number of 52-aa repeats increases over time; six post-2000 strains . Of note, there are β-sheets predicted to encompass most of the repeated region  and scattered within the C-terminal region (aa 235-433).Ssa3 sequences of in 90-12 , and a sMiyayama . There wMiyayama , and Ssa in size . Tandem Illinois . In part 53-196) . ThirteeN. risticii, other than that of N. risticii Illinois have not been determined. Given the strongest similarities between ssa1 of N. risticii Illinois and the unknown ssas from N. risticii strains 25-D (isolated in 1984) and 90-12 (isolated in 1990) [ssa1 fragments were amplified, sequenced, and translated from PA-1 and OH07-1. PA-1 (aa 11-249) and OH07-1 (aa 1-239) Ssa1 fragments were aligned with corresponding regions from N. risticii Illinois Ssa1 (aa 246-469) and the Ssas from 25-D (aa 287-507) and 90-12 (aa 579-817). Ssa1 fragments from PA-1 and OH07-1, which are both post-2000 strains, clustered with the 90-12 Ssa, rather than with the 1980s isolates N. risticii Illinois Ssa1 and 25-D Ssa, suggesting a chronological trend  , two ssad Figure .p51, nsp2, nsp3, and ssa3 are uniquely evolved in Neorickettsia spp. The gene p51 is a single copy gene and demonstrates only loose associations with other proteins of the family Anaplasmataceae [nsps and ssas are both potential operons, consisting of three genes tandemly arranged [nsps belong to pfam01617, and similar to Ehrlichia chaffeensis omp-1 (p28) genes  [nsp2 and nsp3 were strain variable. As seen in the ssas, other members of the family Anaplasmataceae have genes encoding proteins containing strain-variable tandem repeats (involving amino acid variation and changes in the numbers of tandem repeats), including Trp120 (formerly gp120), Trp47 (formerly gp47), and VLPT (variable-length PCR target) from E. chaffeensis and Trp140 (formerly gp140), Trp36 (formerly gp36), and gp19 from Ehrlichia canis [ssas are not homologous to any proteins of the family Anaplasmataceae by blastp. Among p51, the nsps, and the ssas, there have been no signs of intragenomic recombination events, which are seen in the Anaplasma p44/msp2 expression locus [The genes mataceae ,38. The am01617) , the proia canis -52. Of non locus ,54.N. risticii established that P51 is a dominant surface-expressed protein. The recognition of recombinant P51 by PHF horse sera, even by 1:80 IFA titer sera suggests P51 is expressed and highly recognized within the present day naturally-infected horses. Despite P51 amino acid sequence variation among N. risticii strains, this strong universal recognition by horse immune sera suggests rP51 may serve as a defined serodiagnostic antigen. Furthermore, the study suggests that there are immunodominant conserved peptide sequences within P51 which might serve as even more specific PHF diagnostic antigens.Proteomics results performed on two strains of N. risticii strains, with respect to the predicted protein secondary structure, the majority of which are clinical isolates, indicates there are hot spots within the genes with greater strain divergence. These include external loops 2 and 4 in P51, external loop 4 in Nsp2, external loop 2 in Nsp3, and the repeated region of Ssa3. P51 showed strong geographical association; and Nsp2, Nsp3, and Ssa3 showed temporal association. Importantly, N. risticii Illinois (upon which vaccines for PHF are produced) is distinct from most East/Midwest US strains (P51) and most post-2000 strains , which may be a contributing factor in PHF vaccine failure [Sequence comparison of these surface-exposed proteins of  failure ,55.N. risticii strains, such as TN02-1 , KY03-3 (Nsp2), IL01-1 (Nsp3), and OH10-1 (Ssa3), suggest that variation contrary to the popular geographical and temporal influences may be more widespread. When additional contemporary sequences and sequences from more varied geographic regions become available, these analyses are expected to improve.There are outlier strains which do not fit the geographical and temporal patterns. These include 081 ,22, the Neorickettsia include the defective DNA repair systems in both N. risticii and N. sennetsu [Neorickettsia [N. risticii. P51 variation showed substantial geographical association, suggesting these variations were selected under local environmental pressures. It is possible that geographical association of N. risticii sequence variation is due to N. risticii strains being selected within essential reservoir trematode populations. In addition, diverse N. risticii strains may have emerged due to selective pressures inflicted on the infected trematodes and/or on the trematodes' hosts [N. risticii populations. Since Neorickettsia spp. are known (N. risticii and N. helminthoeca) and suspected to be vertically transmitted within their trematode hosts [Neorickettsia in the natural environment.Possible explanations for extensive DNA sequence variation within sennetsu ,38. Thisckettsia , which ws' hosts -9,57-59.de hosts ,60, mammN. risticii survival as a species. N. risticii surface protein genetic diversity revealed in the present study will help in understanding variations in PHF virulence and clinical signs. It may also be possible to use this new molecular knowledge for vaccine development. It would, however necessitate taking into account that the pathogen is an obligate intracellular pathogen, indicating that not only humoral immune responses, but also cell-mediated immunity would play an active role in preventing bacterial infection [Regardless the cause, this genetic variation would result in increased nfection -63.ssa1 from N. risticii Illinois [ssa2 and the non-coding region between ssa1 and ssa2 using blastn. Although both are Maryland isolates, the 25-D strain was isolated six years earlier than the 90-12 strain [N. risticii Illinois using the isogenic Illinois strain sequence data and in N. sennetsu using Miyayama isogenic strain data [Genes encoding the two original Ssas, called P85 (90-12) and P50 (25-D) are most related to Illinois ,31,38,55ain data . It is lN. risticii, and they suggest conservation among geographical regions and time periods. In addition, P51 is implicated as the major surface antigen of N. risticii. These data will be valuable in developing better diagnostic methods and may help in the development of more efficacious vaccines.In conclusion, our data demonstrate the variety present within major surface proteins of The authors declare that they have no competing interests.KEG drafted the manuscript, designed primers, performed PCR and overall sequence analyses, and created secondary structures and dendrograms. GP designed primers, performed PCR, and performed preliminary sequence analyses. SM performed all SDS-PAGE and Western blotting experiments and gathered clinical data. YR edited the manuscript and supervised all research. All authors read and approved the final manuscript.Supplemental Table 1. Primers utilized for PCR amplification. Word document demonstrating primers utilized for PCR amplification of p51, nsp2, nsp3, ssa1, and ssa3.Click here for file


CCNL1 and ADCY5) were identified in a genome-wide association study, the latter of which is also a type 2 diabetes locus. We therefore tested the impact of these genetic variants on birth weight and adult glucose/insulin homeostasis in a large Indian birth cohort.A feature of the Asian Indian phenotype is low birth weight with increased adult type 2 diabetes risk. Most populations show consistent associations between low birth weight and adult type 2 diabetes. Recently, two birth weight-lowering loci on chromosome 3 (near CCNL1) and rs9883204 (ADCY5). Associations were tested for birth weight, anthropometry from infancy to adulthood, and type 2 diabetes related glycemic traits. The average birth weight in this population was 2.79±0.47 kg and was not associated with genetic variation in CCNL1 (p = 0.87) or ADCY5 (p = 0.54). Allele frequencies for the ‘birth weight-lowering’ variants were similar compared with Western populations. There were no significant associations with growth or adult weight. However, the ‘birth weight-lowering’ variant of ADCY5 was associated with modest increase in fasting glucose , 2-hours glucose , and reduced insulinogenic index  and 2-hour insulin .Adults  enrolled in a birth cohort (established 1969-73) were genotyped for rs900400 (near CCNL1 and ADCY5 which implies that non-genetic factors may predominate. However, the ‘birth-weight-lowering’ variant of ADCY5 was associated with elevated glucose and decreased insulin response in early adulthood which argues for a common genetic cause of low birth weight and risk of type 2 diabetes.The low birth weight in Asian Indians is not even partly explained by genetic variants near  CCNL1 and rs9883204 of ADCY5) for low birth weight p = 3×10−26 and 3×10−9, respectively) with birth weight were observed leading to a 113 g difference between homozygous carriers vs. non-carriers for the two respective variants A recent genome-wide association (GWA) study identified two independent loci on chromosome 3  and its effects on birth weight, lending support to the notion that more moderate impairment of insulin secretion provides a link between inheritance of type 2 diabetes susceptibility and reduced weight at birth ADCY5 have been linked to glycemic traits in healthy individuals Genetic variants that influence fetal insulin secretion or insulin sensitivity appear to be important determinants of fetal growth, and subsequent development of type 2 diabetes CCNL1 and rs9883204 of ADCY5) in a longitudinal birth cohort form South India and also test whether the low Indian birth weight is driven, or partly explained, by similar genetic factors as in Europeans. In this replication there are two inherent questions. First, whether the low birth in Indians is due to higher frequencies of ‘birth weight-lowering’ alleles compared to the Western population and secondly, whether the lower average birth weight is modulated by variants in ADCY5 and near CCNL1 in a way similar to the western population. Due to the known associations between birth weight and adult type 2 diabetes and the fact that one of the ‘birth weight-lowering’ alleles (ADCY5) also appears to be a genetic determinant of glycemic traits, we also aimed to investigate possible associations of CCNL1 and ADCY5 genotypes with diabetes related intermediate-traits in addition to investigating the influence of these SNPs on anthropometric traits during longitudinal follow-up from birth to adulthood.Therefore, we aimed to replicate two ‘birth weight-lowering’ alleles  years, drawn from an original birth cohort of 10,691 singleton births during 1969–1973 in one of 24 wards of Vellore town and adjoining rural villages, Tamil Nadu, India. Details of the original cohort are described elsewhere http://www.dtu.ox.ac.uk/homacalculator/index.php. All biochemical measurements were done centrally at the Department of Clinical Biochemistry, Christian Medical College & Hospital, Vellore, on samples that were collected during adulthood (in 2002).Anthropometric measurements included height, weight, waist and hip circumference measured by standard methods during adulthood. Body fat percentage was calculated from four skinfold measurements CCNL1) and rs9883204 (ADCY5), respectively. The TaqMan® genotyping master mix was used following the manufacturer's conditions. Genotyping quality control was tested in 8.6% of the samples (genotyped in duplicate) with 0% difference in genotype. Genotyping failed in 65 (3.0%) for rs900400 and 61 (2.8%) for rs9883204, most probably due to low quality DNA for the platform used.DNA was extracted from peripheral blood using Qiagen kits. The samples were genotyped using 10–20 ng genomic DNA in 384-well format on an ABI 7900 machine at final volume of 4 µl. The genotyping was performed using TaqMan® SNP Genotyping Assays C1860681_10 and C3035719_20 for rs900400  as independent variables, with age, gender, consanguinity and BMI as covariates. Z-scores for selected anthropometric variables were age-adjusted and converted into within-cohort age- and sex-specific Z-scores [(subject mean- cohort mean)/cohort SD] considering variations in ages measured at different time points. The cohort mean and SD were derived from all 4,092 individuals with complete birth and parental measurements. Infant data were included if there was at least one measurement between 1 and 3 months, and the latest available time point used. Childhood data were included if there was at least one measurement between 6 and 8 years, and the average Z-score was used if there were more than one measurement. Adolescent data were included if there was at least one measurement between 10 and 14 years, the Z-score for the age closest to 12 years being selected. The main analysis used all available data at each time point. The absolute effect of genetic variants on Z-scores of anthropometry was assessed using ANOVA after adjustment for gestational age, gender and consanguinity. Effect size of the additive model was computed using regression models adjusted for gestational age, gender and consanguinity. All data were analyzed using STATA (Version 11.0). Subjects with T2DM (n = 55) were excluded from all analysis. All diabetes related traits except 2 hours postprandial glucose and AUC glucose were log transformed to obtain normal distribution before analysis.CCNL1 and 0.1% for ADCY5) as previously described, showed a power of 74% and 32% to detect a variance (r2) explained, for CCNL1 and ADCY5 respectively using adjusted models, calculated at an alpha (0.05), and a sample size of 2,151. The coefficient of variation (CV) of birth weight was 18.8% for CCNL1 and 16.7% for ADCY5 and this was comparable to the effects demonstrated by these variants on birth weight as described by Freathy RM et al A power calculation based on variance explained by both the genetic variants (0.3% for CCNL1) or rs9883204 (ADCY5) with birth weight in the Indian population (p = 0.035) in the cohort. This is a well-known phenomenon in cohorts with a high degree of consanguineous parentage, which was the case in 47% of the rural part of the cohort and 29% in the urban group.There were no significant associations between either rs900400  was observed.Comparing birth weights in extreme homozygotes (CC) for both of the ‘birth weight-lowering’ variants, no statistically significant difference between carriers  and non-carriers .We then compared the birth weights for participants with normoglycemia, IFG, IGT and manifest type 2 diabetes (T2DM) in the entire cohort. The mean birth weight among people with normoglycemia was 2,795±460 g , with IFG (n = 83) 2,754±505 g, with IGT (n = 319) was 2,763±496 g and 2,715±536 g in the diabetes group (n = 55). The difference appeared largest between the normoglycemic group and the groups with manifest type 2 diabetes, but this was not statistically significant   .ADCY5, was significantly and positively associated with increase in 2-hour glucose, fasting glucose, reduced insulinogenic index and reduced 2-hour insulin . The ‘birth weight-lowering’ variants were not associated with calculated index of insulin resistance (HOMA-IR). No impairment of β-cell function was observed for variants at the CCNL1 locus  and 95% confidence was 1.64 (1.08–2.48) p = 0.016, whereas there was no significant effect at the 11 mmol/L cut off .To further analyze the impact on postprandial glycemia in the homozygous carriers of the CCNL1 or in ADCY5 locus are not associated with birth weight in this Indian cohort, but the ‘birth weight-lowering’ variant of ADCY5 was associated with raised glucose and reduced early phase insulin secretion.Genetic variants associated with a birth weight-lowering effect in other populations, near ADCY5 and near CCNL were also the same (ADCY5) or lower (CCNL) than in the original report in Europeans The Indian birth weight is generally about 0.8 kg less compared to the West ADCY5 variant has been associated with increased plasma fasting glucose at a genome-wide level ADCY5 was associated with raised fasting and 2-hour glucose concentrations following an oral glucose tolerance test performed in early adulthood. Although this finding would be in general agreement with the fetal-insulin hypothesis, it is paradoxical compared to a recent study in a large Danish cohort as another ‘birth weight-lowering’ (rs11708067) variant was associated with reduced adult insulin resistance ADCY5 rs11708067 and rs9883204 are in close linkage disequilibrium in the Hapmap CEPH (Utah residents with ancestry from northern and western Europe) population data . rs9883204 is not represented in the Hapmap Indian population (GIH - Gujarathi Indian population), therefore LD cannot be calculated directly within Hapmap. However, a further SNP, rs17361324, which is a proxy for rs9883204 in the CEPH Hapmap data is also a proxy for rs11708067 in the GIH Hapmap data. This may suggest that, the high LD between these two SNPs in the CEPH data is also present in the Indian population and the difference does not appear to be explained by the genetic architecture and will need confirmation in future studies. Additionally, the low birth weight and T2DM risk allele of ADCY5 variant was associated with reduced early insulin response measured by insulinogenic index, consistent with original report by Freathy et al., supportive of a possible role in influencing insulin secretion ADCY5 risk allele may operate by different mechanisms by which they influence birth weight and T2DM risk susceptibility The The loss of heterozygosity of rs9883204 in our population is probably related to the high endogamy which is commonly seen among Indians. It is generally assumed that an association detected by a well-powered GWAS is in LD with the functional variant. The strong signals in GWAS are related to the functional magnitude of the effect and therefore, we assume this holds good in our population despite a considerable degree of consanguinity.CCNL1, but less strong for ADCY5 to explain the variance observed with these two SNPs. The CV for birth weight in our study was comparable to originally described and this further solidifies our power to detect an association. Also the lower mean birth weight, observed in this population, might potentially be associated with a reduced overall variance in the dependent variable, i.e., reduce the effect of the functional variant on birth weight, which could possibly contribute to the lowered power in our study. Although it is evident that a multiple comparison correction would abolish the modest associations with glycemic traits, we believe that the association is biologically relevant and that loss of statistical significance by multiple testing does not necessarily disprove a true association owing to the homogenous population studied within a small geographical region and the similar allele frequencies reported among Caucasians Although, we have studied, the genetic variants associated with birth weight in one of the largest and homogenous birth cohorts from India, our study has limitations. Our study was adequately powered for ADCY5 and near CCNL1 showing strong associations with birth weight in European cohorts appear to have little or no effect in the Indian setting. However, the ‘birth weight-lowering’ variant in ADCY5 was associated with modest glucose intolerance in early adulthood which reinforces the argument for a genetic link between in utero growth and adult type 2 diabetes.In conclusion, the ‘birth weight-lowering’ variants in Table S1Association between rs900400 and rs9883204 and Z-scores of anthropometric traits from birth to adulthood.(DOC)Click here for additional data file.


Testicular epidermoid cyst is extremely rare injury, which in recent studies has been regarded as a tumor. Some authors consider this lesion as a cystic monodermal teratoma, the histogenesis remains controversial. In this study we provide clinical and morphological characteristics of an epidermoid cyst in the left testicle in a 16 years old patient, where apparently a nodular lesion in the left testicle was detected.Cyst; Epidermoid; Intratesticular Intratesticular epidermoid cysts are infrequent tumors that usually appear in young, still very rare in pediatric age, which account for 1% of all testicular tumors. The clinical diagnosis of this tumor poses a major challenge because the clinical course is not different as how it behaves as a malignant tumor [We present an unusual case in a 16 years old male with intratesticular epidermoid cyst.A 16 years old male had a lesion of nodular appearance in the left testicle. The ultrasound shows a hypoechoic lesion, with the analytical and negative markers. Suspecting a tumor, left orchiectomy was performed. Pathology refers to a piece of left orchiectomy of 5.5 cm in length, presenting a cystic soft white content of 1.5 × 1.2 cm in diameter without affecting the epididymis or tunica albuginea.The histopathological study demonstrated a cystic structure lined by a multilayered squamous epithelium, without evidence of atypical changes , 2. SeriImmunohistochemistry was performed on intratesticular epidermoid cyst, showing positive for high molecular weight CK (CK 5-6 and 34bE12) in all of them  with lowEpidermoid cysts are benign lesions that often are located in the skin and are very rare in intratesticular level. At the histological level, they are composed of the same characteristics as those of the skin location. They are solitary lesions in the onset age of 20 - 40 years in white males . AlthougWe considered several histogenetic possibilities for intratesticular epidermoid cyst, such as a source at the expense of metaplasia of the seminiferous tubules or the rete testis; other molecular biology studies consider that in some cases corresponding to a tumor as a cystic monodermal teratoma of less origin has been frequently suggested in the scrotum infundibular cysts , 4.Clinically, lesions appear as firm, well circumscribed, small, solitary, painless, which are sometimes indistinguishable from malignant tumors.There are tools to make a correct diagnosis: (a) absence of the analytical elevation of tumor markers such as alpha fetoprotein and beta-HCG; (b) the ultrasound finding that is very characteristic of this disease, which comprises hyperechoic heterogeneous sonographic pattern which gives the appearance of 'onion skin', accompanied by a peripheral hypoechoic zone; (c) magnetic resonance imaging typically shows the image in 'bull's eye', which consists of a low intensity center because it is composed of dense calcifications and remains, a high-intensity mid-zone which consists of scaly squamous cells, and other peripheral zone of low intensity in T1 and T2 due to compact keratin fibers; (d) with no Doppler vascularization , 6.The histologic diagnosis of epidermoid cyst was described by Price in 1969, a study of 69 cases, 5 therefore established criteria: (a) the injury must be located within the testicular parenchyma; (b) the center cyst composition should be amorphous material keratinized debris; (c) within the cyst, teratoid elements or components of dermal appendages should not exist; (d) there should be no scars in the parenchyma adjacent to the epidermoid cyst .The treatment of choice for intratesticular epidermoid cyst is excision of the cyst with the surrounding testicular parenchyma to check no teratomatous component or malignant germ cell neoplasia accompanying. Postoperative monitoring is recommended .


Studying prenatal influences of early life growth is relevant to life-course epidemiology as some of its features have been linked to the onset of later diseases.size, velocity and tempo. We used data from three contemporary cohorts based in Portugal , Italy , and Chile .We studied the association between prenatal maternal characteristics , smoking, gestational diabetes and hypertension) and offspring weight trajectories in infancy using SuperImposition by Translation And Rotation (SITAR) models, which parameterize growth in terms of three biologically interpretable parameters: size :0.3; 0.5)), others were either found to affect size and velocity , greater velocity ), or to additionally influence tempo , delayed tempo , decreased velocity ).Estimates were generally consistent across the cohorts for maternal height, age, parity and pre-pregnancy overweight/obesity. Some exposures only affected one growth parameter : 0.4% increase in size, velocity and tempo, we found that prenatal maternal characteristics, especially maternal smoking, pre-pregnancy overweight and underweight, parity and gestational hypertension, are associated with different aspects of infant weight growth. These results may offer insights into the mechanisms governing infant growth.By disentangling the growth parameters of  Birth size and early life growth trajectories have been found to be important predictors for the onset and development of a wide range of later diseases A wide-ranging literature exists on the association of prenatal exposures - such as parental age, maternal, environmental and social factors, health status, life-style and pregnancy conditions, - with birth outcomes, mainly birth size and gestational age size, velocity and tempo. SITAR has been used before to model individual growth data In this paper we examine the association between several prenatal maternal exposures with weight trajectories of infants (0–2 years) from three recent cohorts based in countries with diverse socio-economic backgrounds  using the shape-invariant random effects model called SuperImposition by Translation And Rotation (SITAR) All participants to the Generation XXI (GXXI), Nascita e INFanzia: gli Effetti dell'Ambiente (NINFEA) and Growth and Obesity Cohort Study (GOCS) cohorts have read and signed a written informed consent form. Data for this paper were analyzed anonymously. GXXI was approved by the Portuguese Data Protection Authority . NINFEA study was approved by Ethical Committee of the San Giovanni Battista Hospital and CTO/CRF/Maria Adelaide Hospital of Turin . GOCS study was approved by the Institutional Review Board of the Institute of Nutrition and Food Technology (INTA) of the University of Chile. Anonymized data are available upon request to qualified researchers for the purpose of academic, non-commercial, collaborative research.GXXI was established in 2005 in the Porto region of Portugal. All children born of women resident in the region and admitted to one of its five public hospitals for delivery, with a gestational age at birth greater than 24 weeks, were eligible to participate. Recruitment lasted from April 2005 to August 2006. Women were enrolled a few days before their due date and, the majority, completed baseline questionnaires between 24 and 72 hours after delivery. In total the baseline data consist of 8,311 singleton children. Children were actively followed-up through interviewer-administered questionnaires planned at 3, 6, 12–15 and 24 months of age. Due to logistic and financial constraints a restricted time window was allocated for each follow-up occasion and therefore it was not possible to interview every participant at each follow-up visit. The present analyses are based on the information collected at baseline and at the 2-years follow-up, which is available for 786 infants . Maternal and birth characteristics of infants invited to participate in the 2-years follow-up were compared with the rest of the cohort. No systematic differences were found, suggesting that participants who were followed-up at 2 years of age are a representative sample of the whole cohort ; thus rewww.progettoninfea.it) where women complete the first questionnaire (Q1) at any time during their pregnancy. Active follow up is via online questionnaires administered at around 6 (Q2), 18 (Q3), 48 (Q4) months and 7 years (Q5) of age of the child. At Q2 women were asked to report the child's anthropometric measurements at birth, 3 and 6 months, while at Q3 they were asked to report the measures at 12 and 18 months. Revisions of these questionnaires, undertaken after approximately the first 1,500 mothers enrolled, led to inclusion of additional questions on the child's measures at the time of their completion. The analyses involve weight data from birth to around age 2, resulting in a median of 4 (range 1–7) measurements per child. These were obtained from the NINFEA database version 12.03 (downloaded in March 2012) and concern 2,925 singleton children with available data on gestational age at birth, whose mothers were born in Italy, and who, at the time of the data download, were eligible for at least the 6-months questionnaire (Q2). This implies that growth data up to about 6 months of age only are available for those infants who, at the time of the data download, were not eligible for the 18-months questionnaire (Q3). When comparing the distribution of baseline characteristics of infants with/without data from both Q2 and Q3 no systematic differences were observed (data not shown).NINFEA is an on-going Italian web-based cohort study which started in 2005 and aims to recruit pregnant women via the Internet and follow up their children , smoking status during pregnancy and pregnancy complications, namely gestational diabetes and pregnancy hypertension/eclampsia, were instead considered intermediate exposures as their values are likely to be affected by the background variables above. Data on prenatal variables were derived from questionnaires administered during pregnancy in NINFEA, at birth in GXXI, and when the children were approximately 3–4 years old in GOCS. Coding and further details are given in ity be the weight of child i at age t, then SITAR is specified as:h(z) is a natural cubic spline of transformed age z, iα, βi and iγ are subject-specific growth parameters, and εit is the residual error term assumed to have mean zero and constant variance. The three parameters correspond respectively to the size, tempo and velocity of growth specific to each child: iα (size) represents the shift in the weight axis, while βi (tempo) and iγ (velocity) represent the change in location and scale to be applied to the age axis, respectively, in order for all children to share the same shape (mean spline curve h(z)). Size (iα) is expressed in units of weight, tempo (βi) in units of age, while velocity (iγ) is a multiplier, and therefore is scale-free and reported as a percentage. Pizzi et al i = α0+α1iα, where 0α is a fixed parameter, representing the size of a reference child, and 1iα a random, normally distributed variable with mean zero and constant variance, and let similar specifications for βi and iγ, then estimation can be carried out by maximum likelihood as for any (non-linear) mixed effects model 1iα (size) will be positive for heavier children, while β1i (tempo) is related to the timing of maximum growth velocity and therefore will be negative for children whose growth is more advanced at earlier ages (earlier velocity peak), and 1iγ (velocity) will be positive for children with faster growth The observed weight trajectories were modelled using a recently developed shape invariant random effects model. It was introduced by Cole i on subject i can be included in the model by specifying the three growth parameters as follow:αδ, βδ and γδ represent the contribution of the covariate to a child's size, tempo and velocity, respectively. Generalization of A covariate X with observed value xα is to be interpreted as percentage changes in size relative to the reference child β is also expressed in months. The spline function h(z) was defined by placing the internal knots at quantiles of the age distribution, appropriate for each cohort because of varying richness and spread of the available weight measurements . The complexity of the SITAR model relatively to the available data led to imposing constraints on its parameters, namely that the tempo of the standard child, 0β, was zero. Furthermore to be able to compare the three cohorts, βδ, the contribution of each covariate on a child's tempo, was also constrained to be zero. These constraints were relaxed in analyses specific to GXXI as it had more weight growth measurements.Weight was log-transformed to aid meeting the distributional assumptions of the model. As a consequence δcore datasets to allow comparisons between unadjusted and adjusted estimates for each of these variables. Fully-adjusted estimates were obtained by fitting two separate models to the core datasets: (i) the background explanatory variables were mutually adjusted, as well as adjusted for gestational age and gender; (ii) the intermediate explanatory variables were mutually adjusted, as well as adjusted for the background variables, gestational age and gender.Models were initially fitted separately by study. We first included one explanatory variable at a time, with adjustment by gender and gestational age . We used all available data and also just the Models were also refitted on the pooled data from the three cohorts, including a fixed effect for the study indicators and assessing evidence of heterogeneity via significance tests of the interaction between each covariate and the study indicators .There is considerable variation in the distribution of the prenatal exposures across the three birth cohorts ; in partαδ and γδ), by cohort, obtained from models fitted to the core datasets. The minimally-adjusted estimates obtained when fitting the models to each whole cohort are reported in core datasets are likely to be representative of the corresponding whole cohorts. The minimally-adjusted and fully-adjusted estimates in size (iα) being positively associated with maternal height , pre-pregnancy overweight/obesity  and parity , but negatively associated with smoking during pregnancy (fully-adjusted: δα≈−3% in GXXI and NINFEA) and maternal pre-pregnancy underweight (fully-adjusted: δα≈−4% in each cohort). Post-natal growth velocity (iγ) was positively associated with maternal smoking , and possibly maternal underweight , but negatively associated with parity . The results for education were heterogeneous: while in GXXI medium/highly educated women have bigger children who tend to have slower growth velocity, and in GOCS the children from less educated mothers have slower growth compared to those in the reference group, in NINFEA no association was found.α = −6.4%, δγ = 12.8%; similarly in NINFEA), although this pattern was not present in GOCS.The model that examined pregnancy complications showed that, when fully adjusted for the other characteristics, gestational diabetes was not associated with infant weight growth . In contvelocity (iγ)), while children from overweight/obese women have a bigger size (iα) without evidence of decreased postnatal growth rate. Results for maternal education show that less educated mothers have smaller children that however have the same growth velocity (iγ) as children of more educated mothers , with the differences arising from GOCS, unsurprisingly given the results of  mothers .In order to examine whether the observed heterogeneity across the cohorts was due to differences in entry criteria, we replicated cohort-specific analyses on the subset of GXXI and NINFEA children who were born at term and with a birth weight of 2500–4500 grams, using the same entry criteria as GOCS. The results pointed to much more similar effects across the cohorts.tempo, restricting them to the GXXI cohort because of its rich number of repeated weight observations . The results are reported in tempo (βi), and therefore no change in the estimated effects on size (iα) or velocity (iγ). However parity, pre-pregnancy overweight/obesity, and hypertension do influence tempo (βi) of growth. Infants of parous mothers have relatively earlier growth spurts by about 5 days . Allowing for this association ‘explains away’ some of the earlier associations found between parity and size (iα) and velocity (iγ)  by about 8 days  if their mother is overweight/obese. As for parity, given the correlations among the three growth parameters, including maternal overweight/obesity in the specification of tempo (βi) changes its association with size (iα) and velocity (iγ). In particular that for velocity becomes negative  implying that infants of overweight/obese mothers not only have a later peak, but also have slower velocity than that of a reference child. For hypertension the association with size (iα) and velocity (iγ) is reduced when an association with tempo (βi) is allowed. The latter is found to be positive  indicating a delay in peak velocity of almost 10 days  that are influenced by these prenatal factors.To illustrate the effect sizes reported in In this paper we investigated prenatal influences on weight growth in infancy in order to contribute to the understanding of its role in the development of a wide range of later diseases. We used data on children belonging to three contemporary cohorts based in Portugal, Italy and Chile in order to compare effects across socio-economically and geographically diverse populations and gain a more robust understanding of these associations, while accounting for potentially different confounding patterns. The individual weight trajectories were modelled using SITAR size was positively associated with maternal height, parity and pre-pregnancy overweight/obesity, and negatively with pre-pregnancy underweight. Additionally in all cohorts parity negatively affected velocity. In contrast, only for infants from the two European studies, maternal smoking and gestational hypertension were associated with reduced size and increased velocity, while pre-pregnancy underweight was positively associated with velocity. Maternal education was only a moderate predictor of size in the European cohorts and of velocity in the Chilean cohort. When tempo was modeled in terms of covariates in analyses restricted to GXXI, we found that part of the impact on size and velocity observed for parity, maternal overweight/obesity and hypertension was captured by their influence on the tempo dimension. In particular, infants of parous mothers were found to have an earlier timing of growth, while those of overweight/obese or gestational hypertensive mothers to have it delayed. We found instead no evidence of an effect of maternal height, age, or smoking on tempo.Our analyses indicate that prenatal exposures affect different dimensions of the weight trajectories. In all cohorts, size and weight velocity, the positive association between maternal overweight/obesity with increased size Click here for additional data file.Table S2“Minimally-adjusted” estimated coefficients and 95% confidence interval for the association between covariates and size and velocity parameters by cohorts. Gender- and gestational age-adjusted estimates of the association between covariates and size and velocity parameters obtained fitting the models to each whole cohort.(PDF)Click here for additional data file.


LEKR and CCNL1 and in the ADCY5 genes lead to lower birthweight. Here, we study the impact of these variants and social stress during pregnancy, defined as social adversity and neighborhood disparity, on infant birth size. We aimed to determine whether the addition of genetic variance magnified the observed associations.We previously identified via a genome wide association study variants near LEKR and CCNL1) magnified this association in an additive manner. However, likely due to sample size restriction, this association was not significant for the SNP rs9883204 in ADCY5. Birth size difference due to social stress was greater in the presence of birthweight-lowering alleles.We analyzed data from the Northern Finland Birth Cohort 1986 (n = 5369). Social adversity was defined by young maternal age (<20 years), low maternal education (<11 years), and/or single marital status. Neighborhood social disparity was assessed by discrepancy between neighborhoods relative to personal socio-economic status. These variables are indicative of social and socioeconomic stress, but also of biological risk. The adjusted multiple regression analysis showed smaller birth size in both infants of mothers who experienced social adversity  and neighborhood disparity . The birthweight-lowering risk allele (SNP rs900400 near LEKR/CCNL1, the impact of stress on birth size was stronger. These observations give support to the hypothesis that individuals with genetic or other biological risk are more vulnerable to environmental influences. Our study indicates the need for further research to understand the mechanisms by which genes impact individual vulnerability to environmental insults.Social adversity, neighborhood disparity, and genetic variants have independent associations with infant birth size in the mutually adjusted analyses. If the newborn carried a risk allele rs900400 near  Being born small is associated with increased risk of perinatal morbidity and hospitalization LEKR and CCNL1 and in the ADCY5 are associated with birthweight We recently identified in a large-scale genome wide association study (GWAS) that variants near i.e. living in areas where neighborhood financial capacity differs from individual SES, is associated with higher all-cause mortality Neighborhood environment is also known to be associated with lower birthweight and influence morbidity and mortality LEKR and CCNL1 or in ADCY5.Our primary objective was to examine whether social stress and variance in the previously identified birthweight-lowering alleles would contribute in an additive manner to birth size . We used data from the Northern Finland Birth Cohort 1986 (NFBC 1986) and operationalized social adversity as the presence of at least one known environmental factor associated with stress at the individual level The current study is based on data from the NFBC 1986 cohort, which comprises 9362 pregnant women (99% of pregnant population) and 9203 live-born singletons with expected date of birth between July 1985 and June 1986 from the provinces of Lapland and Oulu in Finland Data concerning maternal health and social-demographics were collected via medical records, examinations/interviews by midwives, and data from a self-report questionnaire administered to pregnant women at the first visit to maternity health centers, approximately gestational week 12 and returned by gestational week 24 if still pregnant We had information on maternal social adversity for 9106 mothers and genotype for 5369 children Social stress at the individual level consisted of the sum of three stressors, thus scores ranged between 0 and 3. Young maternal age was defined as being <20 years at time of birth and coded as 1, otherwise as 0 Social stress at the neighborhood level, i.e. neighborhood social disparity, was defined as a discrepancy between the family SES and the neighborhood financial estimate. A disparity score was created by comparing family SES  with neighborhood financial estimate. Maternal and paternal occupation were categorized as 1 = professional, 2 = upper white collar, 3 = lower white collar, 4 = unskilled worker, 5 = farmer/farmer’s wife owning >8 hectares of land, 6 = farmer/farmer’s wife owning <8 hectares of land. Neighborhood financial estimate was based on financial capacity category (FCC) of the neighborhood for the 1982–92 classification by the National Finnish KOUTA database and rated from one (deprived) to six (affluent) LEKR and CCNL1 and in the ADCY5 were genotyped (n = 5369) by Taqman allelic discrimination. No deviation (p≥0.05) from Hardy-Weinberg equilibrium was observed Blood samples were taken when adolescents were 16 years old. The DNA extractions, sample quality controls, biobank up-keeping and aliquotting were performed in the National Public Health Institute, Biomedicum Helsinki, Finland. The rs900400 and rs9883204 single nucleotide polymorphisms (SNPs) near 3)].Data on infant birth size, i.e. birthweight (in kg), head circumference (in cm) and birth length (in cm), were collected at birth by trained medical staff according to standardized procedure, entered into the medical records, and transferred onto the study forms. Ponderal index was calculated using the standard formula . Single-parenthood was the component of the social adversity composite that showed the strongest association with birth size in comparison to infants from two-parent families in a mutually adjusted analysis (data not shown). This association between single parenthood and birth size was significant in males but not in females , birth length −0.40 cm , head circumference −0.34 cm ; females: birthweight −16.87 g  birth length 0.10 cm , head circumference 0.03 cm . In the NFBC 1986 cohort 24.5% of mothers experienced at least one type of adversity during pregnancy, 3.6% two and 0.3% three adversities. Of the women experiencing at least one adversity, 36% had low education (<11years), 5% were single parents, and 4% were young (<20 years) at the time of delivery. 3. The P-value for trend for effect sizes by exposures categories was significant for all birth size outcomes. When stratified by sex  and birth length of −0.12 cm  in the adjusted model . In the CCNL1/LEKR magnified this association. These results provide support for the hypothesis that an individual with a genetic or other biological risk is more vulnerable to environmental adversity. Though the magnitude of the reduction in birth size attributable to variants in genotype is of significant proportion (44 g) we can only stipulate a trend from these results. Though both social stress and the birth weight lowering allele rs9883204 at ADCY5Many human diseases stem from complex interplay between environmental and individual susceptibility. In our study we examined how and whether specific genetic susceptibility modulates the association with adverse outcomes from environmental exposure such as social stress. Our results show that maternal social stress during pregnancy, both at the individual and neighborhood levels, was associated with smaller infant birth size and that carrying the birthweight-lowering rs900400 C allele located near CCNL1/LEKR are linked to lower birth weight, the biology behind this association is still unclear CCNL1/LEKR has not yet been linked to either with type 2 diabetes or adult glycemic traits. On the contrary, a recent study has shown an association between the C-allele of the rs900400 located near CCNL1/LEKR1 and increased insulin response to oral glucose stimulation in non-diabetic individual Though variants near The impact of maternal social stress had on infant birthweight is comparable to mothers smoking two cigarettes per day during pregnancy e.g. head circumference is a measure of occipital-frontal circumference and is subject to some degree of unreliability. However, measurement error is unlikely to be systematically associated with social stress, thus should be considered as random variance and evenly distributed by exposure status.Small head circumference is linked with poor developmental and cognitive outcomes in the offspring Previous studies have shown male fetuses are more vulnerable to intrauterine insults i.e. with increased levels of fetal cord cortisol during late pregnancy We hypothesize that maternal perceived stress as one of the biological pathways through which social stress affects birth size. Perceived stress has been linked to changes in maternal stress hormone levels (i.e. cortisol) during pregnancy Our study also highlights the importance of using three indicators of social adversity in the clinical setting in identifying pregnant women at risk for poor birth outcomes. The factors used to define social adversity are indicative of potential economic hardship and biological risk e.g. health care and other amenities. Interestingly, though these types of neighborhood deprivations are small as compared to other industrialized nations, they still accounted for clinically significant differences in birth size.The association of neighborhood social disparity with smaller birth size is likely to be explained by stress related to access to amenities. In Finland, despite uniformly distributed tax-paid health care, many individuals in rural areas, may require long distance travel to access e.g. in hypothalamic pituitary adrenal-axis functioning. In this regard, it would have been a strength to have maternal blood cortisol samples available. However, cortisol in relation to perceptions of stress is fraught with measurement difficulties Strengths of this study include the prospective data collection with extensive maternal and infant demographic and medical information. The study population is known to be genetically homogenous consisting of white Caucasians, therefore, reducing bias introduced by ethnicity. Moreover, we were able to adjust for major confounders, which has not been possible at this scale in previous studies. The study had, however, limited statistical power to report the additive effects of genetic variants on birth size with higher precision, or to test for any interactions between the genetic variants and social stress. These aspects should ideally be addressed in larger meta-analyses combining several studies, but the availability of such data has become a key issue. Another limitation is that we hypothesized that the objective measures of social stress employed in this study correspond to the biological stress response This study shows that genetic susceptibility magnified the association between social stress,  and birth size. The fact that social stress was associated with smaller birth size even in a society where there is relatively little social inequality as compared with other high-income countries and where tax–paid health care is universally available, is alarming and highlights the strength of the association. Moreover, social stress was a stronger predictor of birth size than having birthweight-lowering alleles, which emphasizes the use of indicators of social stress in clinical settings. It is promising that the addition of the genetic variants made a significant additional contribution which calls for further work in identifying groups of genetic variants and their interaction with environment.Table S1Chi-square tests of associations between maternal characteristics, birth outcomes and social stress in the whole NFBC86 Cohort.(DOCX)Click here for additional data file.Table S2±SD).Genotype frequency by birth size outcomes including means ((DOCX)Click here for additional data file.Table S3CCNL1/LEKR1- rs900400) stratified by sex.Mean differences  in birth size as predicted by the additive effects of social adversity and at least one risk allele ((DOCX)Click here for additional data file.Table S4ADCY5-rs9883204).Mean difference  in birth size as predicted by the additive affects of maternal social adversity and at least one risk allele ((DOCX)Click here for additional data file.


Their LBM, including ALST, was assessed by DXA along with anthropometric measurements. The sample was divided into prediction (60%) and validation (40%) sets. In the prediction set, a number of prediction models were constructed using DXA-measured LBM and ALST estimates as dependent variables and a combination of anthropometric indices as independent variables. These equations were cross-validated in the validation set. Simple equations using age, height, and weight explained >90% variation in the LBM and ALST in both men and women. Additional variables (hip and limb circumferences and sum of skinfold thicknesses) increased the explained variation by 5–8% in the fully adjusted models predicting LBM and ALST. More complex equations using all of the above anthropometric variables could predict the DXA-measured LBM and ALST accurately, as indicated by low standard error of the estimate , as well as good agreement by Bland-Altman analyses . These equations could be a valuable tool in large epidemiological studies assessing these body compartments in Indians and other population groups with similar body composition.Lean body mass (LBM) and muscle mass remain difficult to quantify in large epidemiological studies due to the unavailability of inexpensive methods. We therefore developed anthropometric prediction equations to estimate the LBM and appendicular lean soft tissue (ALST) using dual-energy X-ray absorptiometry (DXA) as a reference method. Healthy volunteers ( The first group of participants  included members of a birth cohort established to assess the long-term impact of early nutrition supplementation provided to pregnant women and young children (Andhra Pradesh Children and Parents Study) . Of these, 108 participants (including 26 women who were pregnant) did not undergo DXA scanning. In addition, data on 36 participants were excluded due to poor scan quality. Finally, data on 2,220 participants who had outcome measurements done by anthropometry as well as DXA were included in the analyses. Demographic information was collected on all study participants using a standardized, interviewer-administered questionnaire.Healthy volunteers (s Study) . The secThese measurements were carried out by two trained investigators using standardized procedures . Weight 2. Corrected arm muscle area  was calculated using the MAC and triceps skinfold measurements with the following formula: CAMA = [MAC − (π × TSF)]2/4π − BA, where MAC is in centimeters, TSF = triceps SFT (in cm), and BA = correction for bone area, which was considered to be 10 cm for men and 6.5 cm for women (Body mass index (BMI) was calculated as weight (kg)/height (m)or women . Anthropor women , 16. Bodor women .A whole-body DXA scan was carried out for each participant using either the Hologic Discovery A model  or Hologic 4500W (10% of scans) on the same day as anthropometry. The scanners were calibrated daily with a phantom, and their performance was monitored as per quality-assurance protocol. During the scan, participants were asked to lie supine on the scanning table with their arms at their sides. Pregnant women were excluded from the DXA scanning. Standard Hologic software options were used to define regions of the body . ALST was calculated as the sum of bone-free lean tissue in arms and legs.t-test. In the prediction data set, two sets of step-wise multiple linear regression analyses were performed to predict LBM and ALST using anthropometric variables as predictor variables. Anthropometric variables  were entered in different combinations as predictors of LBM and ALST. The models were adjusted additionally for the DXA scanner and participant age. Coefficient of determination (adjusted R2), standard error of the estimate (SEE), and Akaike information criterion (AIC) . SFT measurements were log transformed to reduce the skewness of the distribution. Participants were divided randomly into prediction (60%) and validation (40%) groups. To reduce the influence of outliers in the case of the anthropometric and DXA variables, extreme values below the first percentile and above the 99th percentile were adjusted and made equivalent to the first and 99th percentile, respectively. Participant characteristics in the prediction and validation groups were compared using Student's t-test in the validation group. The pure error (PE) was calculated as the square root of the mean of squares of differences between measured and predicted values of the LBM and ALST. A smaller PE value indicated greater accuracy of the equation. The predicted and measured values were also compared using the Bland and Altman method  explained ∼90% of the variation in LBM in the case of men and women. Addition of circumferences at hip, calf, and arm and/or sum of SFT measurements at four sites (Eqs. 2–4) resulted in improved adjusted R2, reduced SEE, and AIC values, indicating better predictive qualities of the models with added variables. An increase in the value of adjusted R2 (from 0.90 to 0.94 in men and 0.91 to 0.92 in women) and a decrease in SEE (from 1.92 to 1.47 kg in men and from 1.84 to 1.63 kg in women) from Eq. 1 to Eq. 4 was, however, marginal. Based on the AIC, Eq. 4, which had the lowest value, can be considered the best fit model.Eq. 1 based on age, height, and weight in the case of men and women were ∼0.28 kg and ∼0.02 kg, respectively  to 0.86 by Eq. 4, which additionally included circumferences and SFTs. SEE and AIC values were lowest with Eq. 4 compared with simpler models proposed by Eqs. 1–3, indicating better prediction quality of the model compared with Eqs. 1–3. Equation 3, which included a derived index of CAMA in addition to height and weight, showed better prediction ability  compared with Eq. 1, indicating that inclusion of this index improved the precision of the estimate.Eq. 1  to Eq. 4  as a result of additional variables added to the model used in Eq. 4. Limits of agreement were also narrower in the case of Eq. 4 compared with Eq. 1. The Bland-Altman plot, comparing the two ALST estimates (measured by DXA and predicted by sex-specific Eq. 4), also showed that the mean estimates by the two methods were similar  that used 24-h urinary creatinine excretion as a reference technique  increased the variation explained to 94%, indicating that equations based on simple measures of height and weight can provide reasonably precise estimates of LBM and ALST. The use of CAMA along with height and weight increased the adjusted R2 value and reduced the SEE .Funding for the study was provided by the The study sponsor had no role in the study design; in the collection, analysis, and interpretation of data; in writing the report; or in the decision to submit the paper for publication. Authors have no potential conflicts of interest.Author contributions: B.K. and H.K. conception and design of research; B.K. and H.K. performed experiments; B.K. analyzed data; B.K., N.M.B., and A.P.H. interpreted results of experiments; B.K. prepared figures; B.K. drafted manuscript; B.K., H.K., A.T., J.C.W., K.V.R., S.K., Y.B-S., G.D.S., S.E., N.M.B., and A.P.H. edited and revised manuscript; B.K., H.K., A.T., J.C.W., K.V.R., S.K., Y.B-S., G.D.S., S.E., N.M.B., and A.P.H. approved final version of manuscript.