Candida albicans cell surface proteins called adhesins mediate binding of the fungus to host cells. The mammalian transglutaminase (TG) substrate and adhesin, Hyphal wall protein 1 (Hwp1), is expressed on the hyphal form of C. albicans where it mediates fungal adhesion to epithelial cells. Hwp1 is also required for biofilm formation and mating thus the protein functions in both fungal-host and self-interactions. Hwp1 is required for full virulence of C. albicans in murine models of disseminated candidiasis and of esophageal candidiasis. Previous studies correlated TG activity on the surface of oral epithelial cells, produced by epithelial TG (TG1), with tight binding of C. albicans via Hwp1 to the host cell surfaces. However, the contribution of other Tgs, specifically tissue TG (TG2), to disseminated candidiasis mediated by Hwp1 was not known. A newly created hwp1 null strain in the wild type SC5314 background was as virulent as the parental strain in C57BL/6 mice, and virulence was retained in C57BL/6 mice deleted for Tgm2 (TG2). Further, the hwp1 null strains displayed modestly reduced virulence in BALB/c mice as did strain DD27-U1, an independently created hwp1Δ/Δ in CAI4 corrected for its ura3Δ defect at the URA3 locus. Hwp1 was still needed to produce wild type biofilms, and persist on murine tongues in an oral model of oropharyngeal candidiasis consistent with previous studies by us and others. Finally, lack of Hwp1 affected the translocation of C. albicans from the mouse intestine into the bloodstream of mice. Together, Hwp1 appears to have a minor role in disseminated candidiasis, independent of tissue TG, but a key function in host- and self-association to the surface of oral mucosa. Specialized  Candida albicans is often found in the oral cavity and gastrointestinal (GI) tract of healthy individuals as a member of the normal flora. Impairment of the immune system allows for the fungus to overgrow, germinate and produce filamentous cells capable of invading the local mucosal surface. Germination and production of filamentous hyphal cells is accompanied by the expression of adhesion proteins that facilitate host cell interactions. Hyphal wall protein 1  [Agglutinin-like sequence 1 protein) and Als3p are involved in the binding of C. albicans to oral epithelial cells [al cells –6. hwp1Δ/Δ strain is unable to cause oropharyngeal candidiasis in mice [-/- mice die of dehydration soon after birth [Hwp1 participates in the formation of covalent bonds to primary amines and to a buccal epithelial cell (BEC) surface protein catalyzed by human transglutaminases (TGs) ,8. TGs a in mice . Howeverer birth . In vitro, both native and recombinant Hwp1 are substrates for guinea pig liver TG2 [hwp1 null strain (hwp1Δ/Δ) in the wild type C. albicans strain SC5314 using the flp recombinase method [URA3-associated confounding effects [hwp1Δ/Δ strain in wild type C57BL/6 and in Tgm2-/- mice, and did not observe a TG2-dependent requirement for Hwp1. We re-tested the virulence of the newly created null strain in BALB/c mice as before [HWP1 at its native locus did not restore full virulence of the reconstituted strain (hwp1Δ/+HWP1). In light of these results, other Hwp1-phenotypes were tested with the newly created deletion strain. Biofilm formation was deficient in the hwp1Δ/Δ strain as reported by others [HWP1 at its native locus. The knock-out strain was also deficient in causing disease in an oropharyngeal model of candidiasis [C. albicans to the bloodstream of C57BL/6 mice using the experimental model previously developed [hwp1Δ/Δ strain was capable of colonizing the mouse gut to equal levels as HWP1+ strains. However, the hwp1 null strain was less virulent relative to the wild type or the reconstituted strain in this model, thus defining a new phenotype for Hwp1 in murine candidiasis.Although a mechanism of adhesion based on transglutaminase activity on BECs could explain the requirement for Hwp1 in oral candidiasis, the functional requirement for Hwp1 in disseminated candidiasis in mice  where ace method  to avoid effects –20. We ts before  and obsey others ,22 and rdidiasis . Lastly,eveloped . This ineveloped –26, and C. albicans for localized infections of the oral mucosa and is less important for disseminated disease. The fungus is still capable of invading kidneys and livers during invasive disease in the absence of Hwp1 or TG2. The wild type genetic background of SC5314 has allowed us to discern subtle in vivo phenotypes that were previously masked, perhaps due to unknown in vivo fitness defects of the previous parental strain . Our reC. albicans-infected mice were closely monitored for signs of distress. Moribund mice exhibiting signs of illness  were humanely sacrificed by CO2 inhalation according to the recommendations of the Johns Hopkins University ACUC. The studies were performed under the ACUC protocol number MO11M261.All animal studies were approved by the Johns Hopkins University Animal Care and Use Committee (ACUC). The approved protocol was in compliance with the Animal Welfare Act regulations and Public Health Service (PHS) Policy. For virulence/survival studies, HWP1 deletion mutants. The hwp1Δ/hwp1Δ strain constructed in CAI4 [OC and cultured in liquid or agar-containing plates of yeast peptone dextrose (YPD) medium at 30°C according to standard methods [Clinical isolate, SC5314 , was use in CAI4 , DD27-U1 in CAI4 , was kinHWP1 using the SAT1 flipper system for gene disruption in C. albicans [HWP1  as follows: the region between bp -491 to +35  was amplified using the PCR from SC5314 genomic DNA with oligonucleotides H1Ap and H2Xh  according to the manufacturer’s recommendations. The 3’ amplicon contained sequences downstream of the polyadenylation site of the HWP1 mRNA [HWP1 rescue fragment was also generated by the PCR (bp -362 to bp 517 downstream of the stop codon) using oligonucleotides H1Ap and H5Xh to add an ApaI site and an XhoI site at the 5’ and 3’ ends of the amplicon, respectively. The disruption plasmid was constructed by first cloning the 3’ HWP1 amplicon between the SacII and SacI sites of pSFS2 to generate pSFSH3. This plasmid was then used to clone both the 5’ HWP1 amplicon between the ApaI and XhoI sites, and the rescue fragment between the ApaI and SalI sites. The cloned sequences were verified by sequencing for authenticity and PCR-induced errors. The disruption plasmid, pSFSH35, contained 5’ and 3’ HWP1 sequences interrupted by the SAT1 flipper cassette, while the rescue plasmid, pSFSH329, contained HWP1 sequences up- and downstream of the coding region followed by the SAT1 flipper cassette and 3’ HWP1 sequences. Gel-purified linear disruption fragment generated by digesting pSFSH35 with KpnI and SacI was used to transform SC5314 by electroporation [HWP1, and were genotypically HWP1/hwp1Δ::SAT1-FLP determined by Southern blot analysis . Both transformants were cultured in YPD for 24 h at 30°C in the presence of 0.4% BSA  to induce loss of the SAT1 cassette via intramolecular recombination at the FRT sites by the site-specific recombinase FLP, and to re-establish nourseothricin sensitivity. Loss of the SAT1-FLP cassette was verified by Southern blotting, and both single disruption derivatives (HWP1/hwp1Δ::FRT) were subjected to a second round of transformation with the linearized deletion construct. Integration of the disruption cassette at the intact HWP1 allele was verified by Southern blotting, and two derivatives were grown in YPD 0.4% BSA to induce loss of the SAT1-FLP cassette at the second HWP1 allele. Nourseothricin sensitive strains were subsequently verified by Southern blotting. The double disruption strain SCH1211  was used as recipients for the rescue DNA fragment harbored in pSFSH329 (digested with BssHII release the rescue HWP1 gene fragment). Integration of the rescue DNA fragment at a single   overnight at 42°C in ULTRAHyb solution (Ambion/Life Technologies). DNA bands were visualized by incubating the membrane with Strepavidin conjugated to alkaline phosphatase and chemiluminescence reagents , followed by exposure of the membrane to X-ray film (Kodak BioMax XAR).Genomic DNA was prepared from overnight cultures using a MasterPure Yeast DNA Purification Kit . Three to five µg of DNA was digested to completion with NdeI  and the fragments separated in an agarose gel as per standard methods. The DNA fragments were blotted onto a BrightStar-Plus (Ambion/Life Technologies) nylon membrane and probed with a biotinylated  C. albicans germ tubes grown in YPD with 10% bovine serum for two hr at 37°C. Five µg of total RNA was separated in a standard formaldehyde gel and transferred to a charged nylon membrane . The membrane was probed with a biotinylated  antisense transcript (riboprobe) containing HWP1 sequences from +11 to +623 relative to the ATG start site generated according to the manufacturer’s recommendations . The membrane was hybridized overnight at 68°C in ULTRAHyb solution (Ambion/Life Technologies). RNA bands were visualized by incubating the membrane with Strepavidin conjugated to alkaline phosphatase and chemiluminescence reagents , followed by exposure of the membrane to X-ray film (Kodak BioMax XAR).Total RNA was prepared using the hot acid phenol method  from C. 7 germ tubes in reaction buffer  for 15 min at 37°C. The reactions were stopped by adding 1.0 mL of 10 mM EDTA, pH 8.0, and the germ tubes washed twice with dH2O. Germ tubes (1 x 105) were spotted onto poly-L-lysine coated slides, allowed to air dry and incubated with Streptavidin conjugated to FITC (Invitrogen/Life Technologies) at 1:50 dilution in phosphate buffered saline . After 30 min incubation at 37°C, the slides were washed in PBS and rinsed with water. A drop of VECTASHIELD mounting medium with DAPI  was added to the germ tubes before observing under epifluorescence using the FITC or DAPI excitation cubes . Cell images were acquired with an Olympus camera running cellSense 1.6 software (Olympus America). Captured images were minimally manipulated with ImageJ64 .Expression of Hwp1 on the surface of germ tubes was visualized by cross-linking 5-(biotinamido)pentylamine (5-BPA)  as before [-/- [Female BALB/c mice (6-8 weeks) were purchased from Charles River Laboratories  one week prior to experiments to allow the mice to acclimatize to the housing at JHU. C57BL/6 Tgm2-/-  breeder C. albicans strains were grown in YPD with vigorous shaking for approximately 24 h at 30°C, washed twice with PBS and adjusted to an OD600 of 0.3-0.32 (3.5-4.0 x 106 cfu/mL) in sterile PBS. We found that adjusting the yeast suspensions to OD600 of 0.3-0.32 (vs. counting by hemocytometer) produced consistent results across experiments. One hundred microliters of the yeast suspensions was injected into the tail veins of mice, and these observed for signs of illness over a 10-14 day period. Moribund mice were humanely euthanized by CO2 inhalation. For histological observations, C57BL/6 and Tgm2-/- mice injected with SCH1211 (hwp1Δ::FRT/hwp1Δ::FRT) were sacrificed at day 3 post inoculation. Kidneys were fixed in buffered formalin, embedded in Paraffin, and sections stained with Periodic-acid Schiff (PAS) to observe the fungal elements at the site of infection. Disseminated candidiasis studies  were perC. albicans was tested in mice as previously described [C. albicans after first reducing the GI bacterial and fungal flora with penicillinG (1500 U/mL) and streptomycin (2 mg/mL), and fluconazole (250 µg/mL) in their drinking water. After verifying the depletion of the gut normal flora, the drinking water was replaced with water containing a suspension of C. albicans at 107 cfu/mL plus penicillinG and streptomycin as above for 5 days. After verifying gut-colonization of C. albicans as CFU/g of stools, the mice were immunosuppressed by four  intraperitoneal injections of cyclophosphamide (150 mg/kg/dose) every other day. Groups of mice (n=7) were observed for signs of illness prior to sacrifice to determine the fungal burden in the livers. Serial dilutions of homogenized livers were spread onto YPD agar with vancomycin at 10 µg/mL and gentamycin at 100 µg/mL, trypicase soy agar (TSA) and MacConkey’s agar plates . Creamy white colonies were enumerated to determine CFU/g of liver.The role of Hwp1 in gut-translocation and dissemination of escribed   test derived from Kaplan-Meier survival curves. Mean biofilm dry weights were analyzed by one-way ANOVA using Tukey’s multiple comparisons test assuming an alpha value of 0.05. Gut colonization levels (CFU/g of stool) were analyzed by one-way ANOVA of log-transformed data and applying Tukey’s multiple comparisons test. For all statistical analyses, a P value <0.05 was considered significant. Statistical analyses were performed using GraphPad Prism .hwp1 null strains in wild type C. albicans SC5314 to avoid unknown in vivo auxotrophic requirements that could complicate virulence studies [in vivo in confounding ways [HWP1 was disrupted in SC5314 at both alleles using the flp recombinase system [hwp1Δ/Δ strains were created (SCH1211 and SCH2822), and one disruptant, SCH1211, was corrected for HWP1 expression at its native locus using the same flp recombinase molecular system  and the amount of protein paralleled the expression of a single allele  at comparable levels reported for SC5314 [HWP1 expression impaired the strain’s ability to establish an infection of the tongue [in vivo biofilm defective phenotype of our hwp1Δ/Δ strain. Published data [URA3 function did not influence the establishment and local invasion of oral tissue in mice further supporting the independent and key role of Hwp1 in OPC. Indeed, CAI4 (ura3Δ/Δ) is capable of producing OPC in immunocompromised gnotobiotic mice [URA3 function is nonessential in this infection model.Pseudomembranous oropharyngeal candidiasis lesions are considered a form of  biofilm , and sev biofilm ,44–46. Toculated  with SCHr SC5314 . Previouhed data  togethertic mice  showing C. albicans from the intestinal tract into the blood stream associated with antibiotic use and hematological immunosuppression or chemotherapy that result in the loss of intestinal barrier function [C. albicans from the GI tract is unknown, and may define a new role for the fungal adhesin. We used the murine model that mimics this route of infection developed by Koh and colleagues [C. albicans in their GI tracts prior to immunosuppression. We found that all three C. albicans strains, SC5314, SCH1211 and HR615, colonized the intestinal track of C57BL/6 mice to similar levels  [7 and 3.94 x 107 CFU/g of stool, respectively; the levels of gut colonization were not statistically different among the groups by one-way ANOVA, Tukey’s multiple comparisons test, alpha=0.5) suggesting that Hwp1 was not influential in gut colonization of mice. Hwp1 is also not a factor in colonizing the GI tract of gnotobiotic mice [A common route of infection in hospitalized patients is the translocation of endogenous function . The reqlleagues . Mice arr levels . The medtic mice . C. albicans into the blood stream and dissemination to the liver. We modified the method of Koh et al. [C. albicans to the liver of mice treated with the published number of cyclophosphamide injections. All of the mice colonized with strains expressing at least one allele of HWP1 (n=14) died by day 8 post-immunosuppression while 2 of the 7 mice colonized with the hwp1Δ/Δ strain survived to the end of the study . These results suggested that wild type hyphal surface levels of Hwp1 were needed for rapid translocation of C. albicans from the mouse gut into the blood stream. However, once translocation had occurred, the lack of Hwp1 did not prevent dissemination to and establishment of C. albicans in the liver.The gut-colonized mice were treated with intraperitoneal injections of cyclophosphamide to induce immunosuppression/mucosal damage, and allow translocation of h et al.  to add ahe study . The surhwp1Δ/Δ strain from death in a Tgm2-/- background relative to wild type mice. However, our results discounted a TG-mediated mechanism in the virulence of C. albicans driven by Hwp1. The equal rates of deaths among the wild type C57BL/6 and Tgm2-/- mice, regardless of the infecting strain, supported the null hypothesis. Further, histological analysis of infected kidneys revealed invasion of tissue by the hwp1Δ/Δ strain independent upon Tgm2 expression status. However, the possibility existed that transamidation-unrelated factors confounded the survival data. Tgm2-/- mice are less sensitive to bacterial endotoxin [-/- mice. The similar rates of death observed in C57BL/6 mice infected with either SC5314 or SCH1211 lessened the association between Hwp1 and virulence (whether the protein participated in transamidation reactions in tissue or not). And although the use of Tgm2-/- mice had limitations, the lack of TG2 activity was not sufficient to improve survival to a significant level even in the absence of Hwp1. Thus, we concluded that host tissue TG2 activity in organs such as the liver and kidneys [C. albicans. Host transamidation activity did not appear to promote dissemination of C. albicans via reactions involving Hwp1. The studies here were initiated to address the role of TG-mediated reactions as a possible mechanism for the requirement of Hwp1 in disseminated candidiasis. We expected to protect mice infected with the ndotoxin , and TG2ndotoxin –53. A dy kidneys  was disphwp1Δ/Δ strains in C57BL/6 mice was an unexpected result of our studies. C57BL/6 and BALB/c are considered to be less susceptible to intravenous C. albicans challenge among mice of different genetic backgrounds tested in this model of candidiasis [C. albicans infections and complicate the interpretation of virulence data [C. albicans infection can be difficult to qualify when survival kinetics reveal an attenuated virulence in a given mouse strain. The inability to observe the same attenuated virulence of SCH1211 in C57BL/6 mice may reflect differences of genetic effects between C57BL/6 and BALB/c mice that govern susceptibility to candidiasis. At least two and perhaps more genetic loci are linked to susceptibility to infection with C. albicans [C. albicans virulence studies. If the role of Hwp1 in disseminated candidiasis was weak at best, this effect was masked in the C57BL/6 genetic background.  The wild type virulence of the didiasis –58, and nce data –63. The C. albicans regardless of Tgm2 expression. The attenuated virulence of the hwp1 null strain observed in BALB/c mice, not corrected by re-introducing a single allele of HWP1, has been noted by others [hwp1Δ/Δ strains [hwp1Δ/Δ strains were created in CAI4 and not corrected for the large deletion that encompasses URA3, a region of IRO1, a putative transcriptional factor, and perhaps the 3’ untranslated region of ORF19.1717  with attenuated virulence BALB/c mice [hwp1Δ/Δ strains in SC5314. Together, the data suggested that strains not fully restored at the URA3 locus confused previous results of virulence studies using hwp1Δ/Δ strains and attributed a greater virulence function to Hwp1. URA3/CAI4-related effects on virulence of C. albicans has been addressed in the literature [in vivo by confounding host and fungal factors. The data also highlighted the complex relationship between C. albicans and the mammalian host, likely defined by the host’s niche and genetic factors that modulate infection. Consideration of the virulence data independently of mouse strain suggested that Hwp1 was not a major factor in dissemination of y others , and fur strains  attribut19.1717  and in host-fungal interactions on the surface of epithelial or endothelial cells  at host niches  where a biofilm phenotype is the dominant “infectious” morphology. However, the role of these proteins to individually promote candidiasis that involves deep tissue invasion in mice appears to be minimal. Finally, C. albicans clinical isolates expressing an allele of HWP1 (HWP1-2) form poor biofilms in vitro but are nevertheless recovered from the blood stream of patients [of media . HWP1 toormation ,22,68. Eal cells . ALS3 andidiasis ,70; prese levels ,42, anote levels . The phepatients , supportC. albicans from the gut into the blood stream and infection of murine livers. However, the hwp1 null strain was able to colonize murine guts at wild type levels similarly to that observed for rbt1 deletion mutants [C. albicans cells found in the cecum of C57BL/6 mice [HWP1 expression has also been detected in fungal cells recovered from the oral cavities of asymptomatic individuals [HWP1 expression originates in yeast cells at these anatomical niches or in low numbers of hyphal cells that are part of the commensal population. C. albicans cells unable to form filaments and express HWP1 (cph1Δ/Δ efg1Δ/Δ) [tup1Δ/Δ) appear more virulent relative to wild type cells in this animal model of candidiasis [C. albicans to undergo morphological transitions or the expression of adhesins  associated with the hyphal morphology as factors affecting gut translocation. Because hwp1 null strains are not deficient in filamentation  a critical amount of C. albicans self-aggregation is necessary to achieve a threshold fungal burden traversing the damaged intestinal mucosa from the lumen into the blood stream. Dissemination to the liver of the hwp1 null strain was delayed as a consequence of decreased cell numbers trafficking from the GI tract to the blood stream. In support of this hypothesis, the flocculent tup1Δ/Δ cells are more virulent in this gut translocation candidiasis model relative to wild type even when gut colonization levels are two logs below wild type [C. albicans to oral epithelial cells in a non-TG dependent manner [Wild type levels of Hwp1 were needed for the rapid translocation of  mutants . Both ofL/6 mice ,74 althoividuals  presumabefg1Δ/Δ) ,77 do nodidiasis . These rild type . (B) AltHWP1 in HR615 restored biofilm formation both in vitro and in vivo but did not fully correct the hwp1 null phenotype in two out of three animal models that involved dissemination via the blood stream. The factors that control wild type dissemination of C. albicans are likely different from those that influence the establishment of superficial biofilms on mucosal surfaces. The results observed with HR615 suggested that Hwp1 may participate in yet undefined functions that aid dissemination via the blood stream that require wild type Hwp1 expression levels. Expression of a single allele of C. albicans with niche-specific properties to form biofilms at oral/mucosal surfaces with localized penetration of the underlying tissue. Hwp1 and other hyphal proteins organize and maintain the three-dimensional structure of the biofilm which is held in place by cross-linking Hwp1 to the surface of oral epithelial cells by the action of host TG. Thus Hwp1 performs the dual function of self-aggregating C. albicans into pathogenic biofilms and adhesion of the biofilm/hyphal cells to the surface of host tissue via TG activity. HWP1 gene expression differs from other niche-specific genes in that HWP1 is normally expressed in hyphal cells regardless of host niche. Many of the currently known differentially-expressed niche-specific genes code for enzymes  [C. albicans as infection takes hold and progresses at given anatomical sites. Hwp1 plays an important niche-defined structural role in C. albicans in vivo colonial morphology that is not specifically associated with fungal fitness or invasive growth advantage. Ultimately, the ability to combine fitness-enhancing niche-specific gene expression with the appropriate infectious colonial morphology adapts C. albicans for survival and growth at ecologically diverse anatomical sites found within its mammalian host. Taken together, our results here support a new functional model for Hwp1 and similar fungal adhesins: Hwp1 engenders roteins) –87 that Table S1HWP1 disruption and reconstitution cassettes.DNA primers used for generating the  Oligonucleotides used to generate the hwp1Δ:SAT1 and HWP1:SAT1 gene cassettes for the construction of deletion and reconstitution (put-back) strains in pSFS1, respectively. Underlined nucleotides introduce an ApaI site at the 5’ end of the amplicon. Double underlined nucleotides introduce XhoI sites; nucleotides in bold introduce a SacII site. The nucleotides in small letters indicate the SacI site in the 3’ region downstream of HWP1.(DOCX)Click here for additional data file.Figure S1hwp1 deletion and reconstituted strains.Construction of the  (A) Schematic of the HWP1 genomic locus (middle construct) with integration of the HWP1-disruption cassette. NdeI restriction sites are shown in bold above the DNA constructs. The PCR amplicon used to detect HWP1 sequences in Southern blots is labeled “HWP1 probe”. The size marker at top indicates 300 nt. Gene designations: CaFLP, sequences coding for the C. albicans flp recombinase; CaSAT1, sequences coding for C. albicans nourseothricin resistance. The arrow above the inducible SAP2 promoter (SAP2p) indicates the direction of transcription of the CaFLP gene. Dashed green lines indicate the double crossover event leading to integration of the disruption cassette (bottom schematic). (B) Reconstitution of HWP1 expression at its native locus. hwp1Δ::FRT at the HWP1 genomic locus (bottom) with integration of the reconstitution cassette (top). Dashed green lines indicate the sites of the double crossovers restoring expression of HWP1. Size marker, 300 nt. The schematics were generated using Gene Construction Kit .(TIF)Click here for additional data file.Figure S2HWP1 deletion and reconstitution strains used in this study.Southern blot analysis of the  SC5314 and subsequent HWP1 deletion/reconstitution derivatives are shown in order from left to right. The expected HWP1-hybridizing NdeI DNA fragments (arrows at left) are as follows: HWP1, 6.3 Kb, hwp1Δ::SAT1, 3.9 Kb, hwp1Δ::FRT, 4.5 Kb, HWP1::SAT1, 6.3 Kb, and HWP1::FRT, 6.7 Kb . The strains in bold lettering were used in the studies described here.(TIF)Click here for additional data file.Figure S3C. albicans germ tubes.Expression of Hwp1 on the surface of  The primary amine, 5-(biotinamido)pentylamine and human recombinant TG2 were used to visualize Hwp1 on the fungal surfaces . The expression level of Hwp1 on the surface of HR615 appeared decreased relative to SC5314 consistent with the introduction of a single copy of HWP1 at its native locus in SCH1211 (see (TIF)Click here for additional data file.Figure S4hwp1Δ/Δ) in C57BL/6 wild type and Tgm2 knock-out mice.Virulence of SCH2283  or Tgm2-/- (n=7) mice injected with a second HWP1 deletion strain, SCH2283. Survival rates between the animal groups were indistinguishable, P=0.86. The median survival for both groups of mice was 3 days.(TIF)Click here for additional data file.


Understanding associated risk for obesity is a prerequisite to develop early life interventions to arrest the increasing epidemic of metabolic syndrome and obesity among preterm born children and adolescents.p = 0.001). Analysis of associated risk for maternal demographics, maternal age, maternal marital status or race, prenatal factors, maternal substance abuse or diabetes, neonatal factors, weight for gestational age or birth weight did not show any statistically significant risk for future obesity. Duration of gestational age  and heavier birth weight  were associated with risk of obesity.A retrospective review of 160 charts was conducted to determine the associated risk of being obese during childhood and adolescent period in preterm children. Birth weight, gestational age, weight gain, demographics, maternal health, socioeconomics, and clinical factors during early neonatal life were evaluated. The number of obese children increased with age and was observed more in the adolescent population. Obese children were significantly heavier at age 24 months old compared to their peers (Among preterm born babies in the study, the highest risk of developing excessive weight during childhood and adolescent periods are babies born at more advanced gestational age. Strong positive association was found between birth weight and body weight in childhood. By 24 months old, there was a distinguished group of toddlers, who were heavier than their peers and remained with excessive weight as they got older. Primary care pediatricians should draw attention to premature babies, overweight infants and toddlers. Obesity is the main focus of healthcare providers and healthcare policy due to a sustained increased in prevalence over the last decade -5. A conLow birth weight (LBW) premature infants demonstrate growth patterns in the early years of their life (catch up growth) which differ from those of large for gestational age (LGA) term and preterm peers, who do not experience that steep weight gain ,8. Very Numerous clinical and experimental studies have confirmed that early developmental influences can lead to cardiovascular, pulmonary, metabolic, and psychological diseases during adulthood regardless of birth weight . ChildreThe objective of the study was to identify significant risk factors in prematurely born children leading to the development of obesity during late childhood and adolescence periods.2 (m). Obesity was defined as a BMI at or above the 95th percentile for children of the same age and sex [All data was abstracted from the charts of general pediatric clinic records for children and adolescents from 10 to 21 years old, who were born at 37 or fewer weeks of gestational age. The study protocol was approved by the Amarillo TTUHSC Institutional Review Board. Charts were pulled by electronic records using ICD-9 code for prematurity. Demographic, maternal, family, neonatal, and postnatal data were abstracted. Maternal and family history included: previous premature births, race, marital status, prenatal care, health insurance, maternal age, maternal substance abuse, maternal illnesses , and previous multiple gestations. History during the neonatal period included severe illness, respiratory distress syndrome, umbilical vein or artery catheter use, oxygen therapy, intracerebral and intra ventricular hemorrhage, bacterial infections, and metabolic diseases, such as hypoglycemia and jaundice. The child’s postnatal data included type of feeding, major illnesses, blood pressure, weight, and height. Body Mass Index (BMI) was calculated as the patient's weight (kg)/height and sex . Term “e and sex .Cases were classified as extremely low birth weight (ELBW) if less than 1000 g, very low birth weight (VLBW) if birth weight was less than 1500 g, and low birth weight (LBW) if less than 2500 g. Small for gestational age (SGA) was defined as a birth weight less than the 10th percentile for the baby's gestational age group, and large for gestational age (LGA) was defined as a birth weight greater than the 90th percentile for the infant’s gestational age group. All charts had multiple visits records and all visits data were abstracted. For children older than one year of age the majority of the charts did not have a detail nutrition description. For hypertension assessment at least three recorded elevated blood pressure measurements have been used.Statistical analysis was performed using Stata, Version 10 to summarize descriptive data and to calculate odds ratios to estimate the association between the prevalence of obesity and each variable of interest. To compare weight gains before 24 months in obese and non-obese groups the t-test was used. Correlation between weight gains during the first three years of life and reported BMI between 10 and 21 years of age was done. Computation of Mann–Whitney U tests for obese subjects (BMI at the 95th percentile or higher) to weight gains was also analyzed.One hundred sixty eligible preterm cases were abstracted from the clinic charts. Among the sample, 18 babies were ELBW  ranged 27.4 ± 2.7), 19 were VLBW (GA ranged 30.7 ± 1.9), 87 were LBW (GA ranged 33.6 ± 2.1) and 36 were NBW (GA ranged 35.5 ± 1.3). Among VLBW babies average BW was 821 ± 138 grams, among LBW - 1983 ± 262 grams, NBW 2869 ± 418 grams. There were 59% male and 41% female.The number of obese children increased with age and this trend was observed more in the adolescent population Figure . Analysivs. odds per 1 SD of body weight increase in childhood . Increased gestational age in preterm babies was also associated with excessive weight during maturation in childhood . Again strong associated risks were noticed between weight and elevated blood pressure, affecting systolic  more so than diastolic .Strong positive association was found between birth weight increase p = 0.001) , positive correlation. Obese adolescents showed significantly higher weight at the age of 24 months as compared with peers who did not have excessive weight later in childhood (Identification of early-life risk factors for developing obesity in prematurely born children is essential for development of early prevention interventions to reduce obesity. The postnatal period is a critical time when nutrition may predispose a child to lifelong metabolic disturbance and obesity. For LBW, premature, and small-for-gestational-age infants more research is needed to determine optimum nutrition needs .Obesity, type 2 diabetes mellitus, hypertension, coronary artery disease and stroke might be the health consequences of being born SGA ,14. Repoet al. also noticed that higher birth weight predicts childhood obesity in preterm infants [The search for associations between the development of excessive weight with numerous pre-, peri-, and post-natal variables in the study showed that preterm born children, who have a longer GA and higher BW were more at risk to become obese during childhood adolescent periods. Gaskins  infants . The mec infants -19 and e infants . Materna infants . Epigene infants ,23 and rThe “postnatal accelerated growth” hypothesis was proposed to explain the association between faster, early postnatal growth and surrogate endpoints in childhood and adolescence indicative of metabolic and cardiovascular risks based on follow-ups of preterm infants in two early neonatal feeding/nutritional intervention trials ,25. A suThe data confirmed that overfeeding in the early stages of childhood is critical to excessive weight gain and obese children had significantly higher weight at age 24 months old compared with peers, who did not have excessive weight at toddler age. This finding was in agreement with the previous finding that children, who show early and complete growth recovery, could be at a higher risk for the occurrence of metabolic syndrome in adulthood . The sigAlthough more focused research is needed to address this issue, a suggestion would be to reassess nutritional habits of all prematurely born children as soon as they achieve growth parameters of their peers during their well child checks. A nutrition model of preterm born children and adolescents with risk factors should be developed.Among preterm born babies in this study, the highest risk of developing obesity during childhood and adolescent period are babies born at more advanced gestational age. Strong positive association was found between birth weight and body weight in childhood. By 24 months old there was a distinguished group of toddlers, who were heavier than their peers and remained with excessive weight with age. Primary care pediatricians should draw attention to premature babies, overweight and obese infants and toddlers. This is a phase where well child checks are more frequent and education regarding adequate nutrition is essential. Timely referral to registered dietitians might help those children, who demonstrate excessive body weight gain during two consecutive well child visits in spite of dietary recommendations from primary health care providers.LBW: Low birth weight; LGA: Large for gestational age; VLBW: Very low birth weight; HTN: Hypertension; SD: Standard deviation; BMI: Basic metabolic index; ELBW: Extremely low birth weight; SGA: Small for gestational age; GA: Gestational age; NBW: Normal birth weight; BW: Birth weight; OR: Odds ratio; CI: Confidence interval.The authors declare that they have no competing interest.TLV designed the research and wrote the paper; CS did primary data collection and organization; AB, SPC and RS organized and analyzed the data; MEO provided consultation and pediatric charts for the research. All authors read and approved the final manuscript.


Low birth weight (LBW), defined as birth weight less than 2500 g, has a complex etiology and may be a result of premature interruption of pregnancy or intrauterine growth restriction. The objective of this study was to provide information on determinants of LBW and contribute to the understanding of the problem in Brazil.A case-control study was conducted in Botucatu city, SP state, Brazil. The study population consisted of 2 groups with 860 newborns in each group as follows: low weight newborns (LWNB) and a control group (weight ≥ 2500 g). Secondary data from 2004 to 2008 were collected using the Live Birth Certificate (LBC) and records from medical charts of pregnant women in Basic Health Units (BHU) and in the Public University Hospital (UH). Variables were as follows: maternal socio-demographic characteristics, pregnancy and birth conditions including quality of prenatal care according to 3 criteria. They were based on parameters established by the Ministry of Health (MH), one of them, the modified Kessner Index. The multivariable analysis by logistic regression was used to evaluate the association between variables and LBW.2 p < 0.001). According to the modified Kessner Index, 64.4% of prenatal visits in the LWNB group were adequate.According to the analysis, the factors associated with LBW were as follows: prematurity , twin pregnancy , maternal smoking , maternal malnourishment , maternal obesity , weight gain during pregnancy less than 5 kg  and weight gain during pregnancy more than 15 kg . Adequacy of prenatal care visits adjusted to gestational age was less frequent in the LBW group than in the control group (68.7% vs. 80.5%, xLWNB are a quite heterogeneous group of infants concerning their determinants and prevention actions against LBW and the follow-up of these infants have also been very complex. Therefore, improvement in the quality of care provided should be given priority through concrete actions for prevention of LBW. Birth weight reflects gestational conditions and development in the fetal period. In 1975, the WHO defined LBW as birth weight less than 2,500 grams, and considered it as a consequence of premature interruption of pregnancy and/or intrauterine growth restriction [Birth weight is an important biological determinant of newborn (NB) survival in adverse conditions. It shows fetal exposition to risk factors such as maternal unfavorable socioeconomic conditions, smoking habit, malnutrition and diseases as well as lack of attention to prenatal care and delivery. In addition, it plays a critical role in estimating newborns at higher risk of death and diseases mainly in the neonatal period -6.LBW has been the object of several studies because, in addition to its important role in infant mortality and morbidity, it poses risk on its growth and development. Studies in Brazil and Latin America have reported low weight as an important indirect cause of newborn deaths and its association with main causes of neonatal death, such as preterm birth, serious infections and perinatal asphyxia ,8.These newborns have schooling and health problems of different types which can impair their physical, cognitive and mental growth. These children are likely to develop nutritional problems during their childhood affecting their growth and development . StudiesMuch research has been conducted aiming at investigating low birth weight, a public health issue, through discussion of either its etiology and population disparities, or its consequences and possible prevention -15.Some studies point out as most frequent factors associated with LBW some maternal conditions such as low education level, the very young or very old ages, inappropriate gestational weight gain, previous malnutrition and smoking habits ,16 and sTherefore, determinants of low birth weight have been controversial in the literature, which explains the difficulties finding factors that are always associated with this event, as different social realities can be distinguished in places where the studies are conducted.The Brazilian overview of the last years reveals an apparently paradoxical behavior, as LBW rates are higher in more developed regions. In the 90 s, a comparative study conducted in a less developed region of the northeast and in a well developed region of the south revealed LBW ratio of 9.71%: 8.08%, respectively. By 2004, this ratio was inverted, 7.57% in the northeast and 8.52% in the south . In an eThis regional difference has been currently attributed to the availability of prenatal and perinatal care rather than to social condition. The latter was the main cause of low birth weight in the last decade in less developed regions in the country .Understanding this local and national realities as well as LWB determinants to both prevent birth and death of these newborns and comprehend possible alterations in their development is crucial for planning actions to enhance the Public Health System, and as a consequence the survival and prognosis of these infants .The Prenatal and Birth Humanization Program (PHPN) was set up in 2000 to offer strategic actions to improve care quality for the pregnant woman and her fetus, and it brought in its core the discussion about prenatal practices and their conceptual basis according to patterns used worldwide. The major strategy of PHPN is to ensure improvement in the access, coverage and quality of prenatal follow-up, delivery and puerperal care for the pregnant woman and newborn, in the perspective of citizenship rights, following specific and well defined actions which must be closely monitored during prenatal care [According to the National Survey on Health and Demography (PNDS) in 2006, Botucatu and most cities in the southeastern region, especially in São Paulo state, were found to have prenatal coverage of 6 or more consults for approximately 80% of pregnant women .Despite good coverage in prenatal visits, a stability in the rates of low birth weight and neonatal mortality could be found in the period of the study in Botucatu ,22. ThesA case-control study with secondary data collection was conducted in the mid-southern region of São Paulo state, Botucatu city, southeastern Brazil. The city has a predominantly urban population (96%) of 127,370 inhabitants . By 2005According to data of the State System Foundation for Data Analysis (SEADE) based on the Information System of Live Births (SINASC) in the years of 2004, 2005, 2006, 2007 and 2008, the number of live births were 1667, 1702,1670, 1675 and 1728 respectively, amounting 8442 live newborns. Data of DATASUS for these same years showed low birth weight rates per year of 9.6%, 9.1%, 10.5%, 12.2% e 9.3%, respectively, amounting 860 children with low birth weight .All live births whose mothers lived in Botucatu at the time of delivery in the period from 2004 to 2008 were included in the study. The newborns of the Case and Control groups were from this population. The Case group consisted of all low weight newborns (with less than 2500 gr) amounting 860 infants, and the Control group consisted of a randomized sample of 860 live newborns weighing ≥ 2500 gr. For each year considered in the study, the same number of NB were selected for the Case group (LBW) and Control group (non LBW).Figure Number of prenatal care visits at gestational age ; clinical breast examination recorded in any medical visit during the gestational period; at least one ultrasound and beginning of follow-up until the 17th week. Prenatal care was considered adequate when having this range of events, and inadequate, when other different situations were present.For analyses of adequacy and prenatal care quality in HBUs, three criteria were considered: 1. the PHPN ,25,26 anFollowing the ethical principles established in the Helsinki Declaration, this study was evaluated and approved by the Research Ethics Committee (REC) of Botucatu Medical School/UNESP (3372/2009), and by the REC of UNIFESP (0280/2010) .Odds Ratio was calculated based on 95% confidence interval in the adjusted and unadjusted model.Chi-square Test was used to evaluate the association among variables. The significance level was 5%  for rejection of the null hypothesis . In the Using data of SEADE Foundation and DATASUS, the prevalence of low birth weight in the period of the study was 10.2% considering all live newborns in the period in the denominator (8442) ,22.Data on 1720 newborns included in the study were obtained in LBC and information on 1049 pregnant women (61%) from medical charts in BHU and UH, in which 48.7% (511) were in the LWNB group and 51.3% (538) in the NB group weighing ≥ 2500 g, as shown in the flowchart , and these NB had higher incidence of deaths in the first year of life  and higher rate of serious and moderate neonatal depression .Distribution of maternal gestational and delivery characteristics as well as those of newborns according to the birth weight group is shown in Additional file p < 0.001) , diagnosis of maternal malnourishment , weight gain lower than the expected one , maternal history of systemic arterial hypertension  and diseases during the current pregnancy .Additional file p < 0.001) and medical visits (p < 0.001) compared to those in the control group. Also, no nursing visits were accomplished during most of these pregnancies (p < 0.001). A high number of ultrasound exams were performed in both groups and the small number of subjects who did not undergo this exam drew our attention . Among the routine exams recommended by the Ministry of Health, breast clinical examination had the lowest frequency in prenatal visits .Additional file p < 0.001) was observed regarding only to the number of adequate prenatal care visits by gestational age, with a higher frequency of inadequate care related to LBW (31.3%) in relation to the control group (19.3%).The analysis of adequacy and quality of prenatal care through the 3 proposed indicators in Additional file p < 0.001), twin birth , maternal malnourishment  or obesity , inadequate weight gain being less than 5 kg  or more than 15 kg  and also smoking during pregnancy  were associated with LBW.Additional file The multivariable analysis by logistic regression including the 3 criteria for evaluation of prenatal care quality was applied and no associations with low birth weight were found considering the adjusted and non adjusted models. No influence of prenatal care quality on the association with other factors and LBW was observed.The study aimed at identifying factors associated with low birth weight over 5 years in a mid- sized city in São Paulo state interior region, Brazil, and expanding the discussion about this theme in the literature. For that purpose, a control group was set up to compare variables and search different data sources for a joint discussion about maternal socio-demographic factors, those related to delivery and to the newborn and in a highly comprehensive manner, the prenatal care provided for these pregnant women.Data and outcomes from this study revealed the reality of the city in the study years concerning characterization of low birth weight newborns, their conditions of prenatal follow-up and birth as well as maternal health and socio-demographic situation, considering that the study analyzed the world of the LWNB and used a sample, for technical feasibility, of non-low weight NB as a control group so that the comparison between them was made.Low birth weight rate for the city of Botucatu has been higher, except for the year 2008, than the rates for São Paulo state , the SouCurrently, reduction in these regional disparities in Brazil has been attributed to prematurity rates, which have been increasing in the country as a whole, and kept low rates of birth weight. Simultaneously, in the last decade, a reduction in term children with low birth weight (intrauterine growth restriction) has been observed. Therefore, low birth weight has been associated rather to conditions of access to health care and services than to socio economic conditions .In this study, factors associated with low weight were prematurity, twin births, maternal malnourishment and obesity, inadequate weight gain less than 5 kilos or more than 15 kilos and maternal smoking. Considering the reality in which the study was conducted, i.e., in a city with good socio-economic conditions, a good score in the HDI classification , high leTherefore, no conclusions can be reached on the association between socio economic and demographic conditions and LBW. This finding is a consequence of different socio economic realities in the study regions, since they are interconnected factors related to life conditions of the population such as, housing, income, basic sanitation, level of knowledge and access to health services which directly influence favorable or unfavorable outcomes in the health-disease process, including pregnancy and birth conditions.A stronger association of LBW with prematurity and not with intrauterine growth restriction is pointed out in this study. Also, twin pregnancies are often associated with shorter period of gestation. The association has already been described in Brazil and worldwide, and currently it has been considered the main factor for keeping low birth weight rates in Brazil ,30,33.The importance of maternal smoking during pregnancy and its relationship with low birth weight is reported in the literature and also confirmed in this study as an important predictor of LBW. This finding was one of the first consequences of this habit, and it is probably related to intrauterine hypoxia leading to fetal malnutrition ,35. SomeThe infant mortality rate of the Low Birth Weight newborn group in our study shows the local reality and represents 60 deaths per 1000 births. No deaths were reported in the control group. However, data analysis from the SEADE Foundation , which rMaternal nutritional status and weight gain during pregnancy have been addressed in many studies. According to Melo et al.  "not onlMany researchers have evaluated the association between previous maternal diseases and gestational diseases and low weight of newborns ,43. SomeFor the city of Botucatu, which has a diversified public health care both in Basic Health Units (BHU) and in the Public University Hospital, as well as good level of maternal schooling, the situation mentioned above is an expected reality considering that pregnant women awareness and the access to health services ensure them an adequate disease control.The Public Health System in Brazil follows organizational principles, and the hierarchical health care is one of them.  The refeData from Brazil show that there have been high coverage rates for prenatal follow-ups with 80.7% of women having 5 or more prenatal consults between 2006 and 2007, and 83.6% of them, starting the prenatal follow-up within the first trimester of gestation. Therefore, the present concern relies on quality evaluation of the care provided.A high coverage rate for prenatal care was also observed in our study, which shows the appropriate understanding of the need for follow-ups by the pregnant women. Moreover, it shows the wide offer of this service in the health care system.Prenatal care is one of the most ancient care practice in the Brazilian Health System, and an optimal condition would be the one in which medical care could be offered to women since the very beginning of the pregnancy diagnosis. For that purpose, health services must have skilled professionals to appropriately welcome and build close ties with the users. Therefore, the main determinant of low quality care provided by the SUS network has been the limited human resources. However, this limitation is qualitative rather than quantitative .Care of pregnant women must include minimal procedures and necessary exams to reduce maternal and fetal morbidity and mortality. This care would be through prevention and early diagnosis of alterations as intrauterine growth restriction and maternal infections which can be vertically transmitted to the offspring . A good According to some studies, neonatal mortality, prevalence of low birth weight and prematurity in Brazil are related to lack of access to routine and basic procedures concerning pregnancy assistance .One of the objectives of this study was to evaluate prenatal care and its relationship with low birth weight. The modified Kessner Index, Adequacy Prenatal Care Utilization Index and Kotelchuck Index are combined measurements more frequently used to a better study of quality of prenatal care utilization ,26. The The increase in inadequacy as a result of the modified Kessner Index utilization, as compared to the analysis which considered only the number of visits at the gestational age, shows that the late start of prenatal follow-up was the contributing factor in this reduction in adequacy.Therefore, when variables are associated for a judicious evaluation of prenatal quality, different results are found compared to those from a more partial or fragmented analysis of care, even when there is no association with LBW as in the present study.Attention to adequate prenatal care should not be limited to prevention of LBW, as this service ensures advising for pregnant women concerning their health, and adoption of healthy life habits for themselves and their infants. Moreover, it identifies risk situations for pregnancy and delivery, interventions are timely applied and unfavorable outcomes for the mother or newborn are avoided. Also, mothers may be enrolled in programs of health care attention to the woman and infant for later follow-ups.Data from this study and from literature show that the risks for LWNB are fully established and they may vary according to the social and economic reality in which the study was conducted. However, this problem must be acknowledged and faced by the Health Public System. Although the widespread knowledge of the risk for low birth weight in Sao Paulo state and Brazil, the specific evaluation of the prenatal follow-up and control of these factors have not been widely and uniformly performed in Brazilian cities. The adequate identification of LBW associated factors must create effective and preventive strategies, and the birth of LWNB must give rise to a necessity of redesigning intra and extra hospital actions to better welcome and follow the triad of the NB/mother/family, reducing risks and promoting better growth and development of this infant.Few limitations were found in this study, as for the object of the study (LBW), all cases in the study period were included. The proposal of identifying prenatal care associated factors and considering births within the Public Health System led to the incorporation of most pregnancies and births occurred in the city. However, because of the great population diversity, the study of prenatal care in private or managed health care settings could bring other contributions to the discussion of this issue.Most factors associated with LWNB in this study would be avoidable if health services were structured to timely recruit pregnant women at the very beginning of their pregnancy, and provide prenatal follow-ups which would include the pregnant women welcome and control of the absentees. This process would enable early detection of risks and promotion of actions through interdisciplinary and inter sectoral approaches aiming at changing habits, diagnosing diseases, providing better nutritional conditions and monitoring gestational diseases. Based on epidemiological characteristics, LBW newborn must be considered rather a sentinel event than a risk indicator. This sentinel event would have a potentially avoidable occurrence: the infant death. A qualitative analysis with individual information examined on a case by case basis could provide further clarification of this Public Health issue which has not been altered in the last decade in Brazil and worldwide.The study shows that LWNB is a very heterogeneous group of children concerning its determinants, and therefore, actions to prevent LBW and follow-up of these children have been a challenge for the Public Health System. This study on risk factors associated with low birth weight in the city of Botucatu as well as others conducted in the country have as their main objective to improve comprehension of this problem so that concrete actions towards it are incorporated. However, lowering these rates of LWNB is not an easy task because of its multifactorial and complex etiology and differentiated display according to the city or region in the study. Cities have different socio-demographic characteristics and health service structure as reported in our study and in the literature.While municipal health programs for pregnancy, delivery and newborn care have been improved as priorities of local and national public policy, structuring health services regarding an adequate follow-up of these children after birth; increasing the number of openings and improving resources in Neonatal Intensive Care Units; providing ward services for an adequate follow-up and prevention of sequelae in these children should also be a political priority.Therefore, the contribution of this study to the area is to show that risk factors for low birth in the city may be overcome. Ultimately, the challenge to be faced by health authorities has currently been political, so that the right to health care has to be ensured to all pregnant women and newborns through improved and already well defined actions towards health care.The authors declare that they have no competing interests.All authors have made substantial contributions to the study and endorsed data and conclusions. CRBF identified the research question, conducted the analyses and wrote the first draft of the article. LRC contributed to the analysis and assisted in editing of the article. RFP, MWLS contributed to identification of the research question, interpretation of findings and writing of the article. All authors have read and approved the final version of the manuscript.Table S1. Distribution of socio-economic characteristics, newborn and gestational conditions, hospital category and mode of delivery.Click here for fileTable S2. Maternal habits, diseases and conditions during gestation and distribution in the study groups.Click here for fileTable S3. Characterization of prenatal follow-up and adequacy indexes of care in the study groups.Click here for fileTable S4. Factors associated with low birth weight and multivariable analysis by logistic regression.Click here for file


The initial observations of David Barker, popularly known as the “Barker hypothesis” or “developmental origins of health and disease,” show that being born with low birth weight, as a result of intrauterine growth restriction produced by maternal undernutrition, is associated with a number of chronic diseases later in life . SubsequOver 346 million people worldwide have diabetes, and according to the estimates of the World Health Organization, the prevalence of type 2 diabetes will double by the year 2030 . SimilarSeveral experimental studies report a relationship between maternal overnutrition and health of the offspring in adulthood. Specifically, maternal body weight, overnutrition, or a high fat consumption during pregnancy is linked to the development of elements of the metabolic syndrome, cardiovascular and renal disease, hypertension, and cerebral dysfunction as well as type 2 diabetes and obesity themselves later in the life of the offspring –12. HoweThe current issue presents 5 clinical research papers as well as 7 review articles. The following is a summary of major points and findings presented in these papers.In overweight/obese mothers, greater % kcal from sweets early in pregnancy is the strongest, independent predictor of higher weight for gestational age at birth as well as after 6 months and higher odds of fetal macrosomia, suggesting that mothers' eating behaviors during pregnancy may have a lasting effect on child weight.In nondiabetic women, maternal glucose levels correlate with the extent and distribution of fetal adiposity and birth weight.In the Jerusalem perinatal study , offspring of mothers with gestational diabetes have higher body mass index and systolic and diastolic blood pressure at 17 years of age compared with offspring of mothers with no gestational diabetes. These data suggest an association between maternal glycemia and cardiometabolic outcomes in the offspring.Interestingly, no association between cardiac function in offspring of type 1 diabetic mothers at 7-8 years of age and maternal glycemic control was found. Whether the cardiac phenotype takes longer to develop in this cohort may need to be examined.At the age of 3 years, offspring of type 1 diabetic mothers were characterized by a delay in cortical evoked responses in both visual and somatosensory systems, suggesting a potential association between maternal glycemia and brain maturation in the offspring.The 7 review articles all provide a comprehensive summary of some of the mechanisms underlying the developmental programming of metabolic syndrome, cardiovascular disease, and obesity later in the life of the offspring. Interestingly, similar mechanisms seem to contribute to different phenotypic outcomes regardless of what the insult was during early development. Specific emphasis is given to the importance of epigenetic changes induced by the maternal environment in predicting later adversity.in utero environment may translate into a health risk in the offspring.In summary, this special issue highlights the fact that maternal health plays a significant role in determining as well as predicating the health of the offspring later in their life. Future studies are warranted to specifically examine the mechanisms by which the perturbation of the Christine Maric-BilkanChristine Maric-BilkanMichael SymondsMichael SymondsSusan OzanneSusan OzanneBarbara T. AlexanderBarbara T. Alexander


Up to now, fiber tractography in the clinical routine is mostly based on diffusion tensor imaging (DTI). However, there are known drawbacks in the resolution of crossing or kissing fibers and in the vicinity of a tumor or edema. These restrictions can be overcome by tractography based on High Angular Resolution Diffusion Imaging (HARDI) which in turn requires larger numbers of gradients resulting in longer acquisition times. Using compressed sensing (CS) techniques, HARDI signals can be obtained by using less non-collinear diffusion gradients, thus enabling the use of HARDI-based fiber tractography in the clinical routine.Eight patients with gliomas in the temporal lobe, in proximity to the optic radiation (OR), underwent 3T MRI including a diffusion-weighted dataset with 30 gradient directions. Fiber tractography of the OR using a deterministic streamline algorithm based on DTI was compared to tractography based on reconstructed diffusion signals using HARDI+CS.HARDI+CS based tractography displayed the OR more conclusively compared to the DTI-based results in all eight cases. In particular, the potential of HARDI+CS-based tractography was observed for cases of high grade gliomas with significant peritumoral edema, larger tumor size or closer proximity of tumor and reconstructed fiber tract.Overcoming the problem of long acquisition times, HARDI+CS seems to be a promising basis for fiber tractography of the OR in regions of disturbed diffusion, areas of high interest in glioma surgery. Due to their rapid growth and infiltrative nature, gliomas remain one of the challenges in neurological surgery, particularly those tumors located in direct vicinity of eloquent cortical areas or fiber bundles. Maximum extent of tumor volume resection has been accepted as the major goal in glioma surgery, being associated with a significant benefit in patient survival nd order tensor. In anisotropic tensors, the main eigenvector encodes for the longitudinal direction of axons in major white matter tracts. For the tractography procedure, algorithms are applied that process the tensor information and reconstruct fiber bundles. Different algorithms have already been implemented and investigated so far nd order tensor model, multiple fiber populations within a voxel cannot be resolved adequately. This causes complications to resolve crossing-, kissing-, diverging or highly curved fibers. Another problem appearing with DTI-based fiber tractography is the resolution of fibers in areas of disturbed diffusion, for example due to tumor or peritumoral edema Due to its practicability, the estimated course and location of white matter tracts in clinical practice is currently performed mainly on the basis of diffusion tensor imaging (DTI)-MRI. DTI is based on a set of diffusion images acquired for at least six different gradient directions and one reference image To overcome these drawbacks, alternative diffusion imaging and reconstruction schemes denoted as High Angular Resolution Diffusion Imaging (HARDI) Using this protocol, HARDI data can be represented using a relatively small number of diffusion-encoding gradients, thus enhancing the feasibility of HARDI-based fiber tractography in the clinical practice. This is implied in compressed sensing techniques (CS) To investigate the feasibility of HARDI+CS for the reconstruction of the optic radiation (OR) in proximity of gliomas in the temporal lobe with associated disturbed diffusion properties, we performed fiber tractography based on DTI and HARDI+CS for eight patients. To our knowledge, this is the first clinical application and comparison of DTI-, and HARDI+CS-based fiber tractography for the OR, showing the potential of HARDI with respect to challenging intra-voxel fiber distributions.3]. Furthermore the minimum distance from the tumor outlines to the fiber object, based on HARDI+CS was calculated in [mm]. All tumors were located in close proximity to the reconstructed OR with less than 20 mm  preoperatively, including T1-weighted 3D images (3D-Magnetization Prepared Rapid Gradient Echo (MPRAGE)): repetition time (TR) 1900 ms, echo time (TE) 2.26 ms, field of view (FoV) 256 mm, matrix 256×256, slice thickness 1 mm, 176 slices, sagittal). The protocol for diffusion imaging was TR 7800 ms, TE 90 ms, FoV 256 mm, matrix 128×128, slice thickness 2 mm, numbers of excitation (NEX) = 1, b = 1000 s/mmEvaluation was performed using the image analysis platform MedAlyVis  For both, DTI-, and HARDI+CS-based fiber reconstruction, the same manually segmented region around the lateral geniculate nucleus (LGN) was used as seed region. The visual cortex was then used as include region, resulting in a fiber object passing through the LGN and terminating in the visual cortex (Brodman areas 17–19). These ROIs were determined by experienced examiners (over six years of expertise), one neurosurgeon and one neurologist.3  35 cm3 .For both DTI and HARDI+CS-based fiber tractography, we found a fiber bundle, reaching from LGN to the visual cortex. However, DTI-based fibers were slender and diffusely orientated. HARDI+CS-based tractography, however displayed a more solid fiber bundle, representing the OR without significant looping of Meyer's loop (ML). Still, all three parts of the OR in relation to the ventricular system are reproducible with both methods , row 1.3 with central necrosis, however with a strong peritumoral edema  in the left temporal lobe, displayed a fiber bundle from LGN to the calcarine sulcus. In this case, the ML does not loop significantly. However, tractography results also include some diffusely running fibers, rather not belonging to the OR . Single-ROI DTI-based fiber tracking results were inconclusively, with only one fiber starting from LGN and terminating in the visual cortex, most likely belonging to the central bundle of the OR , row 3.3 and little peritumoral edema. Tumor localization is in close proximity to the OR with only app. 10 mm minimum distance. Results of DTI-, and HARDI+CS-based fiber tractography vary significantly. With HARDI+CS, the OR is reliably displayed. The ML shows a sharp loop in anterolateral direction. By contrast, using the same starting ROI around the LGN, DTI provides a slight fiber bundle, most likely showing the central bundle of the OR. The ML is not traced sufficiently  of app. 10 cm3, closely located to the OR, given by tractography results , tractography results based on DTI displayed solitary fibers, connecting LGN and visual cortex. However, fibers were not safely traced around the temporal horn of the lateral ventricle. The HARDI+CS-based results displayed a solid OR-object, showing the three bundles of the OR in their course around the temporal horn  accompanied with a large peritumoral edema  compared with DTI. Even in two cases , DTI based fiber tractography fails to generate an OR-object at all.The ML is given in four cases via HARDI-based tractography, in which it can not be reliably displayed by DTI . In all 6 cases , in which HARDI+CS delivered much better tractography results, patients suffered from high-grade tumors that were also associated with significant edema in 4 cases . Also closer location of tumor and OR  resulted in significantly improved results via HARDI+CS compared with DTI.The classical descriptions of the visual pathway, particularly the OR are based on dissection studies by Leuret and Gratiolet Navigation systems are widely used among neurosurgical operating theatres, showing outlines of pre-operatively segmented risk structures and targets in the microscope heads-up display. This requires a previous registration process of physical space and image space The by far most frequently used basis for fiber tractography in the clinical practice is DTI, which is still developed further including the processes of data acquisition, image processing and analysis Although DTI-based tractography results have been shown to contribute to a low postoperative morbidity when integrated in the navigation system Apart from alternative approaches nd order tensor model remain. These can be overcome by using advanced diffusion imaging and reconstruction schemes based on HARDI. Using this approach, multiple intravoxel fiber orientations can be resolved. However, acquisition of HARDI data sets requires a significantly higher number of diffusion gradients, ranging from 60 to 100, with associated data acquisition times of up to 25 minutes as opposed to approximately 4 minutes for DTI (on 3T MRI-systems). In this way, clinical applications of HARDI are still rare although frequently used for theoretical neuroimaging e.g. by Frey et al. Despite these innovations, the previously described intrinsic drawbacks of the 2The disadvantage of increased acquisition times has been alleviated by HARDI+CS, which is based on the theory of sparse representation. Thus, CS enables the reconstruction of HARDI signals from as low as 20 diffusion gradients, although with a low reconstruction error of approximately 1%. In this way, of conventional diffusion weighted data set, which is also used for DTI-based fiber tractography can be used for reconstruction Apart from this, current research also addresses higher practicability for HARDI-based fiber reconstruction in clinical applications. Due to the increased diffusion information provided by HARDI, naïve fiber tracking approaches based on HARDI data are computationally expensive. Several frameworks have been proposed, for example by Prckowska et al. Particularly for reconstruction of neuroanatomically complex fiber bundles, a high impact of advanced diffusion models as base for fiber tractography is likely. We investigated HARDI+CS's possible advantages over DTI on the example of the OR in a neurosurgical patient collective, all patients suffering from gliomas in the temporal lobe with their associated more complex white matter architecture.The basis for interpretation of our results are classical and more recently published dissection studies also mentioned above In all eight cases, tractography results based on HARDI+CS display the OR better compared with DTI-based fiber objects. HARDI+CS-based objects generally display more fibers, thus displaying the OR as a solid tract including its three different bundles. This suggests, according to the recent literature that DTI-based fiber tractography generally underdetermines the extent of a fiber bundle In six out of eight cases however, HARDI+CS-based fiber tractography results in significantly improved fiber objects  compared with DTI. In these cases, the single-ROI DTI-based results only give slender fiber bundles  or even do not display the OR convincingly at all .These obvious advantages of HARDI+CS-based fiber tractography are particularly associated with the following factors:tumor size/size of peritumoral edematumor localization/distance from the ORtumor histopathologytumor morphology in MRI3 and 16 cm3).Thus, in those cases for which HARDI+CS delivered significantly better results, we found a high-grade invasive tumor in six cases , mostly associated with a significant peritumoral edema . Only one of these tumors (patient 3) did not show significant contrast enhancement on the T1-weighted MRI. Furthermore, the tumors were located in direct vicinity to the OR based on HARDI+CS with less than 15 mm in four cases . Compared to this, single-ROI DTI-based fiber tractography resulted in acceptable objects in two patients (1 and 6). Here, both tumors showed no contrast enhancement in T1-weighted MRI, suggesting less invasive behaviour, which was confirmed by histopathology . Furthermore, these tumors were comparatively small according to manual segmentation  is significantly longer with 45 minutes, compared with 5 minutes for DTI-based tractography. In this way, clinical practicability is still reduced, requiring larger effort in time and personnel. Furthermore, the software platform MedAlyVis, on which HARDI+CS is implemented, is not commercially available and strongly emphazises scientific applications. We found, that the teach-in period is significantly longer compared with DTI-tractography applications on clinically orientated and commercially used navigation systems.SlicerFor optimization, we intend to vary parameters  of the diffusion imaging sequence based for the fiber reconstruction via DTI-, and HARDI+CS. This will first be investigated with a software phantom, providing a ground truth fiber bundle to objectively compare the reconstructed fibers. Subsequently, the most promising sequence parameters will be evaluated in a collective of healthy subjects. Furthermore, optimization of tractography data for fibers with neuroanatomically complex course such the language-associated pathways, the optic radiation or the limbic system will be investigated via use of different tractography algorithms applied also to the HARDI data and compared with the tensor deflection algorithm. For future investigations, HARDI+CS-based reconstruction the mentioned fiber bundles should be routinely integrated into the navigation system besides conventional DTI-based tractography to support our hypothesis and evaluate the clinical impact on postoperative morbidity. In this context, it should be the aim to apply HARDI+CS in an open source software platform e.g. To provide an intraoperative fiber-estimation with compensation for brainshift, we suggest matching preoperatively obtained HARDI+CS fibers with intraoperative MRI using non-linear registration or sophisticated pattern recognition techniques With our prospectively conducted case series on eight patients, we can clearly show the potential of HARDI+CS-based fiber tractography compared with conventionally used DTI-based fiber reconstruction. HARDI+CS thus offers high resolution fiber tractography, even for neuroanatomically complex fiber bundles like the OR and in areas of disturbed diffusion patterns, however using low data acquisition times required for clinical use. In this way, a neurosurgical major interest focuses on glioma surgery of the temporal lobe, as in these cases peritumoral diffusion patterns have to be considered disturbed in any case. HARDI+CS seems to be a promising approach, combining the advantages of HARDI's estimation of multiple intravoxel fiber populations in the temporal stem with the clinical feasibility of routinely used DTI image data acquisition in presurgical practice.


Contrary to our predictions, we show effects of maternal birth season on offspring birth weight and head circumference only in recent maternal cohorts born after 1975. Offspring birth weight varied according to maternal birth season from 2.85 to 3.03 kg among women born during 1975–1984 and from 2.84 to 3.41 kg among those born after 1984, but the seasonality effect reversed between these cohorts. These results were not mediated by differences in maternal age or parity. Equivalent patterns were observed for offspring head circumference  and length (not significant), but not for ponderal index. No relationships were found between paternal birth season and offspring neonatal anthropometrics. Our results indicate that even in rural populations living under conditions of relative affluence, brief variation in environmental conditions during maternal early life may exert long-term intergenerational effects on offspring.Environmental conditions experienced in early life can influence an individual's growth and long-term health, and potentially also that of their offspring. However, such developmental effects on intergenerational outcomes have rarely been studied. Here we investigate intergenerational effects of early environment in humans using survey- and clinic-based data from rural Gambia, a population experiencing substantial seasonal stress that influences foetal growth and has long-term effects on first-generation survival. Using Fourier regression to model seasonality, we test whether (i) parental birth season has intergenerational consequences for offspring  Maternal birth weight has been found, inter alia, to be negatively related to offspring adulthood blood pressure ,9 and meet al. [et al. could account for these differences. Painter et al. [in utero famine exposure.Effects of maternal early environment on offspring growth have been studied most extensively in the Dutch ‘hunger winter’ cohorts. Lumey and co-workers –20 studiet al.  studied r et al.  also fouin utero exposure to Ramadan fasting looked separately at effects of exposure during each of the trimesters of pregnancy on four birth outcomes in offspring of both sexes [A few studies of populations other than the Dutch hunger winter cohort examine effects of maternal early environment on offspring growth, and the results are again inconsistent. One paper examining intergenerational effects on offspring growth showed that the offspring of women born during the Chinese ‘Great Leap Forward’ Famine of 1959–1961 had lower height- and weight-for-age . A recenth sexes . The onlth sexes , it doesBecause of the mothers' role in providing an environment and provisioning for offspring from conception until weaning, much of the evidence for intergenerational effects of the early environment on offspring has come through looking at apparent effects of the maternal early environment. However, a mechanism that is being increasingly seen as playing an important role in mediating effects of the early environment on phenotype, and which could operate through both fathers and mothers, is epigenetic programming effects (e.g. methylation patterns), by which gene regulation is permanently altered during early life ,26. DuriThus, the evidence from humans offers some support to the findings from studies of animal models that parental exposure to adverse environmental conditions during critical periods of development can have consequences for offspring phenotype. Unfortunately, few datasets combine measures of parental early environment with offspring outcomes. Changes in environmental conditions as a result of birth season provide one possibility for identifying and comparing groups of individuals experiencing different environmental conditions in early life. In particular, long-term data from traditional societies in which seasonal patterns of farm work, hunger and disease exposure have a significant impact on individual health, and nutritional status may prove more useful in understanding the interactions between the developing human and the environment –34. SuchIn this study, we investigate relationships between maternal and paternal early conditions and offspring neonatal anthropometrics in rural Gambia. Since 1950, residents of several villages in the West Kiang district of the country have been studied and treated by the UK Medical Research Council. Seasonal variation in food supply culminates in an acute shortage of energy when agricultural workload is highest; termed the ‘hungry season’. The consequent energy shortage, compounded by increased exposure to infectious diseases, such as malaria, leads to a marked seasonal reduction in the weights of pregnant and lactating women , and hasWe test two specific predictions: First, we predict that parental birth season will have long-term consequences that impact offspring growth. Second, we predict that changes in the provisioning of medical care over recent years over which the population has been monitored ,39 will 2.Since 1950, residents of three villages in the West Kiang district of The Gambia  have been studied and treated by the UK Medical Research Council. This arrangement was initiated by Ian McGregor, when the population of the largest village, Keneba, numbered approximately 700 inhabitants, mostly of Mandinka ethnicity. Traditionally, the population has subsisted largely on crops of rice, sorghum and millet, with a single cash crop of groundnuts. Over the study period, there has been incremental improvement in the provision of the healthcare to the local population, most notably in the form of increased antenatal and natal care since the 1970s. In 1974, a clinic was established to provide free medical care to residents of the three villages and the surrounding area, and to monitor the health of neonates, infants and children. Overall, under-five mortality rate declined from more than 40 per cent prior to 1970 to less than 10 per cent in the present day ,3,39. FeN = 327 mothers, N = 1442 offspring; 1975–1984: N = 205 mothers, N = 483 offspring; after 1984; N = 49 mothers, N = 57 offspring) and on 942 singleton offspring born to 240 fathers . Tables 3). Not all measures were available for all offspring. Mothers were born between 1950 and 1994, fathers between 1950 and 1984 and offspring between 1976 and 2009. Maternal age at offspring birth ranged from 14 to 47, and paternal age between 12 and 61. Offspring were measured within 24 h of birth by trained staff.We analysed the effects of first maternal, and then paternal, birth season on the neonatal anthropometrics  of a total of 1982 singleton offspring born to 581 mothers ; rβ is the Fourier coefficient of the sine term of the r harmonic; rγ is the Fourier coefficient of the cosine term of the r harmonic and iθ is the point in the annual cycle when the individual i is born expressed in radians .Birth weight split by maternal/paternal birth cohort and two-factor maternal birth season (January–January versus July–December) is shown in the electronic supplementary material, tables S1 and S2. In analysis, season of birth was modelled using Fourier series ,41,42. Fh = 1 . This model also included as predictors maternal or paternal village (three levels); maternal cohort ; maternal age (linear and quadratic); paternal cohort ; paternal age ; maternal parity (two levels: parous and nulliparous) and offspring sex. Bearing in mind the number of interaction terms being considered (see below), in order to avoid producing highly parametrized models, we kept the number of factor levels to a minimum (e.g. two in the case of parity). For maternal/paternal birth cohort, we based our decision on the appropriate number of factor levels on previous research showing substantial difference in mortality rates between the periods as described earlier [For each of the four offspring birth anthropometrics, we produced a linear model that included as predictors the maternal/paternal and offspring birth season Fourier terms with a harmonic number  earlier ,3,39. Ex earlier . Likelihin utero growth have previously been found to be dependent on maternal parity [p > 0.21).In addition to the first-order maternal Fourier terms, we included interactions between maternal/paternal Fourier terms and maternal parity, maternal cohort and maternal/paternal age (linear). This was because (i) effects of maternal early environment on offspring l parity –7,17–20,l parity ,9,45; , so seasonality is parsimoniously described by a continuous variation between two main different seasons per year, not more. We also produced alternative models to constrain fixed effects interacting with the maternal/paternal Fourier terms to modify the amplitude of seasonality [We determined that model fits were not improved either by increasing the harmonic numbers of maternal/paternal and offspring birth Fourier series  and offspring gestational age . 3.(a)p = 0.0009, see the electronic supplementary material, table S3). This indicated that maternal birth season was related to offspring birth weight, but since the suite of maternal birth season terms included in the model comprised interactions between maternal age, maternal parity and maternal cohort as well as the first-order effect of maternal birth season, we removed each term separately and assessed changes to model fits in order to assess which of these were significant. The effect of maternal birth season was only revealed when the interaction between maternal birth season and maternal birth cohort (p < 0.0002) was retained (a), indicating that the effects of maternal birth season changed depending on the year in which a woman was born. Subsequently, we compared models with merged birth cohorts to our base model in order to determine whether different birth cohorts could be pooled without significantly impeding our model fit. The best model fit was obtained when all three cohort levels were retained , demonstrating that the relationship between maternal birth season and offspring birth weight was significantly different among all three maternal cohorts. Assessing the significance of maternal birth season within each cohort revealed no effect in the before-1975 cohort (p = 0.40) but effects in both the 1975–1984 (p = 0.024) and after-1984 (p = 0.0005) cohort. Although the power to detect an effect of maternal birth season differs between cohorts, the trend suggested by the p-values matches the one given by predicted differences in birth weight through the year. We observed almost no difference in the predicted birth weight before 1975 (p = 0.0015), indicating that neither maternal size, gestation length nor the combination of these factors accounted for the results obtained .We analysed birth weights (mean ± s.d. = 2.95 ± 0.42 kg) of 1939 babies born to 576 mothers . Comparison between models that did and did not contain maternal birth season terms showed that the better fit was obtained when maternal birth season terms were included . There was no interaction between paternal birth season and paternal birth cohort  and no first-order effect of paternal birth season .We analysed birth weights (mean ± s.d. = 2.94 ± 0.46 kg) of 913 babies born to 236 fathers . Model comparison showed that, overall, inclusion of paternal birth season terms did not improve model fit ((b)p = 0.36), indicating that maternal birth season did not significantly influence offspring birth length . There were no improvements to model fit as a result of specific inclusion of interactions of maternal birth season with maternal cohort (p = 0.22), or the first-order effect of maternal birth season after removal of the interaction (p = 0.65). However, the non-significant pattern of maternal seasonality in each of the respective cohorts is similar to that found for birth weight (b).We analysed birth lengths (mean ± s.d. = 49.36 ± 2.67 cm) of 1099 babies born to 450 mothers . Exclusion of maternal birth season terms did not reduce model fit . There was no interaction between paternal birth season and paternal birth cohort  and no first-order effect of paternal birth season (p = 0.75).We analysed birth lengths (mean ± s.d. = 49.27 ± 30.20 kg) of 637 babies born to 195 fathers . Model comparison showed that overall, inclusion of paternal birth season terms did not improve model fit ((c)p = 0.0034, see the electronic supplementary material, table S7). As was the case with birth weight, maternal birth season interacted with maternal birth cohort (p = 0.0009 (c) and post hoc analysis revealed that the best model fit was obtained when all three cohort levels were retained . Assessing the significance of maternal birth season in each cohort revealed no effect in the before-1975 cohort (p = 0.14) but effects in the 1975–1984 cohort (p = 0.041) and the after-1984 cohort (p = 0.0022). In the 1975–1984 cohort, a peak of 34.80 cm and a trough of 34.24 cm were predicted for 29 April and 26 October, respectively, while in the after-1984 cohort a peak of 35.72 cm and a trough of 33.79 cm were predicted for 15 October and 14 April. Maternal birth seasonality remained significant even after including in the models the potential mediators maternal infancy weight, maternal adult height and offspring gestational age (p = 0.0038) indicating that neither maternal size, gestation length nor the combination of these factors accounted for the maternal birth season patterns .We analysed birth head circumferences (mean ± s.d. = 34.02 ± 2.54 cm) of 1545 babies born to 512 mothers . Exclusion of all maternal birth season terms reduced model fit overall . There was no significant interaction between paternal birth season and paternal birth cohort  and no first-order effect of paternal birth season (p = 0.96).We analysed birth head circumferences (mean ± s.d. = 34.16 ± 16.46 cm) of 757 babies born to 212 fathers . Model comparison showed that overall, inclusion of paternal birth season terms did not improve model fit ((d)3, mean ± s.d. = 2.48 ± 0.50) of 1065 babies born to 445 mothers . Exclusion of maternal birth season terms did not reduce model fit (p = 0.89), indicating that maternal birth season terms overall did not modify offspring ponderal index . As was the case with birth length, there were no improvements to model fit as a result of the interaction of maternal birth season with maternal cohort (p = 0.69), or the first-order effect of maternal birth season (p = 0.97). The non-significant seasonal pattern for each of the three cohorts is shown in figure 1d.We analysed birth ponderal index  of 619 babies born to 193 fathers . Model comparison showed that overall, inclusion of paternal birth season terms did not improve model fit . Specific inclusion of the interaction between paternal birth season and paternal birth cohort did not significantly improve model fit  and there was no first-order effect of paternal birth season (p = 0.12).We analysed birth ponderal index , 0.43 ± 0.31, p = 0.17 (fathers), see electronic supplementary material, tables S7 and S8). It is therefore entirely possible that comparable effects of paternal birth season do exist in recent cohorts, but insufficient data currently exist to detect this.To our knowledge, our study is the first in humans to examine simultaneously both maternal and paternal putative transgenerational effects of early life environment on offspring outcomes. We did not find any significant relationship between paternal birth season and offspring outcomes. However, because of differences in the distributions of paternal versus maternal birth years, we cannot determine whether or not the birth season effects might be apparent in the offspring of men truly contemporaneous to those women whose offspring displayed an intergenerational effect of maternal birth year. Paternity data are less complete than maternity data, and data are available on fewer offspring sired by men born since 1975 than born to women born over the same period. Some of the coefficients for the interaction between paternal birth season and paternal birth cohort are comparable to significant terms that correspond to mothers  allow effects of early birth environment to be examined [The observation that the environment experienced by women during their own early life can affect the intrauterine growth of their offspring adds to the findings from studies of long-term effects of early life exposure the Dutch hunger winter on offspring anthropometrics ,18,18–21examined ,37,42 wiexamined ,48 and  were 49 per cent, 50 per cent and 54 per cent, respectively. In addition, there was no evidence that maternal heterogeneity, as indexed by anthropometric traits, accounted for the patterns among offspring outcome, as might be expected if it were the case that these effects were caused by selection.The fact that the apparent effects of maternal birth season were not mediated by a mother's own birth/neonatal weight or her adult height indicates that the second generation foetal growth pattern might not be the direct long-term consequence of a first generation growth restriction, as has been found previously ,25,26. HGiven that foetal growth is a complex trait determined by both maternal and foetal characteristic, the effects of maternal birth season described here could be either due to epigenetic programming of maternal somatic or gametic cells, or both. If the results observed in this study are owing to environmental effects on methylation, then the window of exposure that is involved in first generation effects on foetal growth is likely different from that which is relevant for second-generation effects. This is because while any first generation effects are probably owing to constrained third trimester growth during the hungry season ,37, methInterpretation of the inversion of the pattern of the effects of maternal birth season presents another challenge. It is possible that the explanation lies in a combination of different environmental factors. Rates of infection  , exposurin utero growth, with potentially long-term consequences even in rural populations far from starvation. The unexpected emergence of these intergenerational effects of birth season in recent years suggests that early environmental effects can be apparent despite, or perhaps because of, substantial reduction in child mortality. The persistence of these relationships when controlling for measures of maternal size indicate that the mechanisms behind intergenerational effects of maternal early conditions may not be found in a simple model of maternal physical or physiological constraint and point to the need to account for other aspects of maternal phenotype associated with offspring growth such as pelvic dimensions, physiological parameters like insulin metabolism, glucocorticoid levels, hypertension or epigenetic inheritance [To conclude, our results support the idea that brief periods of early environmental variation in mothers can affect their offspring eritance ,3,25,26.


The adenomatoid odontogenic tumor (AOT) is a relatively uncommon lesion which mainly affects females in their second decade of life, exhibiting predilection for the anterior region of the maxilla. The lesion is usually associated with the crown of an enclosed tooth, most commonly the maxillary canine. In this paper we present a case of adenomatoid odontogenic tumor associated with a dentigerous cyst affecting the left maxilla in a 15-year-old female. We also discuss clinical, radiographic, histopathologic, and therapeutic features of the case. Adenomatoid odontogenic tumor (AOT) is comprised of odontogenic epithelium in a variety of histoarchitectural patterns, embedded in a mature connective tissue stroma and characterized by slow but progressive growth. In the WHO classification of 2005, AOT is included under “odontogenic epithelium with mature, fibrous stroma without odontogenic ectomesenchyme” . The age We hereby present the first case of AOT that originated in the wall of a dentigerous cyst of the maxilla in pediatric age group in the Indian subcontinent.A 15-year-old Indian female was referred by her general practitioner for evaluation of a maxillary swelling to Department of Oral and Maxillofacial Surgery, Career Post Graduate Institute of Dental Sciences & Hospital, Lucknow. The medical history was insignificant. The patient was asymptomatic and in good general health. Intraoral examination disclosed a nontender expansion of the left maxilla with missing canine tooth. Covering mucosa appeared normal Figures . The patThe differential diagnosis of dentigerous cyst, calcifying odontogenic cyst, calcifying epithelial tumor, keratocystic odontogenic tumor, and unicystic ameloblastoma was made. The surgical gross measured 4.5 cm × 2.5 cm × 4 cm with a smooth surface and was associated with the canine . No calcAOT was first described by Steensland in 1905 . HoweverRick has reported AOT to occur with many types of cysts and neoplasms including dentigerous cyst, calcifying odontogenic cyst, odontoma, and ameloblastoma. In relation with a dentigerous cyst the AOT may demonstrate, grossly and microscopically, one or more associated cystic cavities. Some of these cysts are lined by nonkeratinized stratified squamous epithelium which is similar to the lining of the dentigerous cyst or lined by less structured membrane that may demonstrate bud-like extensions into the connective tissue . In our In our case, AOT and dentigerous cyst are found in the same lesion. Clinical, radiographic, and macroscopic findings in the present case are consistent with descriptions of the lesion in the dental literature . AOT is The structure of the cyst and its insertion around the crown of an unerupted tooth were typical of a dentigerous cyst. Odontogenesis is a complex process and neoplastic or hamartomatous lesions can occur at any stage of odontogenesis. The secondary development of an ameloblastic proliferation, whether hyperplastic or neoplastic, is well known but remains controversial. In this case, the multifocal cellular proliferation had the structure of an AOT. Its mural development in a dentigerous cyst is not uncommon. The tumor is well encapsulated and shows an identical benign behavior. Therefore, conservative surgical enucleation produces excellent outcome without recurrence. Very few case reports of AOT arising from a dentigerous cyst with histological identification have previously been reported. The present case leaves us with an ambiguity whether we should consider it as an odontogenic cyst, containing both epithelial and mesenchymal components or a hybrid tumor.