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Gene expression in the placenta, assessed by bulk RNA-seq, is a common method to explore placental function. Many rodent studies homogenize the entire placenta, and yet doing so may obscure differences within specific functional regions such as the labyrinth, junctional zone and decidua. Conversely, analysis of the whole placenta could generate apparent differences due to changes in composition (e.g., relative amounts of labyrinth vs junctional zone) rather than differential gene expression. We assess the value of dissecting and separately analysing the labyrinth and junctional zone/decidua by comparing RNA-seq results from the labyrinth, junctional zone/decidua combined, and whole placenta from an experiment examining effects of maternal food restriction and fetal sex in C57BL6/J mice at gestational day 17.5. The number of genes identified as differentially expressed in response to maternal food restriction was substantially higher in the labyrinth (910 genes), than in the junctional zone/decidua (50 genes), which in turn was slightly higher than in the whole placenta (3 genes). Only one gene was differentially expressed in all 3 tissue types, and 20 genes were differentially expressed in both the labyrinth and junctional zone/decidua. The larger number of differentially expressed genes in the labyrinth was due to both larger effect sizes and estimates of effect sizes having smaller standard errors. While dissection to obtain layer-enriched samples is slightly more time-consuming than collection of whole placenta and requires some practice, our results show that layer-enrichment is clearly worth the effort., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: It is hypothesized that male fetuses prioritize growth, resulting in increased mortality, whereas females reduce growth in the presence of adversity. Preeclampsia reflects a chronic condition, in which fetuses have the opportunity to adjust growth. If females reduce their growth in response to preeclampsia, but males attempt to maintain growth at the cost of survival, we predict that differences in birthweight between preeclamptic and non-preeclamptic pregnancies will be greater among females, whereas differences in mortality will be greater among males., Methods: We analysed data from the Centers for Disease Control and Prevention. We compared pregnancies with pregnancy-associated hypertension (PAH) and controls., Results: The difference in birthweight between pregnancies affected by PAH and controls varied by fetal sex and gestational age. Among pregnancies of White individuals, at 34-35 weeks, the difference between PAH and controls was higher among females, as predicted. However, this pattern was reversed earlier in pregnancy and around term. Such variation was not significant in Black pregnancies. In both Black and White pregnancies, early in gestation, males had lower odds of death in PAH pregnancies, but higher odds of death in control pregnancies, counter to our prediction. Later, males had higher odds of death in PAH and controls, although the increased odds of death in males was not higher in PAH pregnancies than in controls. Overall, the difference in birthweight between surviving and non-surviving infants was greater in males than in females, opposite to our prediction., Conclusions: The impact of PAH on birthweight and survival varies widely throughout gestation. Differences in birthweight and survival between male and female PAH and controls are generally not consistent with the hypothesis that males prioritize fetal growth more than females, and that this is a cause of increased mortality in males., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hercus et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Early-life conditions such as prenatal nutrition can have long-term effects on metabolic health, and these effects may differ between males and females. Understanding the biological mechanisms underlying sex differences in the response to early-life environment will improve interventions, but few such mechanisms have been identified, and there is no overall framework for understanding sex differences. Biological sex differences may be due to chromosomal sex, gonadal sex, or interactions between the two. This review describes approaches to distinguish between the roles of chromosomal and gonadal sex, and summarizes findings regarding sex differences in metabolism. The Four Core Genotypes (FCG) mouse model allows dissociation of the sex chromosome genotype from gonadal type, whereas the XY* mouse model can be used to distinguish effects of X chromosome dosage vs the presence of the Y chromosome. Gonadectomy can be used to distinguish between organizational (permanent) and activational (reversible) effects of sex hormones. Baseline sex differences in a variety of metabolic traits are influenced by both activational and organizational effects of gonadal hormones, as well as sex chromosome complement. Thus far, these approaches have not been widely applied to examine sex-dependent effects of prenatal conditions, although a number of studies have found activational effects of estradiol to be protective against the development of hypertension following early-life adversity. Genes that escape X chromosome inactivation (XCI), such as Kdm5c , contribute to baseline sex-differences in metabolism, while Ogt , another XCI escapee, leads to sex-dependent responses to prenatal maternal stress. Genome-wide approaches to the study of sex differences include mapping genetic loci influencing metabolic traits in a sex-dependent manner. Seeking enrichment for binding sites of hormone receptors among genes showing sexually-dimorphic expression can elucidate the relative roles of hormones. Using the approaches described herein to identify mechanisms underlying sex-dependent effects of early nutrition on metabolic health may enable the identification of fundamental mechanisms and potential interventions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Christians and Reue.)

Background: Premature birth and/or low birthweight have long-lasting effects on cognition. The purpose of the present systematic review is to examine whether the effects of prematurity and/or low birth weight on neurodevelopmental outcomes differ between males and females., Methods: Web of Science, Scopus, and Ovid MEDLINE were searched for studies of humans born premature and/or of low birthweight, where neurodevelopmental phenotypes were measured at 1 year of age or older. Studies must have reported outcomes in such a way that it was possible to assess whether effects were greater in one sex than the other. Risk of bias was assessed using both the Newcastle-Ottawa scale and the National Institutes of Health Quality assessment tool for observational cohort and cross-sectional studies., Results: Seventy-five studies were included for descriptive synthesis, although only 24 presented data in a way that could be extracted for meta-analyses. Meta-analyses found that severe and moderate prematurity/low birthweight impaired cognitive function, and severe prematurity/low birthweight also increased internalizing problem scores. Moderate, but not severe, prematurity/low birthweight significantly increased externalizing problem scores. In no case did effects of prematurity/low birthweight differ between males and females. Heterogeneity among studies was generally high and significant, although age at assessment was not a significant moderator of effect. Descriptive synthesis did not identify an obvious excess or deficiency of male-biased or female-biased effects for any trait category. Individual study quality was generally good, and we found no evidence of publication bias., Conclusions: We found no evidence that the sexes differ in their susceptibility to the effects of severe or moderate prematurity/low birthweight on cognitive function, internalizing traits or externalizing traits. Result heterogeneity tended to be high, but this reflects that one sex is not consistently more affected than the other. Frequently stated generalizations that one sex is more susceptible to prenatal adversity should be re-evaluated., (© 2023. The Author(s).)

Males and females have been proposed to have different prenatal growth strategies, whereby males invest more in fetal growth and less in placental development, leaving them more susceptible to early-life adversity. We tested predictions of this hypothesis using data from the National Collaborative Perinatal Project. Male newborns were heavier than females, but there was no difference in placental weight, adjusting for birthweight. Among infants born prior to 33 weeks, the difference in birthweight between males and females was greater among those who did not survive than among those who did, potentially reflecting a strategy whereby males maintained growth in the face of prenatal insults, while females adjusted growth. However, there was no significant difference in mortality between the sexes. Being born small-for-gestational age or very preterm (prior to 33 weeks) was associated with significantly reduced performance for most of the cognitive traits examined at 7 years, although maternal preeclampsia was associated with reduced performance in fewer traits. Generally, these effects of early-life adversity (poor fetal growth, prematurity, and preeclampsia) did not differ between the sexes. However, analyzing the sexes separately (rather than testing the interaction between sex and adversity) resulted in numerous spurious sex-specific effects, whereby the effect of early-life adversity appeared to be significant in one sex but not the other. Overall, we found little support for the hypothesis that males prioritize growth more than females, and that this makes them more susceptible to early-life adversity. Furthermore, our results show that analyzing the sexes separately, rather than testing the adversity by sex interaction, can be highly misleading.

Fetal sex affects the risk of pregnancy complications and the long-term effects of prenatal environment on health. Some have hypothesized that growth strategies differ between the sexes, whereby males prioritize growth whereas females are more responsive to their environment. This review evaluates the role of the placenta in such strategies, focusing on (1) mechanisms underlying sexual dimorphism in gene expression, (2) the nature and extent of sexual dimorphism in placental gene expression, (3) sexually dimorphic responses to nutrient supply, and (4) sexual dimorphism in morphology and histopathology. The sex chromosomes contribute to sex differences in placental gene expression, and fetal hormones may play a role later in development. Sexually dimorphic placental gene expression may contribute to differences in the prevalence of complications such as preeclampsia, although this link is not clear. Placental responses to nutrient supply frequently show sexual dimorphism, but there is no consistent pattern where one sex is more responsive. There are sex differences in the prevalence of placental histopathologies, and placental changes in pregnancy complications, but also many similarities. Overall, no clear patterns support the hypothesis that females are more responsive to the maternal environment, or that males prioritize growth. While male fetuses are at greater risk of a variety of complications, total prenatal mortality is higher in females, such that males exposed to early insults may be more likely to survive and be observed in studies of adverse outcomes. Going forward, robust statistical approaches to test for sex-dependent effects must be more widely adopted to reduce the incidence of spurious results., (© 2021. Society for Reproductive Investigation.)

Background: Lactation results in substantial maternal bone loss that is recovered following weaning. However, the mechanisms underlying this recovery, and in particular the role of insulin-like growth factor 1 (IGF-I), is not clear. Furthermore, there is little data regarding whether recovery is affected by advanced maternal age., Methods: Using micro-computed tomography, we studied bone recovery following lactation in mice at 2, 5 and 7 months of age. We also investigated the effects of reduced IGF-I availability using mice lacking PAPP-A2, a protease of insulin-like growth factor binding protein 5 (IGFBP-5)., Results: In 2 month old mice, lactation affected femoral trabecular and cortical bone, but only cortical bone showed recovery 3 weeks after weaning. This recovery was not affected by deletion of the Pappa2 gene. The amount of trabecular bone was reduced in 5 and 7 month old mice, and was not further reduced by lactation. However, the recovery of cortical bone was impaired at 5 and 7 months compared with at 2 months., Conclusions: Recovery of the maternal skeleton after lactation is impaired in moderately-aged mice compared with younger mice. Our results may be relevant to the long-term effects of breastfeeding on the maternal skeleton in humans, particularly given the increasing median maternal age at childbearing., Competing Interests: The authors have declared that no competing interests exist.

Deficiency of pregnancy-associated plasma protein-A2 (PAPP-A2), an IGF-1 availability regulator, causes postnatal growth failure and dysregulation of bone size and density. The present study aimed to determine the effects of recombinant murine IGF-1 (rmIGF-1) on bone composition and remodeling in constitutive Pappa2 knock-out (ko/ko) mice. To address this challenge, X-ray diffraction (XRD), attenuated total reflection-fourier transform infra-red (ATR-FTIR) spectroscopy and gene expression analysis of members of the IGF-1 system and bone resorption/formation were performed. Pappa2 ko/ko mice (both sexes) had reduced body and bone length. Male Pappa2 ko/ko mice had specific alterations in bone composition (mineral-to-matrix ratio, carbonate substitution and mineral crystallinity), but not in bone remodeling. In contrast, decreases in collagen maturity and increases in Igfbp3 , osteopontin (resorption) and osteocalcin (formation) characterized the bone of Pappa2 ko/ko females. A single rmIGF-1 administration (0.3 mg/kg) induced short-term changes in bone composition in Pappa2 ko/ko mice (both sexes). rmIGF-1 treatment in Pappa2 ko/ko females also increased collagen maturity, and Igfbp3 , Igfbp5 , Col1a1 and osteopontin expression. In summary, acute IGF-1 treatment modifies bone composition and local IGF-1 response to bone remodeling in mice with Pappa2 deficiency. These effects depend on sex and provide important insights into potential IGF-1 therapy for growth failure and bone loss and repair.

Background: Males and females may experience different effects of early-life adversity on life-long health. One hypothesis is that male foetuses invest more in foetal growth and relatively less in placental growth, and that this makes them susceptible to poor nutrition in utero, particularly if nutrition is reduced part-way through gestation., Objectives: Our objectives were to examine whether (1) food and/ or protein restriction in rats and mice has consistent sex-dependent effects, (2) sex-dependency differs between types of outcomes, and (3) males are more severely affected when restriction starts part-way through gestation., Data Sources: PubMed and Web of Science were searched to identify eligible studies., Study Eligibility Criteria: Eligible studies described controlled experiments that restricted protein or food during gestation in rats or mice, examined physiological traits in offspring from manipulated pregnancies, and tested whether effects differed between males and females., Results: Our search identified 292 articles, of which the full texts of 72 were assessed, and 65 were included for further synthesis. A majority (50) used Wistar or Sprague-Dawley rats and so these were the primary focus. Among studies in which maternal diet was restricted for the duration of gestation, no type of trait was consistently more severely affected in one particular sex, although blood pressure was generally increased in both sexes. Meta-analysis found no difference between sexes in the effect of protein restriction throughout gestation on blood pressure. Among studies restricting food in the latter half of gestation only, there were again few consistent sex-dependent effects, although three studies found blood pressure was increased in males only. Meta-analysis found that food restriction in the second half of gestation increased adult blood pressure in both sexes, with a significantly greater effect in males. Birthweight was consistently reduced in both sexes, a result confirmed by meta-analysis., Conclusions: We found little support for the hypotheses that males are more affected by food and protein restriction, or that effects are particularly severe if nutrition is reduced part-way through gestation. However, less than half of the studies tested for sex by maternal diet interactions to identify sex-dependent effects. As a result, many reported sex-specific effects may be false positives.

Obesity increases the risk of a number of pregnancy complications, potentially due to chronic inflammation. We predicted that an obesogenic high-fat diet (HFD) in mice would create an inflammatory environment that would exacerbate the effects of lipopolysaccharide (LPS), an inflammatory insult, administered during pregnancy. Females were placed on a HFD or a low-fat diet (LFD) prior to mating, injected with 2 µg LPS or control on gestational day 7 and collected on day 14. Treatment with LPS increased the odds that a female thought to be pregnant at injection had no conceptuses at day 14 ( p = 0.024), suggesting that injection with LPS was more likely to induce complete abortion. However, there was no effect of diet on the odds of having no conceptuses at day 14 and no interaction between diet and LPS injection. Diet and LPS injection had no effect on the number of viable fetuses in females still pregnant at day 14. For fetal weight, there was a significant interaction between diet and treatment ( p = 0.017), whereby LPS reduced fetal weight in HFD females but not in LFD females. However, LPS treatment of HFD females reduced fetal weight to that observed in control-injected LFD females. Although LPS increased the odds of abortion, there was little evidence that a HFD exacerbated the effects of LPS.

Prepregnancy obesity associates with adverse reproductive outcomes that impact maternal and fetal health. While obesity-driven mechanisms underlying adverse pregnancy outcomes remain unclear, local uterine immune cells are strong but poorly studied candidates. Uterine immune cells, particularly uterine natural killer cells (uNKs), play central roles in orchestrating developmental events in pregnancy. However, the effect of obesity on uNK biology is poorly understood. Using an obesogenic high-fat/high-sugar diet (HFD) mouse model, we set out to examine the effects of maternal obesity on uNK composition and establishment of the maternal-fetal interface. HFD exposure resulted in weight gain-dependent increases in systemic inflammation and rates of fetal resorption. While HFD did not affect total uNK frequencies, HFD exposure did lead to an increase in natural cytotoxicity receptor-1 expressing uNKs as well as overall uNK activity. Importantly, HFD-associated changes in uNK coincided with impairments in uterine artery remodeling in mid but not late pregnancy. Comparison of uNK mRNA transcripts from control and HFD mice identified HFD-directed changes in genes that play roles in promoting activity/cytotoxicity and vascular biology. Together, this work provides new insight into how obesity may impact uNK processes central to the establishment of the maternal-fetal interface in early and mid pregnancy. Moreover, these findings shed light on the cellular processes affected by maternal obesity that may relate to overall pregnancy health., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Spontaneous abortions, intrauterine growth restriction, and preeclampsia are thought to be caused by defective placentation and are associated with increased risk of adverse outcomes in subsequent pregnancies. However, it is not known whether the recurrence of adverse outcomes is associated with the recurrence of placental pathology. We hypothesized that recurrent maternal vascular malperfusion (MVM) underlies the recurrence of adverse outcomes., Methods: Using data from the National Collaborative Perinatal Project, we assessed the recurrence of pregnancy complications and MVM lesions (N = 3865), associations between a history of spontaneous abortions and MVM lesions or adverse outcomes in subsequent pregnancies (N = 8312), and whether the recurrence of pregnancy complications occurred independently of the presence of MVM lesions., Results: The odds of an MVM lesion were higher for a woman who had had an MVM lesion in a previous pregnancy (aOR = 1.6; 95% CI 1.3-1.9), although this was marginally non-significant after adjusting for covariates such as gestational age, race and BMI. The odds of preeclampsia, a small-for-gestational-age infant, premature delivery and early pregnancy loss were 2.7-5.0 times higher if there had been that same adverse outcome in a previous pregnancy. A history of spontaneous abortions was associated with higher risk of a small-for-gestational-age baby (aOR = 2.4; 95% CI 1.7-3.4) and prematurity (aOR = 5.1; 95% CI 2.3-11.5 for extremely preterm), but not preeclampsia. The recurrence of adverse outcomes was significant when restricting analyses to women without MVM lesions. Similarly, associations between adverse outcomes and previous spontaneous abortions were significant when statistically controlling for the presence of MVM lesions, or excluding pregnancies with MVM lesions., Conclusions: Women with adverse outcomes in one pregnancy are at higher risk of complications in subsequent pregnancies. However, there is significant recurrence of adverse outcomes even in the absence of MVM., Competing Interests: The authors have declared that no competing interests exist.

Background: Maternal nutrition during pregnancy has life-long consequences for offspring. However, the effects of maternal overnutrition and/ or obesity on fetal growth remain poorly understood, e.g., it is not clear why birthweight is increased in some obese pregnancies but not in others. Maternal obesity is frequently studied using rodents on high-fat diets, but effects on fetal growth are inconsistent. The purpose of this review is to identify factors that contribute to reduced or increased fetal growth in rodent models of maternal overnutrition., Methods: We searched Web of Science and screened 2173 abstracts and 328 full texts for studies that fed mice or rats diets providing ~ 45% or ~ 60% calories from fat for 3 weeks or more prior to pregnancy. We identified 36 papers matching the search criteria that reported birthweight or fetal weight., Results: Studies that fed 45% fat diets to mice or 60% fat diets to rats generally did not show effects on fetal growth. Feeding a 45% fat diet to rats generally reduced birth and fetal weight. Feeding mice a 60% fat diet for 4-9 weeks prior to pregnancy tended to increase in fetal growth, whereas feeding this diet for a longer period tended to reduce fetal growth., Conclusions: The high-fat diets used most often with rodents do not closely match Western diets and frequently reduce fetal growth, which is not a typical feature of obese human pregnancies. Adoption of standard protocols that more accurately mimic effects on fetal growth observed in obese human pregnancies will improve translational impact in this field.

Objective: In humans, loss-of-function mutations in the gene encoding pregnancy-associated pregnancy protein-A2 cause short stature and slightly reduced bone density. The goal of this study was to determine the effects of Pappa2 deletion on bone in mice., Design: Pappa2 deletion mice and littermate controls were culled at 10, 19 or 30 weeks of age and femurs were analysed by micro-computed tomography. Serum markers of bone turnover and insulin-like growth factor binding protein 5 (IGFBP-5), a proteolytic target of PAPP-A2, were measured by ELISA., Results: At 10 and 19 weeks of age, Pappa2 deletion mice had slightly reduced trabecular parameters, but by 19 weeks of age, female deletion mice had increased cortical tissue mineral density, and this trait was increased by a small amount in deletion mice of both sexes at 30 weeks. Cortical area fraction was increased in Pappa2 deletion mice at all ages. Deletion of Pappa2 increased circulating IGFBP-5 levels and reduced markers of bone turnover (PINP and TRACP 5b)., Conclusions: PAPP-A2 contributes to the regulation of bone structure and mass in mice, likely through control of IGFBP-5 levels. The net effect of changes in bone formation and resorption depend on sex and age, and differ between trabecular and cortical bone., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: Accelerated placental maturation is considered a sign of maternal vascular malperfusion, and is often interpreted as an adaptive, compensatory response by the placenta. We tested this interpretation by comparing outcomes in pregnancies with and without accelerated maturation., Methods: Using data from the National Collaborative Perinatal Project, we categorized preterm placentas (24-34 weeks, inclusive; 2525 births) by whether they showed placental villous hypermaturation (PVH), i.e., had the appearance of a placenta of 37 weeks or over upon microscopic examination. We assessed whether PVH was associated with maternal race, maternal BMI, fetal sex, type of preterm birth, preeclampsia, signs of infection or inflammation or placental abruption. We also assessed whether placentas showing PVH were associated with improved outcomes in terms of survival, Apgar score, or oxygen use., Results: PVH was more common in preeclamptic pregnancies and less common in pregnancies complicated by placental abruption or showing signs of placental infection or inflammation. Adjusting for potentially confounding factors, PVH was associated with reduced odds of fetal death, death between birth and 120 days of age, low Apgar scores and oxygen use. PVH was also associated with higher birthweights for gestational age. When correcting for the effect of birthweight, the association between PVH and reduced fetal and neonatal death remained significant., Discussion: Accelerated placental maturation, as manifested by PVH, is associated with improved outcomes. Our work therefore supports the hypothesis that accelerated maturation is a compensatory response by the placenta to improve its transport capacity in specific pregnancy complications., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction: The ratio of birthweight to placental weight (BW:PW) is often used as a measure of placental efficiency in humans and animals. However, ratios have properties that are known to lead to spurious results. An alternative approach is the use of residuals from regression, which reflect whether birthweight is higher or lower than expected for a given placental weight, given the population pattern. We hypothesized that biologically meaningful measures of placental efficiency would differ between placentas with and without pathology, and between adverse and normal perinatal and postnatal outcomes., Methods: We examined associations between measures of placental efficiency (BW:PW ratio or residuals) and placental pathology, Apgar scores and infant death using National Collaborative Perinatal Project data (4645 preterm births and 28497 term births)., Results: BW:PW ratios and residuals were significantly lower in placentas showing pathologies including signs of large infarcts or hemorrhage, although many of these differences were small. Low BW:PW ratios and residuals were also associated with low Apgar scores and increased risk of postnatal death. Whereas residuals were lower in term placentas that appeared immature by microscopic examination, the opposite was true for BW:PW ratios., Conclusion: The BW:PW ratio produced an artefact whereby histologically less mature placentas at term appeared to be more "efficient" than mature placentas, illustrating a known problem with the use of ratios. For other traits, residuals generally showed differences between placentas with and without pathology that were as great as those seen with BW:PW ratios, and often showed stronger associations with adverse outcomes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Pregnancy-associated plasma protein-A2 (PAPP-A2) is consistently upregulated in the placentae of pregnancies complicated by preeclampsia and fetal growth restriction. The causes and significance of this upregulation remain unknown, but it has been hypothesized that it is a compensatory response to improve placental growth and development. We predicted that, if the upregulation of PAPP-A2 in pregnancy complications reflects a compensatory response, then deletion of Pappa2 in mice would exacerbate the effects of a gene deletion previously reported to impair placental development: deficiency of matrix metalloproteinase-9 (MMP9)., Methods: We crossed mice carrying deletions in Pappa2 and Mmp9 to produce pregnancies deficient in one, both, or neither of these genes. We measured pregnancy rates, number of conceptuses, fetal and placental growth, and the histological structure of the placenta., Results: We found no evidence of reduced fertility, increased pregnancy loss, or increased fetal demise in Mmp9 -/- females. In pregnancies segregating for Mmp9, Mmp9 -/- fetuses were lighter than their siblings with a functional Mmp9 allele. However, deletion of Pappa2 did not exacerbate or reveal any effects of Mmp9 deficiency. We observed some effects of Pappa2 deletion on placental structure that were independent of Mmp9 deficiency, but no effects on fetal growth. At G16, male fetuses were heavier than female fetuses and had heavier placentae with larger junctional zones and smaller labyrinths., Conclusions: Effects of Mmp9 deficiency were not exacerbated by the deletion of Pappa2. Our results do not provide evidence that upregulation of placental PAPP-A2 represents a mechanism to compensate for impaired fetal growth.

Genomic imprinting is essential for normal placental and fetal growth. One theory to explain the evolution of imprinting is the kinship theory (KT), which predicts that genes that are paternally expressed will promote fetal growth, whereas maternally expressed genes will suppress growth. We investigated the expression of imprinted genes using microarray measurements of expression in term placentae. Correlations between birthweight and the expression levels of imprinted genes were more significant than for non-imprinted genes, but did not tend to be positive for paternally expressed genes and negative for maternally expressed genes. Imprinted genes were more dysregulated in preeclampsia (a disorder associated with placental insufficiency) than randomly selected genes, and we observed an excess of patterns of dysregulation in preeclampsia that would be expected to reduce nutrient allocation to the fetus, given the predictions of the KT. However, we found no evidence of coordinated regulation among these imprinted genes. A few imprinted genes have previously been shown to be associated with fetal growth and preeclampsia, and our results indicate that this is true for a broader set of imprinted genes., (© 2017 The Author(s).)

Background: Maternal and neonatal outcomes are influenced by the nature of antenatal care. Standard pregnancy care is provided on an individual basis, with one-on-one appointments between a client and family doctor, midwife or obstetrician. A novel, group-based antenatal care delivery model was developed in the United States in the 1990s and is growing in popularity beyond the borders of the USA. The purpose of this study was to evaluate outcomes in clients receiving interprofessional group perinatal care versus interprofessional individual care in a Canadian setting., Methods: Clients attending the South Community Birth Program (SCBP), an interprofessional, collaborative, primary care maternity program, offering both individual and group care, were invited to participate in the study. Pregnancy knowledge and satisfaction scores, and perinatal outcomes were compared between those receiving group versus individual care. Chi-square tests, general linear models and logistic regression were used to compare the questionnaire scores and perinatal outcomes between cohorts., Results: Three hundred three clients participated in the study. Group care was comparable to individual care in terms of mode of birth, gestational age at birth, infant birth weight, breastfeeding rates, pregnancy knowledge, preparedness for labour and baby care, and client satisfaction. The rates of adverse perinatal outcomes were extremely low amongst SCBP clients, regardless of the type of care received (preterm birth rates ~5%). Breastfeeding rates were very high amongst all study participants (> 78% exclusive breastfeeding), as were measures of pregnancy knowledge and satisfaction., Conclusions: This is the first Canadian study to compare outcomes in clients receiving interprofessional group care versus individual care. Our observation that interprofessional group care outcomes and satisfaction were as good as interprofessional individual care has important implications for the antenatal care of clients and for addressing the projected maternity provider crisis facing Canada, particularly in small and rural communities. Further study of group-based care including not only client satisfaction, but also provider satisfaction, is needed. In addition, research into the role of interprofessional care in meeting the needs and improving perinatal outcomes of different populations is necessary.

Maternal overnutrition and obesity during pregnancy can have long-term effects on offspring physiology and behaviour. These developmental programming effects may be mediated by fetal exposure to glucocorticoids, which is regulated in part by placental 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 and 2. We tested whether a maternal high-fat, high-sucrose diet would alter expression of placental 11β-HSD1 and 2, thereby increasing fetal exposure to maternal glucocorticoids, with downstream effects on offspring physiology and behaviour. C57BL/6J mice were fed a high-fat, high-sucrose (HFHS) diet or a nutrient-matched low-fat, no-sucrose control diet prior to and during pregnancy and lactation. At day 17 of gestation, HFHS dams had ~20% lower circulating corticosterone levels than controls. Furthermore, there was a significant interaction between maternal diet and fetal sex for circulating corticosterone levels in the fetuses, whereby HFHS males tended to have higher corticosterone than control males, with no effect in female fetuses. However, placental 11β-HSD1 or 11β-HSD2 expression did not differ between diets or show an interaction between diet and sex. To assess potential long-term consequences of this sex-specific effect on fetal corticosterone, we studied locomotor activity and metabolic traits in adult offspring. Despite a sex-specific effect of maternal diet on fetal glucocorticoids, there was little evidence of sex-specific effects on offspring physiology or behaviour, although HFHS offspring of both sexes had higher circulating corticosterone at 9 weeks of age. Our results suggest the existence of as yet unknown mechanisms that mitigate the effects of altered glucocorticoid exposure early in development, making offspring resilient to the potentially negative effects of a HFHS maternal diet.

Background: One hypothesis to explain the blue-green colour of the eggs of many bird species is that it is a sexually-selected signal of the laying female's quality, which males use to determine their investment. This hypothesis requires that eggshell pigmentation carries a cost or is otherwise linked to female quality. One potential cost is that biliverdin, a haem derivative and the pigment responsible for eggshell colouration, is limiting. To assess this potential cost, we attempted to manipulate haematocrit and haemoglobin in free-living European starlings (Sturnus vulgaris Linnaeus). Upon collecting unmanipulated first clutches, we treated females with phenylhydrazine (PHZ), a haemolytic agent, and measured the blue-green chroma and reproductive performance of replacement clutches. We also investigated whether eggshell colour was associated with haematocrit or haemoglobin levels in unmanipulated first clutches. To test whether eggshell colour might act as a sexual signal, we examined associations between eggshell colour and reproductive performance, as well as the provisioning rate of the male., Results: PHZ-treatment did not affect eggshell colour in replacement clutches. In unmanipulated first clutches, eggshell colour was not correlated with haematocrit or haemoglobin levels. Eggshell colour was correlated with female mass in unmanipulated first clutches but not replacement clutches. Chicks from eggs with higher eggshell colour had higher haemoglobin levels and longer tarsi just prior to fledging, suggesting that eggshell colour could reflect brood quality. However, eggshell colour was not correlated with the provisioning rate of the male or any other measure of reproductive performance., Conclusions: We found no evidence to support the hypothesis that the availability of resources required for the synthesis of pigment limits eggshell colour in European starlings, or that eggshell colour is used by males to determine their level of reproductive investment. We found little evidence that eggshell colour is correlated with female or offspring quality in this species.

Proper placental development and function is crucial for a healthy pregnancy, and there has been substantial research to identify markers of placental dysfunction for the early detection of pregnancy complications. Low first-trimester levels of a disintegrin and metalloproteinase 12 (ADAM12) and pregnancy-associated plasma protein-A (PAPP-A) have been consistently associated with the subsequent development of preeclampsia and fetal growth restriction. These molecules are both metalloproteinases secreted by the placenta that cleave insulin-like growth factor binding proteins (IGFBPs), although ADAM12 also has numerous other substrates. Recent work has identified ADAM12, and particularly its shorter variant, ADAM12S, as a regulator of the migration and invasion of trophoblasts into the lining of the uterus, a critical step in normal placental development. While the mechanisms underlying this regulation are not yet clear, they may involve the liberation of heparin-binding EGF-like growth factor (HB-EGF) and/or IGFs from IGFBPs. In contrast, there has been relatively little functional work examining PAPP-A or the IGFBP substrates of ADAM12 and PAPP-A. Understanding the functions of these markers and the mechanisms underlying their association with disease could improve screening strategies and enable the development of new therapeutic interventions.

Objective: Pregnancy associated plasma protein-A2 (PAPP-A2) is a protease that cleaves insulin-like growth factor binding protein-5 (IGFBP-5), the most abundant IGFBP in bone. Deletion of Pappa2 reduces postnatal growth and bone length in mice. The aim of this study was to determine whether locally produced PAPP-A2 is required for normal bone growth., Design: We deleted Pappa2 primarily in osteoblasts by crossing conditional Pappa2 deletion mice with mice expressing Cre recombinase under the control of the Sp7 (Osterix) promoter. Effects of disrupting Pappa2 in Sp7-expressing cells were examined by measuring body mass and tail length at 3, 6, 10 and 12 weeks of age and bone dimensions at 12 weeks., Results: Body mass, tail length, and linear bone dimensions were significantly reduced at all ages by osteoblast-specific Pappa2 deletion. Mice homozygous for the conditional Pappa2 deletion allele and carrying the Cre transgene were smaller than controls carrying the Cre transgene, whereas mice homozygous for the conditional Pappa2 deletion allele were not smaller than controls when comparing mice not carrying the transgene. This result unambiguously demonstrates that PAPP-A2 produced by Sp7 expressing cells is required for normal growth. However, constitutive Pappa2 deletion had greater effects than osteoblast-specific Pappa2 deletion for many traits, indicating that post-natal growth is also affected by other sources of PAPP-A2. Immunohistochemistry revealed that PAPP-A2 localized in the epiphysis and metaphysis as well as osteoblasts, consistent with a role in bone growth., Conclusion: Locally-produced PAPP-A2 is required for normal bone growth., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We examined developmental programming studies that reported sex-specific effects published between 2012 and 2014, and examined whether the authors reported a statistical approach to explicitly test whether the effect of treatment differed between the sexes, for example, a sex by treatment interaction term. Less than half of the studies that reported sex-specific effects described explicitly testing whether effects were indeed sex-specific; in most cases, an effect was considered 'sex-specific' if it was significant in one sex but not the other. This is not a robust approach, since significance in one sex and lack of significance in the other sex does not imply a significant difference between the sexes. However, sample size often limits statistical power to detect interactions. We suggest that if the effect is significant in only one sex, but the interaction term is not significant, alternative solutions would be to present the confidence intervals for the effect size for each sex, or using Bayesian approaches to calculate the probability that the effect sizes differ between the sexes. We present a simple example of a Bayesian analysis to illustrate that this approach is reasonably easy to implement and interpret.

Objective: Insulin-like growth factor binding proteins (IGFBPs) are involved in glucose and lipid metabolism, and their actions are modulated by proteases. The aim of this study was to examine the effects of an IGFBP-5 protease, pregnancy associated plasma protein-A2 (PAPP-A2), on glucose metabolism and susceptibility to diet-induced obesity., Design: Postnatal growth, circulating IGF-I, IGFBP-3 and IGFBP-5 levels, and glucose tolerance were measured in Pappa2 deletion mice and littermate controls on a chow diet. Males were subsequently fed a high-fat diet for 8 weeks to measure weight gain and adiposity, as well as glucose tolerance in response to a metabolic challenge., Results: Circulating IGFBP-5 levels were ~2-fold higher in mice with no functional PAPP-A2 than in littermate controls, as expected. In contrast, circulating IGFBP-3 levels were reduced by ~15-fold, and total IGF-I levels were ~60% higher in Pappa2 deletion mice. There was no effect of Pappa2 deletion on fasting blood glucose levels or glucose clearance after intraperitoneal injection of 2g glucose/kg body weight in mice on a chow diet. In males on a high-fat diet, there was no difference between genotypes in weight gain or adiposity, adjusting for differences in initial body weight, or in fasting blood glucose or insulin levels, or in glucose clearance., Conclusions: Despite a dramatic disruption of the balance between circulating IGF-I, IGFBP-3 and -5, we found no effects of Pappa2 deletion on glucose metabolism, weight gain or adiposity on a high-fat diet., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Recent studies have found associations between the gene encoding pregnancy associated plasma protein-A2 (PAPP-A2), a protease of insulin-like growth factor binding protein -5 (IGFBP-5), and measures of female reproductive performance in cattle. The purpose of the present study was to test the effects of Pappa2 deletion on reproduction in mice., Findings: We measured the fertility and offspring growth of Pappa2 deletion females, and also performed reciprocal matings (i.e., deletion males mated to control females) to control for the effects of offspring genotype. Ovarian and testicular IGFBP-5 levels were measured by Western blotting. As expected, deletion of Pappa2 increased ovarian IGFBP-5 levels. However, Pappa2 deletion in females had no effect on the interval between pairing and the birth of the first litter, the interval between the births of the first and second litters, or litter size. Offspring weight was lower in the offspring of Pappa2 deletion females, but effects of similar magnitude were observed in the offspring of Pappa2 deletion males, suggesting that the effects were due to heterozygosity for the deletion in the offspring. Pappa2 deletion in males had no effect on litter size or the interval between pairing and the birth of the first litter. However, the interval between the births of the first and second litters was significantly longer in deletion males., Conclusions: Pappa2 deletion had no effect on female reproductive performance. In contrast, Pappa2 deletion had subtle effects on male fertility, although the underlying mechanism remains to be elucidated.

Background: Adverse gestational outcomes such as preeclampsia (PE) and intrauterine growth restriction (IUGR) are associated with placental insufficiency. Normal placental development relies on the insulin-like growth factors -I and -II (IGF-I and -II), in part to stimulate trophoblast proliferation and extravillous trophoblast (EVT) migration. The insulin-like growth factor binding proteins (IGFBPs) modulate the bioavailability of IGFs in various ways, including sequestration, potentiation, and/or increase in half-life. The roles of IGFBP-4 and -5 in the placenta are unknown, despite consistent associations between pregnancy complications and the levels of two IGFBP-4 and/or -5 proteases, pregnancy-associated plasma protein -A and -A2 (PAPP-A and PAPP-A2). The primary objective of this study was to elucidate the effects of IGFBP-4 and -5 on IGF-I and IGF-II in a model of EVT migration. A related objective was to determine the timing and location of IGFBP-4 and -5 expression in the placental villi., Methods: We used wound healing assays to examine the effects of IGFBP-4 and -5 on the migration of HTR-8/SVneo cells following 4 hours of serum starvation and 24 hours of treatment. Localization of IGFBP-4, -5 and PAPP-A2 was assessed by immunohistochemical staining of first trimester placental sections., Results: 2 nM IGF-I and -II each increased HTR-8/SVneo cell migration with IGF-I increasing migration significantly more than IGF-II. IGFBP-4 and -5 showed different levels of inhibition against IGF-I. 20 nM IGFBP-4 completely blocked the effects of 2 nM IGF-I, while 20 nM IGFBP-5 significantly reduced the effects of 2 nM IGF-I, but not to control levels. Either 20 nM IGFBP-4 or 20 nM IGFBP-5 completely blocked the effects of 2 nM IGF-II. Immunohistochemistry revealed co-localization of IGFBP-4, IGFBP-5 and PAPP-A2 in the syncytiotrophoblast layer of first trimester placental villi as early as 5 weeks of gestational age., Conclusions: IGFBP-4 and -5 show different levels of inhibition on the migration-stimulating effects of IGF-I and IGF-II, suggesting different roles for PAPP-A and PAPP-A2. Moreover, co-localization of the pappalysins and their substrates within placental villi suggests undescribed roles of these molecules in early placental development.

Recent studies have consistently found pregnancy-associated plasma protein A2 (PAPP-A2) to be upregulated in preeclamptic placentae at term. We tested whether first-trimester circulating PAPP-A2 levels differed between complicated and uncomplicated pregnancies. We measured maternal PAPP-A2 levels at 10 to 14 weeks of gestational age in 17 pregnancies resulting in small-for-gestational-age (SGA) infants, 6 which developed preeclampsia (PE), 1 which developed PE and resulted in an SGA infant, and 37 gestational age-matched controls. The concentration of the PAPP-A2 isoform corresponding to the full-length protein was significantly higher in pregnancies that developed PE (35 ng/mL) compared with those that did not (23 ng/mL; P < .044). In contrast, we found no difference in PAPP-A2 levels between pregnancies that did or did not result in an SGA infant. The upregulation of PAPP-A2 that has previously been observed in PE at term appears to begin early in pregnancy, well before the symptoms develop.

Introduction: Recent evidence from chimpanzees and gorillas has raised doubts that preeclampsia is a uniquely human disease. The deep extravillous trophoblast (EVT) invasion and spiral artery remodeling that characterizes our placenta (and is abnormal in preeclampsia) is shared within great apes, setting Homininae apart from Hylobatidae and Old World Monkeys, which show much shallower trophoblast invasion and limited spiral artery remodeling. We hypothesize that the evolution of a more invasive placenta in the lineage ancestral to the great apes involved positive selection on genes crucial to EVT invasion and spiral artery remodeling. Furthermore, identification of placentally-expressed genes under selection in this lineage may identify novel genes involved in placental development., Methods: We tested for positive selection in approximately 18,000 genes using the ratio of non-synonymous to synonymous amino acid substitution for protein-coding DNA. DAVID Bioinformatics Resources identified biological processes enriched in positively selected genes, including processes related to EVT invasion and spiral artery remodeling., Results: Analyses revealed 295 and 264 genes under significant positive selection on the branches ancestral to Hominidae (Human, Chimp, Gorilla, Orangutan) and Homininae (Human, Chimp, Gorilla), respectively. Gene ontology analysis of these gene sets demonstrated significant enrichments for several functional gene clusters relevant to preeclampsia risk, and sets of placentally-expressed genes that have been linked with preeclampsia and/or trophoblast invasion in other studies., Conclusion: Our study represents a novel approach to the identification of candidate genes and amino acid residues involved in placental pathologies by implicating them in the evolution of highly-invasive placenta., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Pregnancy associated plasma protein A2 (PAPP-A2) is a protease of insulin-like growth factor binding protein 5 and is receiving increasing attention for its roles in pregnancy and postnatal growth. The goals of the present study were to characterize the effects of PAPP-A2 deletion on bone size and shape in mice at 10 weeks of age, and to determine whether Pappa2 is the gene responsible for a previously-identified quantitative trait locus (QTL) contributing to natural variation in postnatal growth in mice. Mice homozygous for constitutive PAPP-A2 deletion were lighter than wild-type littermates, and had smaller mandible dimensions and shorter skull, humerus, femur, tibia, pelvic girdle, and tail bone. Furthermore, PAPP-A2 deletion reduced mandible dimensions and the lengths of the skull, femur, pelvic girdle, and tail bone more than would be expected due to the effect on body mass. In addition to its effects on bone size, PAPP-A2 deficiency also altered the shape of the mandible and pelvic girdle, as assessed by geometric morphometrics. Mice homozygous for the PAPP-A2 deletion had less deep mandibles, and pelvic girdles with a more feminine shape. Using a quantitative complementation test, we confirmed that Pappa2 is responsible for the effects of the previously-identified QTL, demonstrating that natural variation in the Pappa2 gene contributes to variation in postnatal growth in mice. If similar functional variation in the Pappa2 gene exists in other species, effects of this variation on the shape of the pelvic girdle might explain the previously-reported associations between Pappa2 SNPs and developmental dysplasia of the hip in humans, and birthing in cattle.

Infections caused by the fungus Aspergillus are a major cause of morbidity and mortality in immunocompromised populations. To identify genes required for virulence that could be used as targets for novel treatments, we mapped quantitative trait loci (QTL) affecting virulence in the progeny of a cross between two strains of A. nidulans (FGSC strains A4 and A91). We genotyped 61 progeny at 739 single nucleotide polymorphisms (SNP) spread throughout the genome, and constructed a linkage map that was largely consistent with the genomic sequence, with the exception of one potential inversion of ∼527 kb on Chromosome V. The estimated genome size was 3705 cM and the average intermarker spacing was 5.0 cM. The average ratio of physical distance to genetic distance was 8.1 kb/cM, which is similar to previous estimates, and variation in recombination rate was significantly positively correlated with GC content, a pattern seen in other taxa. To map QTL affecting virulence, we measured the ability of each progeny strain to kill model hosts, larvae of the wax moth Galleria mellonella. We detected three QTL affecting in vivo virulence that were distinct from QTL affecting in vitro growth, and mapped the virulence QTL to regions containing 7-24 genes, excluding genes with no sequence variation between the parental strains and genes with only synonymous SNPs. None of the genes in our QTL target regions have been previously associated with virulence in Aspergillus, and almost half of these genes are currently annotated as "hypothetical". This study is the first to map QTL affecting the virulence of a fungal pathogen in an animal host, and our results illustrate the power of this approach to identify a short list of unknown genes for further investigation.

Background: Pregnancy-associated plasma protein A2 (PAPPA2) is an insulin-like growth factor-binding protein (IGFBP) protease expressed at high levels in the placenta and upregulated in pregnancies complicated by preeclampsia and HELLP (Hemolytic anemia, Elevated Liver enzymes, and Low Platelet count) syndrome. However, it is unclear whether elevated PAPPA2 expression causes abnormal placental development, or whether upregulation compensates for placental pathology. In the present study, we investigate whether PAPPA2 expression is affected by hypoxia, oxidative stress, syncytialization factors or substances known to affect the expression of PAPPA2's paralogue, PAPPA., Methods: BeWo cells, a model of placental trophoblasts, were treated with one of the following: hypoxia (2% O2), oxidative stress (20 microM hydrogen peroxide), forskolin (10 microM and 100 microM), TGF-beta (10 and 50 ng/mL), TNF-alpha (100 ng/mL), IL-1beta (100 ng/mL) or PGE2 (1 microM). We used quantitative RT-PCR (qRT-PCR) to quantify the mRNA levels of PAPPA2, as well as those of PAPPA and ADAM12 since these proteases have similar substrates and are also highly expressed in the placenta. Where we observed significant effects on PAPPA2 mRNA levels, we tested for effects at the protein level using an in-cell Western assay., Results: Hypoxia, but not oxidative stress, caused a 47-fold increase in PAPPA2 mRNA expression, while TNF-alpha resulted in a 6-fold increase, and both of these effects were confirmed at the protein level. PGE2 resulted in a 14-fold upregulation of PAPPA2 mRNA but this was not reflected at the protein level. Forskolin, TGF-beta and IL-1beta had no significant effect on PAPPA2 mRNA expression. We observed no effects of any treatment on PAPPA or ADAM12 expression., Conclusion: Our study demonstrates that factors previously known to be highly expressed in preeclamptic placentae (PGE2 and TNF-alpha), contribute to the upregulation of PAPPA2. Hypoxia, known to occur in preeclamptic placentae, also increased PAPPA2 expression. These results are consistent with the hypothesis that PAPPA2 is upregulated as a consequence of placental pathology, rather than elevated PAPPA2 levels being a cause of preeclampsia.

Aspergillus fumigatus is an opportunistic fungal pathogen that has recently been found to undergo sexual reproduction. Previous work suggested that invasiveness differs between mating types, and in the present study we tested whether virulence differs between mating types in an in vivo model, i.e., larvae of the wax moth Galleria mellonella. We measured virulence of 20 A. fumigatus isolates; three MAT1-1 isolates of environmental origin, five MAT1-1 isolates of clinical origin, seven MAT1-2 isolates of environmental origin and five MAT1-2 isolates of clinical origin. For each isolate, we measured virulence in six replicates and for each replicate, conidia were grown, harvested, and counted independently, and 2,500 colony forming units were injected into each of 10 G. mellonella larvae. Virulence differed between mating types, with lower survival in larvae injected with MAT1-1 isolates. Virulence also differed between clinical and environmental isolates, but surprisingly larvae injected with environmental isolates had lower survival. Identification of the mechanisms underlying variation in virulence may identify novel targets for the treatment of Aspergillus infections.

Background: Understanding the genetic basis of heritable spinal curvature would benefit medicine and aquaculture. Heritable spinal curvature among otherwise healthy children (i.e. Idiopathic Scoliosis and Scheuermann kyphosis) accounts for more than 80% of all spinal curvatures and imposes a substantial healthcare cost through bracing, hospitalizations, surgery, and chronic back pain. In aquaculture, the prevalence of heritable spinal curvature can reach as high as 80% of a stock, and thus imposes a substantial cost through production losses. The genetic basis of heritable spinal curvature is unknown and so the objective of this work is to identify quantitative trait loci (QTL) affecting heritable spinal curvature in the curveback guppy. Prior work with curveback has demonstrated phenotypic parallels to human idiopathic-type scoliosis, suggesting shared biological pathways for the deformity., Results: A major effect QTL that acts in a recessive manner and accounts for curve susceptibility was detected in an initial mapping cross on LG 14. In a second cross, we confirmed this susceptibility locus and fine mapped it to a 5 cM region that explains 82.6% of the total phenotypic variance., Conclusions: We identify a major QTL that controls susceptibility to curvature. This locus contains over 100 genes, including MTNR1B, a candidate gene for human idiopathic scoliosis. The identification of genes associated with heritable spinal curvature in the curveback guppy has the potential to elucidate the biological basis of spinal curvature among humans and economically important teleosts.

Intrauterine growth restriction (IUGR) and preeclampsia (PE) are leading causes of perinatal and maternal morbidity and mortality. Many studies have found association between low levels of insulin-like growth factor binding protein (IGFBP) proteases in the first trimester maternal circulation and the risk of subsequent development of PE and/or IUGR. These results are generally interpreted to reflect decreased production of the proteases by the placenta, leading to reduced proteolysis of IGFBPs and lower free levels of insulin-like growth factor (IGF), resulting in diminished feto-placental development. However, the association between low circulating levels of placental proteins early in pregnancy and the subsequent development of IUGR and/or PE could be due to low exchange in the placenta and not due to reduced production. In contrast, late in pregnancy, the circulating levels of these proteins and their expression in the placenta are often elevated in PE, which may reflect upregulation to compensate for abnormal placental development, that is an adaptive mechanism to increase IGFBP proteolysis, increase local IGF levels and promote feto-placental growth. Further research into the biological mechanisms underlying these associations will aid the identification of high-risk pregnancies and the development of therapeutic targets for diseases for which there are presently no preventative measures., ((c) 2010 John Wiley & Sons, Ltd.)

Background: Pregnancy-associated plasma protein A2 (PAPPA2) is an insulin-like growth factor binding protein (IGFBP) protease expressed in the placenta and upregulated in pregnancies complicated by pre-eclampsia. The mechanism linking PAPPA2 expression and pre-eclampsia and the consequences of altered PAPPA2 expression remain unknown. We previously identified PAPPA2 as a candidate gene for a quantitative trait locus (QTL) affecting growth in mice and in the present study examined whether this QTL affects placental PAPPA2 expression and, in turn, placental or embryonic growth., Methods: Using a line of mice that are genetically homogenous apart from a 1 megabase QTL region containing the PAPPA2 gene, we bred mice homozygous for alternate QTL genotypes and collected and weighed placentae and embryos at E12.5. We used quantitative RT-PCR to measure the mRNA levels of PAPPA2, as well as mRNA levels of IGFBP-5 (PAPPA2's substrate), and PAPPA (a closely related IGFBP protease) to examine potential feedback and compensation effects. Western blotting was used to quantify PAPPA2 protein. Birth weight was measured in pregnancies allowed to proceed to parturition., Results: PAPPA2 mRNA and protein expression levels in the placenta differed by a factor of 2.5 between genotypes, but we did not find a significant difference between genotypes in embryonic PAPPA2 mRNA levels. Placental IGFBP-5 and PAPPA mRNA expression levels were not altered in response to PAPPA2 levels, and we could not detect IGFBP-5 protein in the placenta by Western blotting. The observed difference in placental PAPPA2 expression had no significant effect on placental or embryonic mass at mid-gestation, birth weight or litter size., Conclusions: Despite a significant difference between genotypes in placental PAPPA2 expression similar in magnitude to the difference between pre-eclamptic and normal placentae previously reported, we observed no difference in embryonic, placental or birth weight. Our results suggest that elevated PAPPA2 levels are a consequence, rather than a cause, of pregnancy complications.

Pregnancy-associated plasma protein-A and -A2 (PAPPA and PAPPA2) are proteases that cleave IGF binding proteins (IGFBPs) and thereby increase the bioavailability of growth factors. PAPPA has long been recognized as a marker of fetal genetic disorders and adverse pregnancy outcomes. In contrast, although PAPPA2 is also highly expressed in human placenta, its physiological importance is not clear. To establish whether mice will be a useful model for the study of PAPPA2, we compared the patterns of expression of PAPPA2 in the placentae of mouse and human. We show, for the first time, that Pappa2 is highly expressed in mouse placenta, as is the case in humans. Specifically, it is expressed at the interface of the maternal and fetal layers of the mouse placenta at all gestational stages studied (10.5-16.5 days post coitum). Similarly, PAPPA2 is expressed in the syncytiotrophoblast layer of human placental villi and is also detected in some invasive extravillous trophoblasts in the first trimester. These results are consistent with a model whereby PAPPA2 cleaves IGFBPs produced in the maternal decidua to promote feto-placental growth, and indicate that this protein may play analogous roles in human and mouse placenta. PAPPA2 protein is detectable in the circulation of pregnant mice and humans during the first trimester and at term, raising the possibility that PAPPA2 may be a useful biomarker of placental dysfunction. Pappa2 expression also shows specific localization within the mouse embryo and therefore may play roles in fetal development, independent of its action in the placenta.

In fungi, microsatellites occur less frequently throughout the genome and tend to be less polymorphic compared with other organisms. Most studies that develop microsatellites for fungi focus on dinucleotide and trinucleotide repeats, and thus mononucleotide repeats, which are much more abundant in fungal genomes, may represent an overlooked resource. This study examined the relative probabilities of polymorphism in mononucleotide, dinucleotide and trinucleotide repeats in Aspergillus nidulans. As previously found, the probability of polymorphism increased with increasing number of repeating units. Dinucleotide and trinucleotide repeats had higher probabilities of polymorphism than mononucleotide repeats, but this was offset by the presence of numerous long mononucleotide repeats within the genome. Mononucleotide microsatellites with 20 or more repeating units have a probability of polymorphism similar to dinucleotide and trinucleotide microsatellites, and therefore, consideration of mononucleotide repeats will substantially increase the number of potential markers available., (© 2009 The Authors. Journal compilation © 2009 Blackwell Publishing Ltd.)

In mammals, sex steroid hormones influence spatial learning and memory abilities but there are few data regarding such effects in birds. We investigated whether non-invasive sex steroid hormone treatment would affect spatial memory task performance of great tits (Parus major). For five consecutive days, birds were fed wax moth larvae injected with either 80 microg testosterone or 80 microg estradiol carried in peanut oil immediately prior to behavioral testing. During the 5 days prior to and the 5 days following hormone treatment, birds were fed vehicle-injected larvae. Both hormone manipulations resulted in an elevation of circulating hormone levels within 5 min of larva ingestion. This elevation was sustained for at least 30 min but had no short-term (<1 day) effect on spatial memory performance. However, performance tended to increase during the first 5 days of vehicle treatment and during both sex steroid treatments whereas it decreased during the 5 days of vehicle treatment following either hormone treatment. These results suggest that both hormones led to some improvement in spatial memory that declined once treatment ended. The great tit hippocampus was found to express androgen and estrogen receptors which would provide a direct site of sex steroid action.

A recurring issue in studies of quantitative trait loci (QTLs) is whether QTLs that appear to have pleiotropic effects are indeed caused by pleiotropy at single loci or by linked QTLs. Previous work identified a QTL that affected tail length in mice and the lengths of various bones, including the humerus, ulna, femur, tibia, and mandible. The effect of this QTL on tail length has since been found to be due to multiple linked QTLs and so its apparently pleiotropic effects may have been due to linked QTLs with distinct effects. In the present study we examined a line of mice segregating only for a 0.94-Mb chromosomal region known to contain a subset of the QTLs influencing tail length. We measured a number of skeletal dimensions, including the lengths of the skull, mandible, humerus, ulna, femur, tibia, calcaneus, metatarsus, and a tail bone. The QTL region was found to have effects on the size of the mandible and length of the tail bone, with little or no effect on the other traits. Using a randomization approach, we rejected the null hypothesis that the QTL affected all traits equally, thereby demonstrating that the pleiotropic effects reported earlier were due to linked loci with distinct effects. This result underlines the possibility that seemingly pleiotropic effects of QTLs may frequently be due to linked loci and that high-resolution mapping will often be required to distinguish between pleiotropy and linkage.

Identifying genes responsible for quantitative variation remains a major challenge. We previously identified a quantitative trait locus (QTL) affecting body size that segregated between two inbred strains of mice. By fine mapping, we have refined the location of this QTL to a genomic region containing only four protein-coding genes. One of these genes, PAPPA2, is a strong candidate because it codes for an enzyme that cleaves insulin-like growth-factor-binding protein 5 (IGFBP-5), an important stimulator of bone formation. Among littermates that segregate only for the four-gene region, we show that the QTL has a significant effect on the circulating levels of IGFBP-5 and IGFBP-3 (the latter subject to limited degradation by PAPPA2), but not on levels of IGFBP-2 and IGFBP-4, which are not cleaved by PAPPA2. There are 14 nonsynonymous SNPs among QTL alleles, which may affect the activity of the translated protein. The refinement of the target region to four genes and the finding that the QTL affects IGFBP-5 levels suggest that PAPPA2 may be involved with normal postnatal growth. Our mapping results also illustrate the potentially fractal nature of QTL: as we mapped our QTL with increasing resolution, what appeared to be a single QTL resolved into three closely linked QTL (previous work), and then one of these was further dissected into two in this study.

The genetic basis of variation in complex traits remains poorly understood, and few genes underlying variation have been identified. Previous work identified a quantitative trait locus (QTL) responsible for much of the response to selection on growth in mice, effecting a change in body mass of approximately 20%. By fine-mapping, we have resolved the location of this QTL to a 660-kb region containing only two genes of known function, Gpc3 and Gpc4, and two other putative genes of unknown function. There are no non-synonymous polymorphisms in any of these genes, indicating that the QTL affects gene regulation. Mice carrying the high-growth QTL allele have approximately 15% lower Gpc3 mRNA expression in kidney and liver, whereas expression differences at Gpc4 are non-significant. Expression profiles of the two other genes within the region are inconsistent with a factor responsible for a general effect on growth. Polymorphisms in the 3' untranslated region of Gpc3 are strong candidates for the causal sequence variation. Gpc3 loss-of-function mutations in humans and mice cause overgrowth and developmental abnormalities. However, no deleterious side-effects were detected in our mice, indicating that genes involved in Mendelian diseases also contribute to complex trait variation. Furthermore, these findings show that small changes in gene expression can have substantial phenotypic effects.

Identifying genes that influence phenotypic variation is extremely difficult, especially when the allelic variants only have a small effect. A recent study has used a novel approach to identify a gene that affects the behaviour of mice.

The aim of this study was to examine the effects of a QTL in different genetic backgrounds. A QTL affecting body mass on chromosome 6 was identified in an F(2) cross between two lines of mice that have been divergently selected for this trait. The effect of the QTL on mass increased between 6 and 10 weeks of age and was not sex-specific. Body composition analysis showed effects on fat-free dry body mass and fat mass. To examine the effect of this QTL in different genetic backgrounds, the high body mass sixth chromosome was introgressed into the low body mass genetic background and vice versa by repeated marker-assisted backcrossing. After three generations of backcrossing, new F(2) populations were established within each of the introgression lines by crossing individuals that were heterozygous across the sixth chromosome. The estimated additive effect of the QTL on 10-week body mass was similar in both genetic backgrounds and in the original F(2) population (i.e., approximately 0.4 phenotypic standard deviations); no evidence of epistatic interaction with the genetic background was found. The 95% confidence interval for the location of the QTL was refined to a region of approximately 7 cM between D6Mit268 and D6Mit123.

Obesity is proving to be a serious health concern in the developed world as well as an unwanted component of growth in livestock production. While recent advances in genetics have identified a number of monogenic causes of obesity, these are responsible for only a small proportion of human cases of obesity. By divergent selection for high and low fat content over 60 generations, we have created Fat (F) and Lean (L) lines of mice that represent a model of polygenic obesity similar to the situation in human populations. From previous crosses of these lines, four body fat quantitative trait loci (QTL) were identified. We have created congenic lines (F(chr15L)), by recurrent marker-assisted backcrossing, to introgress the QTL region with the highest LOD score, Fob3 on Chr 15, from the L-Iine into the F-line background. We have further mapped this QTL by progeny testing of recombinants, produced from crosses between the F-line and congenic F(chrl5L) mice, showing that the Fob3 QTL region is a composite of at least two smaller effect QTL-the proximal QTL Fob3a is a late-onset obesity QTL, whereas the distal Fob3b is an early-onset obesity QTL.

Previous work identified a QTL affecting murine size (particularly tail length) in a cross between C57BL/6J and DBA/2J mice and refined its location to an 8-cM region between D1Mit30 and D1Mit57. The present study used recombinant progeny testing to fine map this QTL. Individuals from a partially congenic strain carrying chromosomes recombinant between D1Mit30 and D1Mit57 were mated to DBA/2J, generating 942 progeny. Two QTL affecting 10-week tail length were identified in this population: one at 9.7 cM distal to D1Mit30 (the position estimated in previous work), and another of smaller effect near D1Mit30. A second population ( n = 787) was generated by mating siblings from the progeny test population that were heterozygous for the same segment of chromosome, including only recombinants between D1Mit265 and D1Mit57. In the latter population, two QTL were also identified: one at 10.2 cM distal to D1Mit30, and another of smaller effect at the distal end of the mapped region (at D1Mit150). When the two populations were analyzed together, the estimated location of the central QTL was 10.2 cM distal to D1Mit30 and there was marginally significant evidence of the distal QTL. The central QTL explained approximately 7% of the phenotypic variance, and the 95% confidence interval for its position (determined by bootstrapping) was a 1.4-cM region, approximately the region from D1Mit451 to D1Mit219. The central QTL also affected tail length and body mass at 3 and 6 weeks of age, but to a lesser degree than 10-week tail length.

Previous work identified a tail length QTL on Chromosome (Chr) 1 in an F(2) population of C57BL/6J x DBA/2J mice. The goals of the present study were to (1) refine the position of this QTL by additional genotyping of samples from the original study; (2) confirm the effect of this QTL by producing a partially congenic strain carrying the C57BL/6J allele against the DBA/2J background; and (3) examine the effect of the QTL on skeletal dimensions. The presence of the QTL was confirmed in a new F(2) population (N = 431) derived from the partially congenic strain, and estimates of its additive effects were similar to those from the original F(2) population (N = 901) in both sexes, i.e., the C57BL/6J chromosomal segment increased tail length, the additive effect (half the difference between homozygotes) being 0.5-0.8 standard deviations. The QTL region was more than halved, relative to that in the previous study, to an 8-cM region between D1Mit30 and D1Mit57. Among a subsample of individuals (N = 30) from the new F(2) population that were not recombinant within the QTL region, there was a significant additive effect of the QTL on the length of the humerus, femur, tibia, mandible, scapula, pelvic girdle, and a tail bone; the direction of the effect was the same as for tail length. No significant effect was found on the number of bones in the tail or on the dimensions of the ulna, skull, or first vertebra.

Identifying genes that influence phenotypic variation within species has proven to be more difficult than expected. A recent study has achieved exceptional success in yeast, and demonstrates how complex genetic architecture can be.