Advances in neonatal care allow survival of extremely premature infants, who are at risk of handicap. Neurodevelopmental follow up of these infants is an essential part of ongoing evaluation of neonatal care. The neonatal care in resource limited developing countries is very different to that in first world settings. Follow up data from developing countries is essential; it is not appropriate to extrapolate data from units in developed countries. This study provides follow up data on a population of very low birth weight (VLBW) infants in Johannesburg, South Africa.The study sample included all VLBW infants born between 01/06/2006 and 28/02/2007 and discharged from the neonatal unit at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH). Bayley Scales of Infant and Toddler Development Version 111 (BSID) 111 were done to assess development. Regression analysis was done to determine factors associated with poor outcome.178 infants were discharged, 26 were not available for follow up, 9 of the remaining 152 (5.9%) died before an assessment was done; 106 of the remaining 143 (74.1%) had a BSID 111 assessment. These 106 patients form the study sample; mean birth weight and mean gestational age was 1182 grams (SD: 197.78) and 30.81 weeks (SD: 2.67) respectively. The BSID (111) was done at a median age of 16.48 months. The mean cognitive subscale was 88.6 (95% CI: 85.69 - 91.59), 9 (8.5%) were < 70, mean language subscale was 87.71 (95% CI: 84.85 - 90.56), 10 (9.4%) < 70, and mean motor subscale was 90.05 (95% CI: 87.0 - 93.11), 8 (7.6%) < 70. Approximately one third of infants were identified as being at risk (score between 70 and 85) on each subscale. Cerebral palsy was diagnosed in 4 (3.7%) of babies. Factors associated with poor outcome included cystic periventricular leukomalacia (PVL), resuscitation at birth, maternal parity, prolonged hospitalisation and duration of supplemental oxygen. PVL was associated with poor outcome on all three subscales. Birth weight and gestational age were not predictive of neurodevelopmental outcome.Although the neurodevelopmental outcome of this group of VLBW infants was within the normal range, with a low incidence of cerebral palsy, these results may reflect the low survival of babies with a birth weight below 900 grams. In addition, mean subscale scores were low and one third of the babies were identified as "at risk", indicating that this group of babies warrants long-term follow up into school going age. Advances in neonatal care allow survival of extremely preterm infants, who are prone to a range of long term complications in comparison to their term counterparts -4. TheseOngoing long term neurodevelopmental follow up of preterm infants and current outcome data , with anThe aim of this study was to determine developmental outcome in a cohort of VLBW infants at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH), as measured by the Bayley Scales of Infant and Toddler Development (third edition) (BSID 111)  and to dAll VLBW infants born between 2006/06/01 and 2007/02/28 treated at the CMJAH neonatal unit who survived to discharge were invited to participate in the follow up study. The following infants were excluded: babies who were transferred to step-down facilities before discharge, those whose parents were relocating to other areas and would not be available for follow up at CMJAH, babies who were put up for adoption and those whose parents refused consent. The study was approved by the Ethics Committee of the University of the Witwatersrand for Research on Human Subjects. Written informed consent was obtained from each baby's parents before being entered into the study.All babies were admitted into the neonatal unit of CMJAH and care was according to standard protocols. Resuscitation at birth refers to the need for bag mask ventilation with or without chest compressions. Gestational age was assessed by attending staff using a combination of maternal history and Ballard scoring. The need for nasal continuous positive airways pressure (NCPAP), surfactant therapy and intubation with mechanical ventilation was at the discretion of the attending physician. Intermittent positive pressure ventilation (IPPV) was used. High frequency oscillation and jet ventilation were not available in the unit at the time. Ventilatory support, including NCPAP, with or without surfactant therapy, was only given to babies with a birth weight above 900 grams. Babies below 900 grams were given all other care, but not ventilation. This cutoff for ventilation is determined by limited health resources and is well established in neonatal practice in South Africa. All infants were resuscitated at birth if needed, regardless of birth weight.Patent ductus arteriousus (PDA) was confirmed on echocardiogram when suspected clinically. Intraventricular haemorrhage (IVH), graded according to Papile , and cysThe hospital records of each patient were reviewed and maternal obstetric information, details of the mode and place of delivery, labour room information and details of the infant's hospital stay were all recorded.Infants were seen by a paediatrician at a dedicated follow up clinic at 3 monthly intervals until a corrected age of 15 to 18 months was attained. For purposes of the study, the chronological age of the baby was corrected for the degree of prematurity, using a gestational age of 40 weeks as term. Children with health and/or developmental problems were followed up for longer and referred to appropriate specialist clinics as needed. Parents/caregivers were provided with a small transport allowance at each follow up visit. Routine follow up included an interval history, systematic examination and growth monitoring at each visit.A major concern was the anticipated failure of patients to return for follow up after one year. Potential reasons for loss to follow up in this population included parents unable to afford repeated absenteeism from work to attend clinic, children sent to rural areas to live with grandparents, financial constraints and the perception that children are well and do not need follow up. For this reason, two developmental assessments were done - one between the ages of 8 and 12 months and another 6 months later.The developmental assessment was done using the BSID 111, by one of two neurodevelopmentally trained physiotherapists (NH/JP) who were blinded to the details of the patient's birth and hospital admission. Inter observer standardization was done between the two testers; a 98% agreement was achieved between the results.Defaulters were contacted both telephonically and by mail to encourage them to return for follow up. The reasons for defaulting and the child's general condition were obtained if possible. If a defaulter returned to the follow up clinic, the developmental assessment was done at that visit.Stata Statistical Software: Release 10. College Station, TX: StataCorpLP). Frequency tabulations and percentages for categorical data such as gender, mode of delivery and HIV status were produced to describe patient characteristics. For continuous data, summary measures such as mean and standard deviation (SD) or 95% confidence intervals for normally distributed data or median and inter-quartile range (IQR) for non-normally distributed data were presented. Cross-tabulations of patient characteristics with each of the abnormal Bayley scales are also presented. The BSID 111 does not have a single composite outcome. There are 3 separate subscales - motor, cognitive and language. An abnormal outcome on each subscale is a score < 70, and those at risk are considered to have a score of between 70 and 85. In line with standard reporting an abnormal score for regression analysis would be considered as a score of ≤ 85. Associations between patient and clinical characteristics with each abnormal outcome were investigated using the Chi-squared test at 5% level of significance.Descriptive statistics and analysis to determine factors associated with developmental outcomes were performed using STATA version 10  died before discharge, 44 14%) were transferred to regional step-down facilities and 178 (56.6%) were discharged home. Details by birth weight category are shown in Table 4% were tThe latest Bayley assessment was done at a median corrected age of 16.48 months (range 8 to 22 months). The overall results for each subscale are shown in Table The mean birth weight and mean gestational age of the study patients was 1182 grams (SD: 197.78) and 30.81 weeks (SD: 2.67) respectively. The median maternal age was 26 years  and the median Apgar score was 8 . The mean duration of hospital stay was 40.28 days (SD: 15.06) and the median duration of intensive care was 6 days . Babies were predominantly black African, the majority of the babies, 61 (58%), were female and 49 (46%) were SGA. Details of delivery, labour room and hospital stay are shown in Table Although most of the cross-tabulations in Table All factors which were significantly associated with poor outcome at univariate level were considered in the multivariable logistic regression. Such factors included gender and blood transfusion for abnormal Bayley cognitive score; 5 minute Apgar score, resuscitation at birth, syphilis results and antepartum haemorrhage for abnormal motor score; and duration of hospital stay, duration on supplemental oxygen and resuscitation at birth for an abnormal language score. For example, at univariate level analysis, babies were more likely to be abnormal on the Bayley motor scale if they were resuscitated at birth  but less likely if their 5 minute Apgar score was more than 6 .Although not significant and could be due to chance, the results of the multiple logistic regression show that female babies were 1.76  times more likely to have an abnormal Bayley cognitive score whereas babies who had blood transfusion were less likely  compared to those who had not although these adjusted results were not significant.The adjusted analysis for the Bayley motor scale showed that resuscitation at birth was not significant . Logistic regression results for language scale showed that duration of hospital stay and resuscitation at birth (p = 0.039) were statistically significant factors. Those who stayed in hospital between 30 to 40 days were 7.41 times  more likely to have an abnormal language score than those who stayed for less than 30 days. Those who were resuscitated at birth  and with a maternal parity of 4  were at higher risk of an abnormal language score than those who were not resuscitated and those with a maternal parity of 1 respectively.Various multiple linear regression analyses which included only factors univariately associated with the outcome variables were performed. Hypothermia (p = 0.007) and PVL (p = 0.044) were significant risk factors for the cognitive score as outcome.For the BSID 111 motor subscale, we found that PVL (p = 0.008) was a significant factor and that duration on oxygen (p = 0.064) was a borderline significant risk factor for BSID 111 motor score. Only PVL was statistically significantly associated with BSID 111 language score (p = 0.014).The present study provides information on neurodevelopmental outcome in a cohort of VLBW infants in Johannesburg, South Africa. The mean BSID 111 scores at a median corrected age of 16.48 months age were within normal limits for the cognitive, motor and language subscales. Furthermore, considering 70 as the cutoff for the BSID 111 score, only 15.1% of infants had an abnormality on a single subscale, while 3.7% were affected in all 3 areas. However, the mean score on each subscale was less than the anticipated population mean of 100. Almost one third of patients were considered to be "at risk", with a BSID 111 score between 70 and 85, indicating that this is a high risk group of children likely to have long term developmental problems who warrant ongoing monitoring and intervention. Currently long term follow up of ex preterm infants in developing countries is frequently limited to those with obvious handicap due to insufficient resources.This group of VLBW infants represents a select group, as ventilatory support was not offered to infants with a birth weight less than 900 grams, resulting in a very low survival of infants of lower birth weight. Although other neonatal units in developing countries may not apply strict birth weight cut offs for ventilation, or the birth weight cut offs may be different, the challenges of being unable to ventilate all preterm infants who require support will be very similar.The neurodevelopmental outcome results in the present study are similar to those of Cooper and Sandler  at ChrisIn the present study, cystic PVL was associated with poor cognitive, motor and language function. Further, duration of supplemental oxygen, prolonged hospitalisation, resuscitation at birth and increased maternal parity were associated with poor outcome. Duration of intensive care showed a trend towards worse outcome. These findings were similar to those reported in other studies where NEC -28, gendThe rate of cerebral palsy (CP) is of great importance when considering the outcome of preterm infants. The reported number of CP cases in the present study is low (3.7%). This could be an underestimation of the true rate of cerebral palsy in this population as the age of assessment in the present study is too low to reliably report on the rate of cerebral palsy. The diagnosis of cerebral palsy in many children can only be made reliably after the age of 2 years . A signiAlthough the rate of follow up achieved of 74.6% is acceptable and comparable to other reports, it is possible that a number of handicapped children were lost this way. The most common reason for non compliance was relocation of the parents to their place of origin, some as far afield as Malawi and Tanzania. Parents also return to work and find it difficult to bring their children for follow up after the first few visits. Transport and hospital strikes also resulted in the loss to follow up of some patients. Five patients returned to follow up on the incorrect day when a physiotherapist was unavailable, so did not have a Bayley assessment; these children were all clinically normal.The BSID 111 has been tested on black South African children, between 0 and 18 months of age, who did not have risk factors or pre-existing conditions. The results showed that these children performed well and were often above average , confirming that the BSID 111 is suitable for use in line with previous studies in this population ,52.Another limitation of the study is that children had assessments done at different ages for the reasons outlined above. Ideally all assessments should have been done at the same age on all patients. As previously noted, the age of assessment influences the result obtained in the BSID scores. For this reason and high missing data on follow-up visits, we reported the latest BSID 111 in each patient as the outcome variable and did not conduct a repeated measures analysis, which would have been ideal.This study provides neurodevelopmental outcome data in a group of VLBW infants in Johannesburg, South Africa. The prevalence of cerebral palsy and severe handicap is low and is similar to that reported from other developing countries. However, this low rate of handicap may reflect the low survival rate of infants with a birth weight below 900 grams. Also, the mean scores on each of the BSID 111 subscales although within normal limits, were relatively low and one third of the patients were identified as being at risk of developmental problems, with a BSID 111 score of between 70 and 85. Thus, VLBW infants in this setting are a high risk group of patients likely to have learning and other difficulties at school going age and warrant long-term follow up.The following abbreviations are found in the article: BSID 111: Bayley Scales of Infant and Toddler Development (Version 111); CMJAH: Charlotte Maxeke Johannesburg Academic Hospital; CP- cerebral palsy; ELBW: Extremely low birth weight (≤ 1000 grams); IPPV: intermittent positive pressure ventilation; KMC: kangaroo mother care; NCPAP: nasal continuous positive airways pressure; NEC: Necrotising enterocolitis; PDA: patent ductus arteriosus; PVL: Cystic periventricular leukomalacia; VLBW: Very low birth weight (≤ 1500 grams)The authors declare that they have no competing interests.DEB was the main researcher, conceptualized and conducted the follow up study, collated the data and wrote up the manuscript. JP advised on the study design, conducted the BSID 111 on the patients and reviewed the manuscript. TC conducted the statistical analysis and reviewed the manuscript. NH conducted the BSID 111 and reviewed the manuscript. PC assisted with the study design, follow up of patients and review of manuscript. The final submission of the manuscript was approved by all authors.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/12/11/prepub


Varanus) are known from diagnostic remains beginning in the early Miocene (Varanus rusingensis), although extremely fragmentary remains have been suggested as indicating earlier Varanus. The paleobiogeographic history of Varanus and timing for origin of its gigantism remain uncertain.Varanidae is a clade of tiny  to giant (>600 mm PCL) lizards first appearing in the Cretaceous. True monitor lizards  of Samos, Greece is described. The holotype consists of a partial skull roof, right side of a braincase, partial posterior mandible, fragment of clavicle, and parts of six vertebrae. A cladistic analysis including 83 taxa coded for 5733 molecular and 489 morphological characters (71 previously unincluded) demonstrates that the new fossil is a nested member of an otherwise exclusively East Asian Varanus clade. The new species is the earliest-known giant (>600 mm PCL) terrestrial lizard. Importantly, this species co-existed with a diverse continental mammalian fauna.A new Varanus includes, by far, the largest limbed squamates today. The only extant non-snake squamates that approach monitors in maximum size are the glass-snake Pseudopus and the worm-lizard Amphisbaena. Mosasauroids were larger, but exclusively marine, and occurred only during the Late Cretaceous. Large, extant, non-Varanus, lizards are limbless and/or largely isolated from mammalian competitors. By contrast, our new Varanus achieved gigantism in a continental environment populated by diverse eutherian mammal competitors.The new monitor is larger (longer) than 99% of known fossil and living lizards.  Varanus komodoensis), and are famous for reaching large body sizes, the earliest monitors were small- to medium-sized lizards  Estesia mongoliensis and Palaeosaniwa canadensis) Saniwa ensidensVaranus priscus) Monitor lizards (Varanidae) are known to have originated by the Late Cretaceous Varanus) species are known from across Africa, Eurasia, and Australia Varanus fossil record is sparse and most fossil species are represented only by isolated vertebrae More than 70 extant true monitor lizard  known for its mammalian diversity and its combination of European, Asian, and African faunal influences In addition to being diagnostic as a new species, the new taxon is a nested member of an otherwise exclusively Asian AMNH, American Museum of Natural History ; BMNH PR, Natural History Museum, London ; GM, Geiseltal Museum of the Martin-Luther Universität in Halle/Saale (Germany); UF, University of Florida, Florida State Museum .PLoS One, 185 Berry Street, Suite 3100, San Francisco, CA 94107, USA along with a check for $10 (to cover printing and postage) payable to “Public Library of Science.”The electronic version of this document does not represent a published work according to the International Code of Zoological Nomenclature (ICZN), and hence the nomenclatural acts contained in the electronic version are not available under that Code from the electronic edition. Therefore, a separate edition of this document was produced by a method that assures numerous identical and durable copies, and those copies were simultaneously obtainable (from the publication date noted on the first page of this article) for the purpose of providing a public and permanent scientific record, in accordance with Article 8.1 of the Code. The separate print-only edition is available on request from PLoS by sending a request to http://zoobank.org/. The LSID for this publication is urn:lsid:zoobank.org:pub:2E46BE4B-A001-4879-BD9D-4F203743D1DB.In addition, this published work and the nomenclatural acts it contains have been registered in ZooBank, the proposed online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix Clade names follow recent phylogenetic definitions Squamata Oppel 1811Anguimorpha Fürbringer 1900Varanidae Gray 1827Varanus White 1790Varanus (Varaneades) amnhophilis, gen. et sp. nov.Varaneades) subgen. nov.(ZooBank LSID urn:lsid:zoobank.org:act:FCD0FAC3-D8C5-4308-912D-7B0DBA0C8837Varanus (Varaneades) amnhophilis sp. nov.ZooBank LSID urn:lsid:zoobank.org:act:65B2D84B-B7FB-48A0-80F2-5E268C6E1E5DAMNH FR 30630; right side of braincase , right quadrate, partial right coronoid, partial palate, skull roof fragments, glenoid region of right mandible, a small fragment of the right clavicle, and five-and-a-half presacral vertebrae , 3.Varaneades (subgenus) from “Varanus” modern true monitor lizards, and “Neades” a group of mythical beasts from Samos, Greece. Amnhophilis from Greek “amnos” (“αμνόζ”) or “amnhos,” archaic Greek for “lamb;” and “-philis” (“φιλiζ”), meaning “a lover of.” The name alludes to the propensity of large-bodied Varanus to take mammalian prey. Note, also, that the American Museum of Natural History (AMNH) is the repository for the specimen.Mytilini Formation  of Samos, Greece; dated at 6.9–7.6 million years old Varanus (Varaneades) amnhophilis is diagnosed by the presence of the following combination of apomorphies: Pterygoid teeth absent  is relatively well developed anteriorly and posteriorly, but is very weakly developed at the level of the trigeminal notch. More posteriorly, near the level of the posterior margin of the spheno-occiptial tubercle and extending to a level near the posterior margin of the fenestra ovalis, the prootic crest possesses a pronounced ventrolateral flange. This process, here termed the anterostapedial process, is absent in some odoensis , among ogalensis ), or as igularis ). Becauspresence .Near the posterior base of anterostapedial process, the prootic is developed into a robust paroccipital process that is strongly sutured to, but not fused with, the paroccipital process of the otooccipital . The proSome features of the otic capsule and surrounding anatomical structures are visible on the medial view of the prootic . The medVaranus amnhophilis lacks the posterolateral flanges of the parabasisphenoid . The right basipterygoid process is well preserved and extends anterolaterally as in most squamates. It is narrowest at its base with a gentle distal expansion. The expansive pterygoid facet and faces dorsolaterally.We refer to the compound structure formed by fusion of the dermal parasphenoid and the endochondral basisphenoid as the parabasisphenoid, following some recent usage sphenoid ; a condiVaranusShinisaurus crocodilurusA robust crista sellaris extends directly mediolaterally in anterior view, rather than being dorsally concave. The anterior openings of the abducens, carotid, and Vidian canals are typical of those in other The inferior process of the parabasisphenoid is largely overlaid by the complementary process of the prootic . The proThe right lateral part of the basioccipital is preserved. Half of the parabasisphenoid contact is preserved, as are the contacts with the right prootic, and the lateral and ventral contacts with the right otooccipital . No remnThe fused exoccipital-opisthotic unit is here referred to as the otooccipital, following recent usage The otooccipital constitutes the dorsal part of the posterolateral braincase walls. Anteriorly, it preserves most of the crista interfenestralis, which separates the fenestra ovalus from the occipital recess and demonstrates that this crest possesses the posterolateral expansion described above. The contact between the prootic and the otooccipital is vertical and extends from the spheno-occipital tubercle dorsally to the crista interfenestralis. The crista interfenestralis extends posteriorly and slightly posterodorsally from a point dorsal to the posterior margin of the spheno-occipital tubercle to the paroccipital process .Varanus. As in many Varanus (but not Varanus griseus and Varanus acanthurus), the crista tuberalis possesses a distinct, ventrolateral, paroccipital tuberosity  clade and Varanus flavescens  . The dorosphenes , and theosphenes . The preavescens . The minSaniwa ensidensVaranus exanthematicus and Varanus albigularis (Zygosphenes-zygantra or structures similar to them (pseudozygosphenes) are characteristic of some varanids, such as igularis .Shinisaurus crocodilurus as a representative shinisaur outgroup  were chosen to represent the basal condition in that clade, based on their position in recent phylogenetic analyses Varanus than to Shinisaurus crocodilurus. These taxa included Paravaranus angustifrons, Proplatynotia longirostrata, Lanthanotus borneensis, Cherminotus longifrons, Aiolosaurus oriens, Ovoo gurvel, and the necrosaurs ‘Saniwa’ feisti, Necrosaurus cayluxi, Necrosaurus eucarinatus, Saniwides mongoliensis, and Telmasaurus grangeri. A full list of comparative material can be found with the  In addition to taxa included in the earlier analysis Varanus amnhophilis (AMNH FR 30630) based on additional preparation of the specimen and further study of the material (see the description above). We provide the morphological codings for the matrix below in the  T.N.T: Tree analysis using new technologyWe used MesquiteAfter tree construction and consensusing, we mapped biogeographic data in the computer program Varanus amnhophilis consists of cranial, mandibular, and vertebral elements, along with one pectoral fragment  have relatively short tails compared to their overall lengths, whereas others  have relatively long tails Precaudal lengths (PCL) was used as a proxy for body size because the tails of monitor lizards make up a variable amount of their total body length; that is, some monitors  and length of posterior dorsal vertebrae (DVL). Lateral braincase length is measured as the length from the anterior tip of the basipterygoid process to the posterolateral tip of the paroccipital process. Dorsal vertebra length was measured from the anterior limit of the centrum at the midline  to the posteromedial condylar lip (not extending onto the articular surface of the condyle).Varanus amnhophilis and Varanus priscus . The equation is as follows:For estimating sizes based on dorsal vertebrae, we used the data in measurement of interest * (average PCL for relevant extant taxa/average measurement of interest for relevant extant taxa)For the purposes of our study, we include only non-snake squamates (‘lizards’) for size comparison. Snakes (Serpentes) are, of course, just one of many radiations of ‘lizards’ that show extreme limb reduction and limblessness. However, snakes are much more speciose and diverse than any other single group of limbless squamates. It is noteworthy that snakes include some of the longest extant reptiles—certainly the longest extant squamates, suggesting that they (or some sub-group of them) may have evolutionary innovations causing them to operate under a slightly different set of biological pressures. Although there is the lingering idea that snakes may be closely related to varaniform squamates The shortest tree-length recovered by the analysis had a length of 10,325 steps, consistency index excluding uninformative characters of 0.2614, and a retention index of 0.4550 . The anaBecause we included some very fragmentary taxa, a strict consensus of our analysis recovered little resolution . FollowiSaniwa-Varanus clade exclusive of the Eocene-Oligocene varanids from Egypt  spp. are united to the exclusion of Varanus amnhophilis by the presence of a prootic contribution to the posterior Vidian canal opening and a ventrolaterally expanded paroccipital process tubercle.Our analysis recovers a id teeth  and abseid teeth . The claid teeth  by one msphenoid  and 28 uVaranus” sivalensis is a poorly known taxon represented only by a fragmentary humerus and two vertebrae Varanus and were not included in the current analysis.“Varanus has been suggested based, in part and most recently, on some Egyptian Paleogene fossils attributed to Varanus or its ‘stem’ (i.e. Varaninae) Saniwa-Varanus dichotomy  spp. together constitute the basal-most Varanus radiation and occur in Africa. Varanus griseus is recovered as a phylogenetic intermediate between the Polydaedalus-group and other Varanus, and is known from Africa and western Asia. Other Varanus come from eastern Asia and Australia Varanus as Asia.Many of the most proximal Varanus in which those measurements were available is 17.68 with a standard deviation (SD) of 2.322 . The PCL/BCL for Varanus komodoensis is 16.76 (SD of 0.474) , and for the Indo-Asian A clade it is 18.57 .The average PCL/BCL ratio for all Varanus is 37.84 . The average PCL/DVL for Varanus komodoensis is 39.93  and for the Indo-Asian A clade it is 36.91 .The average PCL/DVL for all measured Varanus amnhophilis is 18 mm; its BCL is 38.38 mm. The DVL of Varanus priscus (AMNH FR 1486) is 53 mm. The BCL of Varanus priscus (BMNH 39965) is estimated at 106 mm based on an incomplete braincase The DVL of Varanus allow us to estimate PCL in our new fossil  based on braincase length, or approximately 664.5 mm (±36.89 mm) based on dorsal vertebra length . However, except for the mosasaurs, which were marine and could reach total lengths of as much as 17 m Varanus , 2.Varanus amnhophilis has a PCL greater than approximately 99% of known, non-snake, squamates ever.Varanus priscus ; dorsal vertebra length = 52.97 mm], we estimate its PCL at 2116 mm (±169.6 mm) . Our metric and the available Varanus priscus data suggest that at least AMNH FR 6304 was much larger than that, but considerably smaller than many historical estimates Varanus exanthematicus may be 151 percent of the mean, large Varanus rudicollis may be 175 percent of the mean, Varanus komodoensis may be 181 percent of the mean, and the largest Varanus salvator salvator measured nearly 225 percent of the mean size for the subspecies Varanus priscus might have exceeded 3000 mm PCL. This is similar to some of the larger body-size estimates for the species. It is also possible that Megalania is a species-complex, not a species We also estimated the size of 69.6 mm) . A recen69.6 mm)  —within two percent of the reported SVL Varanus-Saniwa dichotomy.These metrics are also applicable to proximal Varanus rusingensis (the Rusinga Monitor) and Varanus amnhophilis (the Samos Dragon) are the earliest-known demonstrable Varanus. Varanus amnhophilis offers the first evidence of a Miocene divergence for major Varanus clades beyond the basal dichotomy between the African Polydaedalus and all other true monitors , the Dendrovaranus-Soterosaurus clade, the Australasian clade, the Indo-Asian B clade, the lineage including Varanus griseus, and the Polydaedalus clade.monitors . Given tVaranus across Africa, Arabia, Eastern Europe, and Asia by the late Miocene. This is further supported by fragmentary material of uncertain phylogenetic affinity, such as Varanus ‘cf. bengalensis’, Varanus marathonensis, and others Varanus with subsequent major diversification perhaps occurring in southern Asia.In addition to the morphological diversity suggested by the presence of these clades, we hypothesize a broad distribution for VaranusPolydaedalus clade and all other Varanus. However, our analysis differs from some recent molecular analyses Varanus griseus is not found to be a part of Polydaedalus radiation. Given this hypothesis, the distribution of Varanus griseus requires some further attention. One interpretation would be that Varanus griseus, like all non-Polydaedalus monitors, is of a primarily Asian origin with a later invasion of northern Africa. Aridification during the Miocene, which probably made the Sahara a more significant biogeographic barrier Varanus yemenensis  on the Arabian Peninsula would represent a dispersal across the Red Sea, perhaps at its southern end where Arabia approaches Africa and the Indian Ocean.As with some recent analyses of Varanus is of African origin and subsequently dispersed northward and eastward to its present range, Varanus griseus and its lineage might represent an early emigration. However, this would require complete extirpation of non-Polydaedalus Varanus from Africa, with no remnant of their presence. Our data more strongly support the Eurasian origin for Varanus.An alternate interpretation would be that the out-of-Africa hypothesis is correct. If Varanus lizards alive today  are limbless and have adopted some degree of fossoriality. Cyclura evolved large (if not giant) sizes on isolated islands. This is similar to some of the larger varanids, including Varanus komodoensis, Varanus salvator, and Varanus mabitang.Varanidae originated in the Cretaceous and soon exploited small body-size ecomorphs Varanus is unique in that it contains giant lizards that co-occur with endemic mammalian competitors. Numerous species of Varanus appear alongside marsupials in Australia Varanus (Varanus niloticus and Varanus ornatus) co-occur with numerous eutherian mammals similar to the competitors Varanus amnhophilis would have encountered Boidae and Pythonidae include the longest and heaviest extant squamate species Varanus amnhophilis lived in an environment including mixed forest and open areas with freshwater streams, and possibly strong seasonality including some flooding Varanus amnhophilis belongs  are largely terrestrial, but are often strong climbers and swimmers Geological data from Samos suggest that Varanus amnhophilis, shared its environment with mustelids , hyaenids , and suids (Microstonyx) Varanus eggs. Despite this, Varanus (Varaneades) amnhophilis is the earliest-known giant limbed lizard and the first to establish the upper one-percent of lizard sizes on land.The Samos Dragon, DATASET S1MORPHOLOGICAL CHARACTER-BY-TAXON MATRIX. Here, we include the full morphological data matrix, including the character scorings for those characters described in earlier analyses. Note that some taxa were coded for only molecular characters (see text). Those taxa have a “?” for each coding but are included here for ease of the reader should she or he desire to reproduce our matrix.(DOC)Click here for additional data file.TEXT S1COMPARATIVE MATERIAL. Observations on the following specimens were used for this study. Institutional abbreviations: AMNH, American Museum of Natural History; BSP, Bayerische Staatssammlung für Paläontologie und Geologie; BMNH PR, Natural History Museum, London (Great Britain); GM, Geiseltal Museum of the Martin-Luther Universität in Halle/Saale (Germany); UF, University of Florida, Florida State Museum; ZPAL, Zoological Institute of Paleobiology, Polish Academy of Sciences, Warsaw (Poland).(DOC)Click here for additional data file.TEXT S2CHARACTERS USED IN THE PHYLOGENETIC ANALYSIS. Morphological characters and character-states used in the phylogenetic analysis.(DOC)Click here for additional data file.TEXT S3FULL CONSENSUS TREES FOR THE ANALYSES. Below we include the strict and the Adams consensus results for the results of the TNT Varanus’).(DOC)Click here for additional data file.


Sam68 wild type and knockout mouse testes and comparison of their protein content by mass spectrometry indicated that Sam68 ablation did not cause overall alterations in the CB proteome. Lastly, we found that SAM68 interacts with DROSHA and DICER in secondary spermatocytes and early round spermatids and that a subset of miRNAs were altered in −/−Sam68germ cells. These results suggest a novel role for SAM68 in the miRNA pathway during spermatogenesis.The chromatoid body (CB) is a unique structure of male germ cells composed of thin filaments that condense into a perinuclear organelle after meiosis. Due to the presence of proteins involved in different steps of RNA metabolism and of different classes of RNAs, including microRNAs (miRNAs), the CB has been recently suggested to function as an RNA processing centre. Herein, we show that the RNA binding protein SAM68 transiently localizes in the CB, in concomitance with the meiotic divisions of mouse spermatocytes. Precise staging of the seminiferous tubules and co-localization studies with MVH and MILI, two well recognized CB markers, documented that SAM68 transiently associates with the CB in secondary spermatocytes and early round spermatids. Furthermore, although SAM68 co-immunoprecipitated with MVH in secondary spermatocytes, its ablation did not affect the proper localization of MVH in the CB. On the other hand, ablation of the CB constitutive component MIWI did not impair association of SAM68 with the CB. Isolation of CBs from  Mvh, Miwi, Grth, Tdrd6 and Tdrd7 in mice impairs spermatogenesis and male fertility Miwi, Grth, Tdrd6 and Tdrd7 knockout germ cells exhibits morphological abnormalities Mvh knockout mice prevented this analysis Spermatogenesis is characterized by a complex regulation of gene expression. Male germ cells have to face an extended period of silencing of the genome, which occurs during meiotic homologous recombination and during morphological differentiation of haploid spermatids into spermatozoa −/−Sam68 male germ cells. Together with the observation that SAM68 interacts with DROSHA and DICER, these results suggest a novel role for SAM68 in CB-linked RNA processing events and in the miRNA pathway during spermatogenesis.Pioneer studies using cell labelling and histochemistry had already suggested the presence of RNA and ribonucleoproteins in the CB few decades ago SAM68 shuttles between nucleus and cytoplasm in differentiating germ cells SAM68 was reported to accumulate in cytoplasmic stress granules upon several cellular stresses These results indicate that SAM68 transiently accumulates in perinuclear granules during the meiotic divisions and the early phases of spermiogenesis.To test whether the SAM68-containing granule is the CB, we co-stained purified male germ cells with anti-SAM68 and anti-MVH antibodies or anti-MILI antibodies. SAM68 and MVH localize to different compartments in primary spermatocytes and round spermatids. Namely, SAM68 was found exclusively in the nucleus of meiotic and post-meiotic cells, whereas MVH accumulated in perinuclear structures of primary spermatocytes and in the CB of round spermatids . HoweverTo corroborate the association of SAM68 with CB components, we performed immunoprecipitation experiments from purified germ cells. Extracts obtained from a germ cell fraction enriched in secondary spermatocytes Sam68 wild-type and knockout testes to high-resolution morphological analysis by transmission electron microscopy (EM). We found that the CBs in −/−Sam68 late round spermatids (steps 6–8) were often smaller and/or appeared incorrectly assembled  piRNA band . However, since the Sam68−/− CB samples were less concentrated, it is likely that less abundant CB components could not be identified reliably.The purified CBs from wild type and knockout germ cells were then subjected to analysis by mass spectrometry. The main constituents of the CB are MVH, MIWI, TDRD6, TDRD7, GRTH and PABPC3 These results suggest that SAM68 expression is not essential for the CB structure.Sam68 ablation selectively affected 12 miRNAs, the majority of which (9 out of 12) were upregulated in knockout germ cells  is normal. In late round spermatids (steps 6–8) from knockout testes we often observed smaller and/or incorrectly assembled CBs as compared to those of wild type germ cells. Nevertheless, it is likely that these abnormalities are indirect consequences of the morphological and molecular defects occurring in Sam68−/− haploid germ cells, which ultimately lead to apoptosis (18). This interpretation is also supported by our mass spectrometry analysis of purified CBs. We found that, although less material was obtained from Sam68−/− testes, the main CB protein constituents were all correctly recruited in the absence of SAM68. This unbiased analysis also confirmed that SAM68 was present in the CB purified from wild type germ cells, thus corroborating the immunofluorescence results. Our study also showed that some of the less abundant CB proteins were absent in the CBs of knockout samples, whereas piRNAs were present in lower amounts. However, since a general lower yield in CB material was obtained from the knockout germ cells, we suggest that these differences are mainly attributable to the lower concentration of these proteins than to their selective differential recruitment.SAM68 is a ubiquitously expressed KH-type RBP, whose expression levels and localization vary during cell differentiation in several tissues −/−Sam68 male germ cells. Twelve miRNAs were differentially expressed between wild-type and knockout germ cells. The majority of them were up-regulated, whereas only three miRNAs were down-regulated in −/−Sam68 germ cells. Notably, a similar increase of more than 50 miRNAs was also observed in Tdrd6 knockout testis Several reports have recently linked the CB to components of the miRNA pathway The expression of miRNAs can be regulated at the post-transcriptional level by modulating nuclear and cytoplasmic processing events. For instance, the KH-type splicing regulatory protein KSRP binds to the terminal loop of its target miRNAs, thereby promoting their processing In conclusion, our study identifies SAM68 as a novel CB component that transiently associates with this structure and provide evidence for the involvement of SAM68 in the miRNA pathway during spermatogenesis.Sam68 knockout colony was maintained by crossing Sam68 heterozygous mice. Genotyping of the mice was performed as described previously Sam68 wild-type and knockout mice were used to obtain pachytene spermatocytes, secondary spermatocytes, and round spermatids by elutriation technique as described previously The Mouse germ cells were fixed in 4% paraformaldehyde (PFA) and washed three times in PBS. Cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min and then incubated for 1 h in 0.5% BSA. Cells were washed three times in PBS and incubated for 2 h at room temperature (RT) with antibodies against SAM8 , MVH  or MILI , followed by 1 h incubation with cy3-conjugated anti-mouse IgGs (Alexa Fluor) or FITC-conjugated anti-rabbit IgGs (Alexa Fluor). After washes, slides were mounted with Mowiol reagent (EMD) and analyzed by microscopy at RT as described g for 10 min, pre-cleared for 2 h on protein A–Sepharose beads (Sigma-Aldrich) in the presence of rabbit IgGs (5 μg) and 0.05% BSA. After centrifugation for 1 min at 1000 g, supernatant fractions were incubated with rabbit anti-SAM68, anti-DROSHA (Abcam) or control IgGs (5 μg) for 3 h at 4°C under constant rotation. After washing, the beads were eluted in SDS sample buffer for Western blot analysis with anti-MVH antibody (1∶500).Isolated mouse germ cells were washed in PBS and homogenized in lysis buffer  supplemented with 0.5% Triton X-100. Soluble extracts were separated by centrifugation at 10000 Sam68 wild-type and knockout mice were digested in PBS containing 0.5 mg/ml of collagenase for 60 minutes at room temperature. Digested tubules were filtered through 100 μm filter (BD biosciences) and then centrifuged for 5 minutes at 300 g, 4°C. After two washes in PBS, cells were crosslinked in 0.1% PFA for 20 min. The reaction was stopped by adding 0.25 M glycine, pH 7. Cells were washed in PBS, resuspended in RIPA buffer  and sonicated with Bioruptor UCD-200 sonicator . The cell lysate was centrifuged for 10 minutes at 500 g. The pellet containing the CBs was filtered and resuspended in RIPA buffer and used for immunoprecipitation Testes from adult C57BL6 Small pieces of testis were cut, fixed in 5% glutaraldehyde and treated with a potassium ferrocyanide-osmium fixative. The samples were embedded in epoxy resin , sectioned, stained with 5% uranyl acetate and 5% lead citrate, and visualized on a JEOL 1200 EX transmission electron microscope.Proteins were shortly separated with NuPAGE Novex Gel System (Invitrogen) followed by in-gel digestion with Trypsin (Promega) and the extraction of peptides. The peptides were analyzed with LC-MS/MS using an Agilent 1200 series nanoflow system (Agilent Technologies) connected to a LTQ Orbitrap mass-spectrometer  equipped with a nanoelectrospray ion source .+/+Sam68 (n = 4) and −/−Sam68 (n = 4) mice was extracted kit (QIAGEN). RNA samples were tagged for labeling with Cy3 and Cy5 dyes, respectively using QIAzol lysis reagent and the MIRNeasy mini, by using the miRCURY Labeling Kit (Exiqon) for use with Exiqon miRCURY LNA Array (v 10.0). The array consists of probes for the mature forms of all miRNA present in the miRBase 14.0 release of the miRNA registry. Probes corresponds to 1891 mature miRNAs, each represented twice on the microarray. The locked-nucleic acid miRNA probes are highly sensitive and optimized to minimize cross-hybridization between similar mature miRNAs. Two biological replicates for each sample were analysed. For each sample, 4 μg of total RNA was labeled and hybridized to the microarray. Sample RNA quality control was performed using Bioanalyser2100. Microarray data were normalized using the global Lowess  regression algorithm (Exiqon).RNA from isolated male germ cells  from adult Validation of selected miRNA expression levels was performed using the Taqman® miRNA Expression Assays (Applied Biosystem). RNA was reverse-transcribed using specific miRNA stem-loop primers and the Taqman® miRNA reverse transcription kit (Applied Biosystems). Mature miRNA expression was measured with Taqman® microRNA assays (Applied Biosystems) according to the manufacturer's instructions and normalized for the expression level of the snRNP U6 RNA.


Substantial subnational variation in newborn mortality also exists. Our objective is to measure the extent to which trends and subnational variation in early neonatal mortality reflect differences in the prevalence of risk factors  compared to differences in clinical care.Between 1990 and 2010, the U.S ranking in neonatal mortality slipped from 29Observational study using linked birth and death data for all births in the United States between 1996 and 2006. We examined health service area (HSA) level variation in the expected early neonatal mortality rate, based on gestational age (GA) and birth-weight (BW), and GA-BW adjusted mortality as a proxy for clinical care. We analyzed the relationship between selected health system indicators and GA-BW-adjusted mortality.The early neonatal death (ENND) rate declined 12% between 1996 and 2006 (2.39 to 2.10 per 1000 live births). This occurred despite increases in risk factor prevalence. There was significant HSA-level variation in the expected ENND rate (Rate Ratio: 0.73–1.47) and the GA-BW adjusted rate (Rate ratio: 0.63–1.68). Accounting for preterm volume (defined as <34 weeks), the number of neonatologist and NICU beds, 25.2% and 58.7% of the HSA-level variance in outcomes was explained among all births and very low birth weight babies, respectively.Improvements in mortality could be realized through the expansion or reallocation of clinical neonatal resources, particularly in HSAs with a high volume of preterm births; however, prevention of preterm births and low-birth weight babies has a greater potential to improve newborn survival in the United States. First, the studies reporting risk-adjusted outcomes have largely been restricted to selected populations, e.g. hospitals, regions or states and for selected years. The most recent study to examine risk-adjusted outcomes at the national level over time was conducted using data between 1950 and 1975 The objective of our study is two-fold. The first is to describe trends and subnational variation in early neonatal mortality, the expected early neonatal mortality based on gestational age (GA) and birth weight (BW), and GA-BW adjusted early neonatal mortality in the US between 1996 and 2006. The second is to quantify the relationship between health system indicators and subnational variation in risk-adjusted mortality.We used linked birth and death data from the National Vital Statistics System (NVSS) for the years 1996 to 2006, the latest available year, from the US Centers for Disease Control and Prevention. We used the birth cohort linked data for 1996 to 2003 and created cohort data by linking period-based birth and death data using gestational age, birth weight, year of birth and county of birth for the years 2004 to 2006. We excluded neonates weighing less than 500 g due to differential classification of live-birth status based on weight We purposefully excluded covariates that may be correlated with quality of care, such as socioeconomic status and race. Similarly, the presence of an APGAR score reflects the process of care and thus may be correlated with quality of care. Data on congenital anomalies were also excluded due to inconsistent coding.The primary outcome of the analysis was early neonatal death (ENND), defined as death occurring within 7 days of birth. We assessed subnational variation by Health Services Area (HSA). HSAs are a single county or cluster of contiguous counties which are relatively self-contained with respect to hospital care We developed our first-stage GA-BW adjustment model of ENND estimated using multivariate logistic regression to control for the two primary biological predictors of ENND: gestational age  and birth weight . We also control for temporal trends using 1996 as the reference year. With the large number of observations (44 million), to facilitate computation, we estimated the model coefficients based on a random sub-sample and then applied the estimated coefficients to the full dataset. We used a random sub-sample of 800,000 observations from each year from 1996 to 2006– a total of 8.8 million records. To ensure that our model wasn’t unduly influenced by regions with a high prevalence of premature births, which may result from the transfer of high risk births, we conducted a sensitivity analysis of the coefficients sampling only from HSAs among the lowest two quintiles of ENND.We used the linear prediction to determine the expected early neonatal mortality rate based on the distribution of gestational age and birth weight for each HSA-year. We regressed the variance of expected early neonatal mortality rate among HSAs on year to assess if there was a relationship between risk factor disparity and time. We also used the prediction for each record as a covariate in a second-stage logistic regression model where we include a random effect by HSA, . The random effect quantifies the remaining variation in early neonatal mortality at the HSA-level after accounting for differences in the distribution of gestational age and birth weight, i.e. it represents the GA-BW adjusted mortality rate. This second stage model was run using three sequential years of pooled data across the time period, i.e. 1996–1998, 1997–1999 to 2004–2006. We regressed the variance of the HSA random intercept on year to assess if there was a relationship between variation in clinical care and time.Using the pooled data from 2004 to 2006 , we compared the variances of the random effect in the base case model to models where we include along with the linear prediction, fixed effects for a range of HSA-level covariates, such as the presence of a NICU or the volume of pre-term births . We sepaThere were 44,015,582 births weighing at least 500 grams from 1996 to 2006. Among these births there were 97,914 ENND which accounted for 71.2% of all neonatal deaths. The odds of ENND, after controlling for birth weight and gestational age, has declined steadily over this time period. There was a statistically significant 15% lower odds of early neonatal mortality in 2000 compared to 1996, after controlling for birth weight and gestational age. By 2005–2006, there was a 24% lower odds of ENND compared to 1996 . The obsWe consider spatial patterns to refer to geographic clustering of rates. There were strong spatial patterns in the expected ENND rate . The mapGA-BW adjusted mortality did not exhibit strong spatial patterns . There wSignificant reductions in the odds of ENND were only present in HSA’s with at least 2.39 obstetricians-gynecologists per 1,000 births among all births and more than 1.86 obstetricians-gynecologists per 1,000 births among VLBW births. HSA’s with neonatologists present were associated with a significant reduction in the odds of ENND compared to HSAs without neonatologists. There was evidence of a larger effect with increasing quintiles of neonatologists per birth among very low birth weight babies. Higher volumes of preterm births were associated with a reduced risk of death, however these finding were only statistically significant among those HSAs that also contain NICU facilities.In our multivariate analysis, preterm volume, the number of neonatologists and the number of NICU beds explained 25.2% of the variance in GA-BW adjusted outcomes among all births and 58.7% of the variance among VLBW births.Our findings indicate that part of the reason for the lack of progress in reducing neonatal mortality in the United States compared to other countries is that the prevalence of high-risk births has steadily increased. This has offset improvements through clinical care that reduced GA-BW adjusted early neonatal mortality by about one-quarter between 1996 and 2006. As a result, the observed early neonatal mortality rate over the same period declined by only about one-tenth. We found subnational variation in both the prevalence of high risk births and GA-BW adjusted mortality with the variation in high-risk births but not GA-BW adjusted mortality increasing over time. Health system indicators of obstetric and neonatal care explained around one-quarter and more than one-half of the variation in risk adjusted mortality, for all births and VLBW births, respectively.in utero nutrition could be correlated with both poor early neonatal outcomes as well as a higher prevalence of adult conditions such as type II diabetes, as postulated by the fetal origins hypothesis The reasons for the increasing prevalence and variation in earlier gestational age and lower birth weights are complex. The spatial patterns of high-risk births – with higher prevalence in Southern states such as Mississippi, Georgia, Louisiana, Alabama, North Carolina and South Carolina – mirror other health indicators such as life-expectancy GA-BW adjusted mortality has improved over time, indicating that the quality of obstetrical and neonatal care is improving. Likely mechanisms of improved obstetric and neonatal care include the expanded use of surfactant replacement therapy Consistent with our findings, an earlier national study found that neonatal mortality was not improved with an increased number of neonatologists or NICU beds While the increasing prevalence of preterm birth seems to be driving the decline in US rankings, a review of approximately 2000 studies, found only 2 interventions that were effective in preventing preterm birth, namely smoking cessation and progesterone Effective policy targeted at reducing neonatal mortality will differ depending on the region. Parts of North East Minnesota have a very low expected ENND rate and a GA-BW adjusted rate that is higher than expected. This suggests that policy should target improvements in the quality of care – potentially through the regionalization of birthing facilities. In contrast, in regions in south-eastern Florida, policies targeting smoking cessation, progesterone use for high-risk pregnancies, increases in birth spacing as well as interventions designed to reduce mortality in preterm deliveries are likely to be more effective. Our findings should be interpreted with several limitations in mind. Firstly, our analysis was conducted at a regional not institutional level. This means that while an HSA may contain a NICU, or other resources, we cannot assess whether an individual birth used those resources. Secondly, we were only able to explain a fraction of the variation in GA-BW adjusted mortality. This reflects the fact that we only had relatively crude indicators of quality of care and were not able to capture differences related to factors such as how efficiently resources are being used, training and skills of providers and other aspects of an individual’s process of care. Remaining variation may also be a reflection of differences in the presence of congenital anomalies. The data on congenital anomalies was poorly recorded and inconsistent in the data and we were not able to control for this risk factor. In addition, it’s likely that there are regional differences in the rate of abortion and this will confound our results if pregnancy termination is correlated with unadjusted risk factors for ENND. Finally, as we only had information about risk factors at the time of birth it was not possible to separate out the mortality impacts of obstetrical care versus neonatal care, although we show a stronger relationship between GA-BW adjusted mortality and measures of neonatal care resources compared to obstetric resources.A shift towards higher-risk births has offset gains in the quality of obstetric and neonatal care services. Substantial subnational variation in both mortality related to gestational age and birth weight and GA-BW adjusted mortality remain. Efforts to improve neonatal outcomes should consider the relative contributions of risk factors and quality of care at the local level. At the national level, there is some scope to improve early neonatal outcomes through expansion or reallocation of clinical neonatal resources towards regions with high volumes of preterm infants. There is the potential for more significant improvement in outcomes through risk factor prevention; however, the pathways to achieving this are less clear which highlights the need for further research on interventions to prevent preterm birth and low birth weight babies.Table S1Random intercept by State, HSA and County.(XLSX)Click here for additional data file.


We compared the efficacy of microdissection testicular sperm extraction (microdissection TESE) and conventional TESE in patients with non-obstructive azoospermia (NOA) and related the positive sperm recovery to certain variables: follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, testicular volume and histology.Sperm retrieval rates (SRR) in patients with NOA who underwent microdissection TESE (n = 65) or conventional TESE (n = 68) were compared and related to the different variables.SRR by microdissection TESE (56.9%) was significantly higher than conventional TESE (38.2%). There was a positive relation between the SRR and increased testicular volume or decreased FSH levels. No effect of Testosterone or Prolactin levels on SRR by using either technique was observed. Sperm were recovered from those with hypospermatogenesis in 84% and 92.9% by conventional and microdissection TESE, respectively (P = 0.3). In cases of maturation arrest the SRR was 27.3% and 36.4%, respectively (P = 0.6). In cases of Sertoli-cell-only syndrome (SCOS) the SRR was 6.2% and 26.9%, respectively (P = 0.03). No major operative complications occurred in any patient in either group, and no patient required post-operative hormone replacement to treat hypogonadism.Microdissection TESE significantly had twice better probability of success of SRR when compared to conventional TESE. No secure pre-operative prognostic elements of sperm recovery exist for NOA patients. Microdissection TESE appears to be recommendable in cases of atrophied testicles, high FSH concentration, or when SCOS with high FSH concentration can be predicted.Microdissection TESE; Sperm retrieval; Non-obstructive azoospermia; Histopathology; FSH concentration; Orchidometry Non-obstructive azoospermia (NOA) refers to absence of spermatozoa in semen analysis due to minimal or no production of fully developed spermatozoa in the testicles. Etiologies for testicular failure include genetic disorders such as sexual chromosomal abnormalities, translocations and microdeletions of the Y chromosome, cryptorchidism, testicular torsion, radiation and toxins .Approximately 1% of all men and 10% of infertile men are affected by testicular failure as a result of NOA . TesticuDirect vision with the operating microscope in microdissection TESE is of great advantage as larger, more opaque, whitish tubules, presumably containing more germ cells with active spermatogenesis, can be identified. This procedure is currently the best method for the certain identification of sperm, resulting in a high spermatozoa retrieval rate (SRR) and minimal postoperative complications. Histological findings are important in any comparison, since a relationship between SRR and testicular histopathology has been reported in the context of conventional TESE , 7, 8.In our present study we compared SRR by microdissection TESE with that obtained by conventional multiple TESE in patients with histological findings including hypospermatogenesis, maturation arrest (MA), and Sertoli cell-only syndrome (SCOS). Diagnostic biopsy specimens were reviewed in all cases. As far as is known, there are few studies that compare conventional and microdissection TESE and relate the positive sperm recovery to certain variables, FSH and LH concentrations, testicular volume and testicular histology, which are all clinically relevant for NOA patients. We hope our study would add some information to support the previous reports.A consecutive series of 133 men referred with a diagnosis of azoospermia were involved in the current study. All patients were diagnosed with NOA on the basis of a complete history, physical examination, endocrine profile, and chromosomal analysis before being scheduled for TESE with sperm freezing. Those with abnormal karyotyping were excluded from analysis. All patients underwent ejaculated semen examination at least 3 times before surgery. Patients also underwent careful evaluation by urologists concerning the duration of sterility, medical history, sexual function and results of a gynecologic evaluation of the spouse. Ultrasonography was performed to measure testicular volume and to determine the status of the epididymis and testis. Serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were measured by immunoradiometric assay, while testosterone was measured by radioimmunoassay. Patients included in the study were allocated, according to the waiting list on the basis of the general operative theatre plan, after informed consent including explanations about results in the literature and invasiveness of the two procedures. Every operated testicle was classified according to the following variables: (i) testicular volume (V), categorized according to volume ; (ii) FSH concentration, categorized into two groups according to multiples of the normal range (N) ; (iii) testicular histology based on the most advanced pattern of spermatogenesis present such as hypospermatogenesis, maturation arrest (MA) and Sertoli cell-only syndrome (SCOS). Institutional Review Board (IRB) approval was obtained.Conventional multiple TESE ordinarily was performed under general or local anesthesia. Through a small vertical incision in the median scrotal raphe, the skin, dartos muscle, and tunica vaginalis were opened to expose the tunica albuginea. The tunica albuginea was incised for about 4 mm at the upper pole near the head of the epididymis. If no sperm were seen in the initial sample, subsequent samples were taken from other locations, in the middle of the testis and at the lower pole opposite the rete testis, and subsequently from the contralateral testis. The procedure was terminated when sufficient spermatozoa were retrieved.Microdissection TESE was performed under general anesthesia according to the procedure reported previously . After the tunica albuginea was opened widely along the antimesenteric border, direct examination of the testicular parenchyma was performed under the operating microscope. An attempt was made to identify individual seminiferous tubules that were larger, more opaque and whiter than other tubules in the testicular parenchyma, which were considered to contain spermatozoa. The procedure was terminated when sperm were retrieved or further biopsy was thought likely to jeopardize the blood supply of the testis. If all tubules were seen to have an identical morphological appearance, at least three samples  that were equivalent to those from multiple TESE were obtained. The procedure was terminated when a sufficient volume of spermatozoa had been retrieved for ICSI. At the same time of testicular intervention in both procedures, a small tissue specimen was placed in Bouins solution and sent for histopathological examination.Each sample was placed in a Petri dish filled with 0.5 ml of sperm wash media , minced and shredded using sterile glass slides. Then, each sample was examined immediately by placing a small droplet of dispersed tissue suspension on the slide under a phase microscope using x 200 magnification for the presence of the testicular sperm. Further, all testicular samples for ICSI with 5 ml of media were subjected to centrifugation at 2000 x gravity for 10 minutes with careful, extended examination to determine the presence of even a single sperm after TESE procedure.The results were statistically evaluated using Mann-Whitney and Pearson’s Chi Square tests for comparison of the baseline data. A binary logistic regression was used to assess the adjusted odd ratios for the success of sperm retrieval in both methods of treatment. P-values less than 0.05 were considered statistically significant. All data were analyzed using the Statistical Package for the Social Sciences program .Unexplained nonobstructive azoospermia was diagnosed in 68 men undergoing conventional and in 65 undergoing microdissection TESE. Two patients who underwent microdissection TESE had a history of intensive chemotherapy. Another 4 patients had bilateral undescended testes and 2 each underwent conventional and microdissection TESE. Pre-operative patient characteristics in the two groups including endocrine data and histopathological diagnosis were considered nearly identical .Age did not have significant effect on sperm retrieval in either microdissection or conventional TESE. The success rate of sperm retrieval in patients with NOA was significantly higher for the microdissection than for the conventional TESE procedure  patients with FSH ≥ 2N (24 mIU/mL) ; in 53/94 (56.4%) patients with < 2N (< 24 mIU/mL) . SRR was significantly higher by microdissection TESE for those with FSH ≥ 2N . The testis volume and histology were shown to play a significant but less important role.In addition, major postoperative complications, such as acute epididymitis, scrotal hematoma, and testicular hydrocele, were not seen significantly in this study. Only 5 (7.4%) patients from the conventional group and 3 (4.6%) patients from the microdissection group developed scrotal wall hematoma that resolved shortly during follow-up. In addition, no patient required hormone replacement therapy for treatment of post-operative hypogonadism.The identification of areas in which spermatogenesis still occurs represents the background for the addition of magnification to TESE. Individual seminiferous tubules can be seen under the microscope allowing the identification of larger, whitish and opaque tubules in which spermatogenesis is active in opposition to tubules where no sperm production occurs . This stSome authors compared the results obtained by microdissection and conventional TESE in NOA patients  and repoSeveral studies have compared the two techniques. Okada et al.  in theirWe also investigated the SRR by microdissection TESE compared to that by conventional TESE of each type of testicular histology in patients with NOA. Furthermore, SRR by microdissection TESE for patients with hypospermatogenesis was 92.9%, whereas that by conventional TESE was 84%. We also found SRRs by microdissection TESE for maturation arrest and SCOS were 36.4% and 27.3%, respectively, whereas those by conventional TESE were 26.9%, and 6.2%. Only those of SCOS showed significantly higher SRR by microdissection TESE. Recently, Ramasamy et al.  reportedUnlike these studies, there have been other studies showing no difference in the SRR between the two techniques . There wMicrodissection TESE also avoided such complications as hematoma, fibrosis, and androgen decline, which otherwise might have been caused by conventional TESE in the patients with atrophic testes. We performed microdissection TESE on 17 patients whose testes were atrophic (testis volumes were ≤ 8 mL). Despite this effort, the SRR by microdissection was only 29.4% in this group which was still non-significantly higher than the conventional group . The small number of the cases may influence our statistics workup.Clinically, testicular volume is correlated with spermatogenesis. Testicular volume had been found to have poor predictive value for successful TESE, however, because topographical variations in testicular pathology, independent of testicular volume, can occur . Indeed,Therefore, it can be suggested that the patients with NOA whose testis volumes are lower should be informed about the low SRR with conventional or microdissection TESE. The high amounts of removed testicular tissue may cause testicular insufficiency with a decrease in testosterone levels, especially in the hypoplastic/atrophic testicles. Schlegel  and AmerIn our study, increase in FSH levels showed significant failure of sperm retrieval in general which was more significant in conventional TESE by odds of 0.82 (P < 0.001) versus the odds of 0.96 for microdissection TESE (P = 0.05). Although previous studies revealed a negative correlation between increased FSH levels and the SRR, recent studies showed no significant relation between FSH levels and the SRR . Even inRegarding histopathology, only SCOS significantly affects the SRR of the two procedures. We and other authors found FSH value and the surgical procedure were the two variables significantly predicting positive sperm retrieval . IncreasThe combination of different factors in microdissection TESE  significantly increased the success of SRR.Microdissection TESE was reported to cause significantly fewer acute and chronic complications than conventional procedures based on post-operative ultrasonography . However Microdissection TESE significantly had twice better probability of success rate for sperm retrieval when compared with conventional TESE. All things considered, performing microdissection instead of conventional TESE is still the most effective treatment alternative in terms of high SRR and fewer complications. There was a relation between the SRR and testicular volume and FSH levels. Levels of testosterone or prolactin had no effect on the success rate of sperm retrieval using either method. Microdissection TESE appears to be recommendable especially in cases of atrophied testicles, high FSH concentration, or when SCOS with high FSH concentration can be predicted on the basis of the pre-operative prognostic data.


Objective: The effect of ghrelin on growth of the newborn has long been argued, but not fully clarified. In this study, we aimed to investigate the relationship between ghrelin levels and growth parameters in the first 3 months of life.Methods: The study included 60 babies (27 girls and 33 boys) born at gestational ages between 38-42 weeks. The newborns were divided into three groups according to the Lubchenco curves as: small for gestational age (SGA), appropriate for gestational age (AGA) and large for gestational age (LGA). The relationship between ghrelin levels and growth parameters in the third month was investigated. Results: Ghrelin concentrations were significantly higher in SGA (2.4±2.6 ng/dL) babies than in AGA (1.3±0.9 ng/dL) and LGA (1.0±0.8 ng/dL) babies. The lowest ghrelin levels were in the LGA group. In SGA infants, ghrelin concentrations were inversely correlated with change in weight , change in length , and change in head circumference .Conclusion: The results show that at age 3 months, SGA infants had higher ghrelin levels than AGA and LGA infants. Our findings indicate that ghrelin may be involved in the process of catch-up growth in these infants.Conflict of interest:None declared. Ghrelin is a peptide hormone, which has an effect on growth hormone secretion and energy balance. It also has a central effect on eating behavior and regulation of body weight. When first discovered, it has been considered as a growth hormone secretagogue. In recent publications, attention has been drawn to its effects on appetite and weight regulation . More reGhrelin may be implicated in the stimulation of appetite and growth hormone release in both rodents and humans , 4. HumaRecent studies have reported the potential role of ghrelin in adaptation to intrauterine malnutrition . Lower gThe aim of this study was to investigate the relationship between ghrelin and growth parameters at birth and at age three months in healthy infants.Newborn infants born in GATA Medical Faculty Hospital between July 2005 and March 2006 by normal spontaneous vaginal delivery were enrolled in the study. Gestational age was assessed by maternal last menstrual date and confirmed by Dubowitz scoring . We exclSmall for Gestational Age (SGA): This group included 20 babies with birth weights <10th percentile. Appropriate for Gestational Age (AGA): This group included 20 babies with birth weights between 10th and 90th percentiles.Large for Gestational Age (LGA): This group included 20 babies with birth weights >90th percentile.th minute Apgar scores were ≥8 and their physical examinations were normal. Anthropometric measurements  were made on each infant and blood samples were taken. All newborns included in the study were healthy infants and products of consecutive deliveries. Their mothers had had no remarkable complications during pregnancy. None of the infants had congenital malformations, chromosomal abnormalities, or intrauterine infections. Their 5th session). Informed consent was obtained from each mother before the blood samples were taken from their infant. Birth weight and birth length were obtained from each neonate immediately after birth. The infants were weighted naked on an electronic scale. For length measurements a measuring board  was used. Measurement of head circumference was done on day 2 to allow for resolution of edema and head molding. Head circumference measurements were made by passing a non-stretch measuring tape from the glabella through the occipital protuberance. Approval for the study was obtained from the GATA Medical Faculty Ethics Committee on December 1, 2005 , between 9.00 am and 9.30 am. All samples were assayed together at the end of the study, so measurements of growth were recorded blind to the hormone levels. The samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes. Within 1 hour, the chilled samples were centrifuged, the plasma was removed and stored at -70°C until being assayed. Ghrelin levels were measured with Ghrelin Human EIA kit  by using ELISA (Enzyme-linked immunosorbent assay) method. A Microplate Reader RT-2100 C device was used in the readings.The blood samples  from each infant were taken in the early neonatal period (first 7 days) and in the 3Statistical AnalysisThe data were expressed as means±SDs. The Kolmogorov-Smirnov test was applied separately for the groups to check the normality of the variables. Differences in the means of variables were tested using both parametric and non-parametric tests depending on the distribution of the variables between the groups. The changes in anthropometric measurements  and serum ghrelin levels between baseline values and values obtained at 3 months were separately tested for the three groups using the paired-sample t-test. Pearson’s correlations were used to examine relationships among clinical growth-related parameters and hormone levels. Statistical significance was taken as p<0.05. All statistical analyses were performed using the Statistical Package for Social Sciences .While there were no significant differences between their gestational ages, as expected, significant differences existed between the groups at baseline in their anthropometric indices . GhrelinAt age 3 months, weight gain was significantly higher in the AGA group babies than in the other groups . However, the percentage of weight gain was significantly higher in SGA babies than in LGA babies (124% vs. 64%). The change in ghrelin levels was significantly higher in SGA (2.4±2.6 ng/dL) babies than in AGA (1.3±0.9 ng/dL) and LGA (1.0±0.8 ng/dL) babies. The least change in ghrelin levels was in the LGA group. Ghrelin concentrations were inversely correlated with change in weight , change in length  and change in head circumferences  in the SGA group, but not in the other groups.Ghrelin levels and changes are also depicted in Ghrelin has been proposed to be a hormone involved in pre- and postnatal growth, and its serum levels have been considered of prognostic value for postnatal catch-up growth. In some studies ghrelin levels were reported to be associated with anthropometric parameters , 10. AlsHigh ghrelin concentrations sustained by a continuous infusion have been associated with increased weight and adiposity in mice . HoweverGhrelin levels might be increased to induce appetite and to form a positive energy balance after birth-at a time when energy supply from the placenta ceases and breast-feeding starts. Yokata et al  and FarqThere are reports which indicate that ghrelin levels may be affected by acute changes as well as long-term changes in energy balance. Plasma ghrelin levels vary as a consequence of the adaptive response to these circumstances. Bellone et al  demonstrIn conclusion, our findings indicate that in SGA babies, higher ghrelin levels at birth are associated with faster weight gain in the first 3 months of life.


Trace elements play important nutritional roles in neonates. Our objective was to examine whether there are differences in maternal/neonatal serum trace element concentrations between mature infants and premature infants.P<0.05 were considered statistically significant.During 2012, 44 infants born at National Center for Global Health and Medicine, Tokyo, Japan, were enrolled. Serum samples were collected to measure serum iron, zinc, copper, and selenium concentrations 5 days after birth. Maternal serum samples were obtained before delivery and cord blood was taken at delivery to measure the same trace elements. We compared the results between term group whose birth weight were ≥2500 g and gestational age were ≥37 weeks and premature group whose birth weight were <2500 g or gestational age were <37 weeks. Variables significantly different between two groups were included in linear regression models to identify significant predictors of birth weight. Values of P = 0.001). Maternal serum selenium concentrations were also significantly lower in the mothers of premature group than in the mothers of term group . There were no significant differences in neonatal or maternal iron, zinc, or copper concentrations between two groups. Multivariate linear regression analysis showed that, except for gestational age, only maternal serum selenium was significantly associated with birth weight (P = 0.015).Serum selenium concentrations were lower in premature group than in term group (43.3±7.0 µg/L vs. 52.0±8.9 µg/L, Serum selenium concentrations were lower in premature group and their mothers compared with the term group. The maternal serum selenium concentration was positively correlated with birth weight. These results suggest that maternal serum selenium concentration may influence neonatal birth weight. Although trace elements are an essential nutritional component of humans, their specific roles are still poorly understood, especially in the perinatal period. Several micronutrients are thought to affect the growth of fetuses and infants. Some studies have also examined the effectiveness of micronutrient supplementation during pregnancy. One study in Nepal found that a multiple micronutrient supplement taken during pregnancy resulted in increases in infant weight that were sustained until ≥2 years of age Some studies have examined whether micronutrient interventions during pregnancy can improve birth weight and reduce early infant mortality in developing countries In Japan, very few studies have examined the effects of individual trace elements. Kon et al. reported a correlation between serum iron concentrations and developmental outcomes in preterm infants This study was approved by the Ethics Committee of the National Center for Global Health and Medicine (NCGM), Tokyo, Japan. Participating women were interviewed and tested after providing written informed consent.The study population consisted of 44 neonates who were born at the NGCM  between January and December 2012, forming a retrospective cohort. 44  out of 358 neonates  were enrolled. Because our study protocol required maternal serum samples and aimed to avoid unnecessary blood drawing with the sole purpose of research, only mothers whose blood samples had already been obtained before delivery for some other reasons were regarded eligible. Blood samples were collected from the neonates at 5 days of age to measure serum Fe, Zn, copper (Cu), and selenium (Se) concentrations because all neonates born in Japan are performed blood testing for newborn screening at 5 days of age. Maternal serum samples were obtained before delivery  and cord blood was obtained at delivery. These samples were also used to measure the concentrations of Fe, Zn, Cu, and Se. The mothers underwent medical interviews on admission to determine their age, body mass index (BMI) before pregnancy and before delivery, medical history, smoking status, and alcohol consumption. Birth weight and gestational age at delivery were obtained retrospectively from the medical records. Gestational age was calculated by last menstrual period. The infants and their mothers were divided into a term group  or a premature group .t test or the Mann–Whitney U test was used to compare continuous variables between the two groups. Variables that differed between the two groups at P<0.10 were included in univariate and stepwise multivariate linear regression models to identify independent predictors of birth weight. To exclude the possibility of a false-positive association, multicollinearity of predictors was assessed using the variance inflation factor (VIF), and predictors with VIF>5 were removed from the final model. For linear regression, values of P<0.05 were considered statistically significant.Statistical analyses were performed using SPSS software version 20.0. Fisher's exact test was used to compare categorical variables between the mature and LBWI groups. Student's Of the 44 infants, 14 were classified into premature group. 8 were <2500 g, 13 were <37 weeks gestational age and 2 were intrauterine growth retardation (IUGR). The mean ± standard deviation gestational age was 35.6±2.3 weeks in the premature group and 39.5±1.4 weeks in the term group. The mean birth weight was 2388.1±465.0 g in the premature group and 3043.4±321.2 g in the term group. There were no significant differences in the background characteristics of the mothers. .P = 0.001). However, there were no significant differences in Fe, Zn, or Cu between these infants. Maternal serum Se concentrations were also significantly different between these two groups . The cord blood of term group had slightly higher Se and Cu concentrations compared with cord blood of premature group. The cord blood Se concentration was 52.8±9.7 µg/L in premature group and 59.4±10.0 µg/L in term group (P = 0.071). The cord blood Cu concentration was 28.3±10.0 µg/L in premature group and 36.4±11.9 µg/L in term group (P = 0.056). . , 3, 4P = 0.015). The results of the univariate and multivariate regression analyses are presented in Stepwise linear regression analysis was performed, in which the insignificant independent variables were removed one by one. Other than gestational age, only one variable, maternal Se, was independently associated with birth weight. The regression coefficient was 10.985 . These findings suggest that maternal serum Se concentrations are a predictor of neonatal birth weight, independent of maternal nutritional status.Multiple factors, including maternal nutritional status, can influence birth weight. BMI before pregnancy is often used as a marker of the mother's nutritional status, and can affect the birth weight of infants Our study provides further evidence for a positive association between Se concentrations and neonatal birth weight. If maternal Se concentrations are a potential risk factor for low birth weight, independent of maternal nutritional status, measuring maternal serum Se concentrations may be useful to identify LBWIs or preterm delivery, even in women with a normal BMI.There are some limitations to our study. First, the similar characteristics of the enrolled subjects might be due to the small sample size and the study design. Our database included only 44 infants born in 2012 at a single institute. Although we tried to exclude known confounding factors that may reflect maternal nutritional status, some confounders may remain. Furthermore, we have no robust hypothesis to explain why Se concentrations can affect neonatal birth weight, and very few studies have examined the potential mechanisms. Although it can be inferred that maternal trace elements are transported through the placenta, there were no significant differences in cord blood concentrations of the four trace elements between term infants and premature infants, which may undermine our conclusion. Therefore, we think that determining the mechanism by which maternal Se affects neonatal birth weight is an important topic for future research.Generally speaking, Se is incorporated into proteins to make selenoproteins, including the glutathione peroxidase antioxidant enzymes, thioredoxin reductases, and selenoprotein-P. Most of required Se values in which worldwide difference exist has been determined from the intake believed necessary to maximize the activity of the antioxidant glutathione peroxidase in plasma. Therefore low serum concentration of Se may be considered as a potential indicator of Se deficiency, although the level at which symptoms due to Se deficiency become present has not been obvious yet.Hiten and Paula insisted that recurrent early pregnancy loss has been associated with reduced serum selenium concentrations compared to healthy controls in some studies We found that serum Se concentrations were lower in premature infants than in mature infants. Additionally, maternal serum Se concentrations were positively correlated with neonatal birth weight. However, we found no association between maternal BMI or gestational age and serum Se concentrations. These results suggest that maternal serum Se concentrations may have an effect on neonatal birth weight, and that serum Se concentrations are an independent predictor of neonatal birth weight that is independent of the mother's nutritional status. Further studies are necessary to confirm the generalizability of our results.


The role of the placenta in fetal programming has been recognized as a highly significant, yet often neglected area of study. We investigated placental size in relation to psychopathology, in particular attention deficit hyperactivity disorder (ADHD) symptoms, in children at 8 years of age, and later as adolescents at 16 years.Prospective data were obtained from The Northern Finland Birth Cohort (NFBC) 1986. Placental weight, surface area and birth weight were measured according to standard procedures, within 30 minutes after birth. ADHD symptoms, probable psychiatric disturbance, antisocial disorder and neurotic disorder were assessed at 8 years (n = 8101), and ADHD symptoms were assessed again at 16 years (n = 6607), by teachers and parents respectively. We used logistic regression analyses to investigate the association between placental size and mental health outcomes, and controlled for gestational age, birth weight, socio-demographic factors and medical factors, during gestation. There were significant positive associations between placental size  and mental health problems in boys at 8 and 16 years of age. Increased placental weight was linked with overall probable psychiatric disturbance , antisocial behavior  and ADHD symptoms . No significant associations were detected among girls.Compensatory placental growth may occur in response to prenatal insults. Such overgrowth may affect fetal development, including brain development, and ultimately contribute to psychopathology. Mental health problems, in particular attention deficit hyperactivity disorder (ADHD) symptoms, are a significant cause of functional disability in children and adolescents Here we analysed prospective data from a large, longitudinal cohort to investigate whether characteristics related to placental size – placental weight, surface area and placental-to-birth-weight ratio, are associated with mental health, in particular ADHD symptoms, in childhood and adolescence. We investigated sexual dimorphism, due to well-established sex differences in behavior and the placenta. Besides the higher prevalence of behavioral problems among boys compared to girls The ethics committee of Northern Ostrobotnia Hospital District approved the study, and both parents and adolescents gave written informed consent.Data were obtained from The Northern Finland Birth Cohort (NFBC) 1986, which consists of 9479 children born in Oulu and Lapland provinces, who were studied prospectively from early pregnancy until 16 years of age . ChildrePregnant women were recruited at maternity health centres, at their first antenatal visit , and provided background information using structured self-report questionnaires, which were returned by gestational week 24, if still pregnant. Antenatal clinical and birth outcome data were obtained from maternity health centres and hospital medical records (completed by midwives during pregnancy and at birth), and abstracted onto study forms.After excluding multiple birth children (226 twins and 6 triplets) and those who had not consented use of their data (n = 249), data from 8954 liveborns (4596 boys) were available for analysis . When chWithin 30 minutes following delivery, placentas were washed with water, cleaned from blood clots, and then weighed to the nearest gram. Placental weight included membranes and the umbilical cord, cut approximately 5 cm from the neonate. Whilst spread out on a plane, placental breadth and length were used to measure placental surface area  in centimetres squared. The placental-to-birth-weight ratio was calculated by dividing placental weight by birth weight, and then multiplying by 100 to produce a percentage of placental weight relative to birth weight.Teachers assessed child behavior at the age of 8 years using the Rutter B2 scale th percentile of the distribution of mean scores on each subscale was used as a cut-off point to identify adolescents with ADHD symptoms as probable clinical cases. The cut-off values for the inattention, hyperactive-impulsivity and combined subscales were respectively 0.625, 0.125 and 0.277, previously described in detail At 16 years of age, parents reported adolescents' behavior using the Strengths and Weaknesses of ADHD symptoms and Normal behavior (SWAN) scale 2 [m2]) (included as a continuous variable), gestational weight gain , and smoking during pregnancy .We considered potential confounders which may be related to placental size and mental health outcomes, as indicated by our descriptive analysis as well as the literature. These were gestational age (weeks) 2 increments, respectively. To facilitate clinical interpretation of the results, the main analysis included placental weight and surface area as continuous variables in 100 g and 10 cm2 increments, respectively. All the mental health outcomes were dichotomized, coded as 1 to indicate the presence of mental health problems (and 0 if absent), according to the criteria of each screening instrument. We controlled for gestational age, birth weight, socio-demographic factors , and medical factors .We initially performed correlation and ANOVA analyses to identify any relations between placental size and potential confounders. Logistic regression analyses were used to investigate the association between placental size  and mental health outcomes in children and adolescents. The predictors – the placental characteristics, were included as continuous variables. Placental weight and surface area were initially analysed continuously in 1 g and 1 cmth percentile), 550–719 g (represents 25th–75th percentiles) and >720 g (represents >75th percentile).To investigate non-linear associations, we repeated the analysis with stratified placental data . Furthermore, to assess frequency distributions for descriptive purposes, placental weight was stratified according to its distribution in the study population, such that three categories were formed: <550 g , birth weight , maternal socio-demographic factors (except for education): age , family structure at birth , social class by occupation , and medical factors: parity , pre-pregnancy BMI , gestational weight gain  and smoking during pregnancy . 2) and placental-to-birth-weight ratio, increased placental weight (100 g) was the strongest predictor of mental health problems in boys at both 8 and 16 years of age. Linear analyses indicated that a respective increase of 1 g and 1 cm2 in placental weight and surface area were significantly associated with mental health problems in boys. A 1 g increase in placental weight was associated with probable psychiatric disturbance at 8 years (OR  = 1.001 [95% CI  = 1.000–1.002]) and combined inattention-hyperactivity at 16 years (OR  = 1.002 [95% CI  = 1.000–1.003]), and a 1 cm2 increase in placental surface area was associated with inattention-hyperactivity symptoms at 8 years (OR = 1.002 [95% CI = 1.001–1.004]) and inattention symptoms at 16 years (OR = 1.003 [95% CI = 1.000–1.005]). A 10 g increase in placental weight i.e. within 1 SD from the mean, was positively associated with mental health problems in boys . In girls, no significant associations were detected between placental size and mental health outcomes at either 8 years or 16 years of age, as shown in Both unadjusted and adjusted logistic regression analyses revealed positive associations between placental size  and mental health outcomes in boys at 8 and 16 years of age, as shown in After repeating the analysis using stratified placental data, we did not find evidence for a U-shaped association, as small placental size was unrelated to the mental health outcomes.This is the first study to investigate the relation between placental size and mental health outcomes in children and adolescents. We identified a positive association between placental size  and mental health problems in boys; increased placental size was linked with overall probable psychiatric disturbance, ADHD symptoms and antisocial disorder at 8 years of age, and ADHD symptoms at 16 years, after adjusting for known confounders. It is important to note that the magnitude of the associations were small; however, because psychiatric disorders are highly complex – in terms of both etiology and diagnosis, it is expected that any single variable will contribute a small portion of the variance. Our results suggest that variation in placental size may play a role in the causal pathway leading from prenatal exposures to later mental health problems, but further work is required to determine causality.Placental overgrowth may occur as a compensatory mechanism in response to various maternal prenatal insults Variation in the size of the placenta affects aspects of its function, in particular the ability to transfer nutrients to the fetus via changes in the exchange surface area It is interesting to note that placental size was related to a range of mental health problems  in this study. This may correspond to the understanding that placental size is sensitive to various maternal influences, and thus possibly represents an archive of gestational insults, which could affect the developing brain non-specifically.While there is evidence for a U-shaped relation between placental size and various health outcomes In the present study, large placental size was associated with psychiatric problems only in boys. Male placentas may be more sensitive to prenatal insults, and more readily undergo compensatory growth in response to such disturbances The present study has a number of strengths, including use of prospective data, derived from a large, longitudinal, population-based cohort. Placental measurements were performed according to standard procedures, by medical personnel at the time of birth. Furthermore, mental health outcomes were assessed twice over an 8-year period, using validated screening instruments. Nevertheless, the findings should be viewed in light of the following limitations. First, placental weight included membranes and the umbilical cord, and since these components are not involved in nutrient exchange, this may in particular affect measurement of the placental-to-birth weight ratio. Second, placental size provides only limited insight into the role of the placenta in fetal programming In conclusion, this study shows that placental size was associated with mental health problems in boys during childhood and adolescence. Placental enlargement may occur in response to chronic adverse intrauterine conditions, and could lead to altered fetal brain development, with long-term effects on mental health. Future work is required to determine whether deviation in placental size is causal or lies on the causal pathway linking prenatal exposures to child psychopathology.