The initiation of puberty is driven by an upsurge in hypothalamic gonadotropin-releasing hormone (GnRH) secretion. In turn, GnRH secretion upsurge depends on the development of a complex GnRH neuroendocrine network during embryonic life. Although delayed puberty (DP) affects up to 2% of the population, is highly heritable, and is associated with adverse health outcomes, the genes underlying DP remain largely unknown. We aimed to discover regulators by whole-exome sequencing of 160 individuals of 67 multigenerational families in our large, accurately phenotyped DP cohort. LGR4 was the only gene remaining after analysis that was significantly enriched for potentially pathogenic, rare variants in 6 probands. Expression analysis identified specific Lgr4 expression at the site of GnRH neuron development. LGR4 mutant proteins showed impaired Wnt/β-catenin signaling, owing to defective protein expression, trafficking, and degradation. Mice deficient in Lgr4 had significantly delayed onset of puberty and fewer GnRH neurons compared with WT, whereas lgr4 knockdown in zebrafish embryos prevented formation and migration of GnRH neurons. Further, genetic lineage tracing showed strong Lgr4-mediated Wnt/β-catenin signaling pathway activation during GnRH neuron development. In conclusion, our results show that LGR4 deficiency impairs Wnt/β-catenin signaling with observed defects in GnRH neuron development, resulting in a DP phenotype.
Alessandra Mancini, Sasha R. Howard, Federica Marelli, Claudia P. Cabrera, Michael R. Barnes, Michael J.E. Sternberg, Morgane Leprovots, Irene Hadjidemetriou, Elena Monti, Alessia David, Karoliina Wehkalampi, Roberto Oleari, Antonella Lettieri, Valeria Vezzoli, Gilbert Vassart, Anna Cariboni, Marco Bonomi, Marie Isabelle Garcia, Leonardo Guasti, Leo Dunkel
Changes in maternal immunity during pregnancy can result in an altered immune state and, as a natural perturbation, this provides an opportunity to understand functional interactions of the immune system in vivo. We report characterisation of maternal peripheral immune phenotypes for 33 longitudinally sampled normal pregnancies, using clinical measurements of complete blood counts and major immune cell populations, as well as high parameter flow cytometry for 30 different leukocyte antigens characterising 79 cell populations, and monitoring of 1305 serum proteins using the SomaLogic platform. Cellular analyses characterised transient changes in T cell polarization, and more persistent alterations in T and B cell subset frequencies and activation. Serum proteomic analysis identified a novel set of 7 proteins that are predictive of gestational age: DDR1, PLAU, MRC1, ACP5, ROBO2, IGF2R, and GNS. We further show that gestational age can be predicted from the parameters obtained by complete blood count tests and clinical flow cytometry characterizing 5 major immune cell populations. Inferring gestational age from this routine clinical phenotyping data could be useful in resource limited settings which lack obstetric ultrasound. Overall, both the cellular and proteomic analyses validate previously reported phenotypic immunological changes of pregnancy, and uncover new alternations and predictive markers.
Richard Apps, Yuri Kotliarov, Foo Cheung, Kyu Lee Han, Jinguo Chen, Angelique Biancotto, Ashley L. Babyak, Huizhi Zhou, Rongye Shi, Lisa A. Barnhart, Sharon M. Osgood, Yasmine Belkaid, Steven M. Holland, John S. Tsang, Christa Zerbe
Few therapeutic methods exist for preventing preterm birth (PTB), or delivery before completing 37 weeks of gestation. In the US, progesterone (P4) supplementation is the only FDA-approved drug for use in preventing recurrent spontaneous PTB. However, P4 has limited effectiveness, working in only approximately one-third of cases. Computational drug repositioning leverages data on existing drugs to discover novel therapeutic uses. We used a rank-based pattern-matching strategy to compare the differential gene expression signature for PTB to differential gene expression drug profiles in the Connectivity Map database and assigned a reversal score to each PTB-drug pair. Eighty-three drugs, including P4, had significantly reversed differential gene expression compared with that found for PTB. Many of these compounds have been evaluated in the context of pregnancy, with 13 belonging to pregnancy category A or B — indicating no known risk in human pregnancy. We focused our validation efforts on lansoprazole, a proton-pump inhibitor, which has a strong reversal score and a good safety profile. We tested lansoprazole in an animal inflammation model using LPS, which showed a significant increase in fetal viability compared with LPS treatment alone. These promising results demonstrate the effectiveness of the computational drug repositioning pipeline to identify compounds that could be effective in preventing PTB.
Brian L. Le, Sota Iwatani, Ronald J. Wong, David K. Stevenson, Marina Sirota
Adequate iron supply during pregnancy is essential for fetal development. However, how fetal or amniotic fluid iron levels are regulated during healthy pregnancy, or pregnancies complicated by intraamniotic infection or inflammation (IAI) is unknown. We evaluated amniotic fluid and fetal iron homeostasis in normal and complicated murine, macaque, and human pregnancy. In mice, fetal iron endowment was affected by maternal iron status but amniotic fluid iron concentrations changed little during maternal iron deficiency or excess. In murine and macaque models of inflamed pregnancy, the fetus responded to maternal systemic inflammation or IAI by rapidly upregulating hepcidin and lowering iron in fetal blood, without altering amniotic fluid iron. In humans, elevated cord blood hepcidin with accompanying hypoferremia was observed in pregnancies with antenatal exposure to IAI compared to those that were non-exposed. Hepcidin was also elevated in human amniotic fluid from pregnancies with IAI compared to those without IAI, but amniotic fluid iron levels did not differ between the groups. Our studies in mice, macaques, and humans demonstrate that amniotic fluid iron is largely unregulated but that the rapid induction of fetal hepcidin by inflammation and consequent fetal hypoferremia are conserved mechanisms that may be important in fetal host defense.
Allison L. Fisher, Veena Sangkhae, Pietro Presicce, Claire A. Chougnet, Alan H. Jobe, Suhas G. Kallapur, Sammy M. Tabbah, Catalin S. Buhimschi, Irina A. Buhimschi, Tomas Ganz, Elizabeta Nemeth
During pregnancy the maternal pancreatic islets of Langerhans undergo adaptive changes to compensate for gestational insulin resistance. Kisspeptin has been shown to stimulate insulin release, through its receptor, GPR54. The placenta releases high levels of kisspeptin into the maternal circulation, suggesting a role in modulating the islet adaptation to pregnancy. In the present study we show that pharmacological blockade of endogenous kisspeptin in pregnant mice resulted in impaired glucose homeostasis. This glucose intolerance was due to a reduced insulin response to glucose as opposed to any effect on insulin sensitivity. A β cell–specific GPR54-knockdown mouse line was found to exhibit glucose intolerance during pregnancy, with no phenotype observed outside of pregnancy. Furthermore, in pregnant women circulating kisspeptin levels significantly correlated with insulin responses to oral glucose challenge and were significantly lower in women with gestational diabetes (GDM) compared with those without GDM. Thus, kisspeptin represents a placental signal that plays a physiological role in the islet adaptation to pregnancy, maintaining maternal glucose homeostasis by acting through the β cell GPR54 receptor. Our data suggest reduced placental kisspeptin production, with consequent impaired kisspeptin-dependent β cell compensation, may be a factor in the development of GDM in humans.
James E. Bowe, Thomas G. Hill, Katharine F. Hunt, Lorna I.F. Smith, Sian J.S. Simpson, Stephanie A. Amiel, Peter M. Jones
With multifactorial etiologies, combined with disease heterogeneity and a lack of suitable diagnostic markers and therapy, endometriosis remains a major reproductive health challenge. Extracellular vesicles (EVs) have emerged as major contributors of disease progression in several conditions, including a variety of cancers; however, their role in endometriosis pathophysiology has remained elusive. Using next-generation sequencing of EVs obtained from endometriosis patient tissues and plasma samples compared with controls, we have documented that patient EVs carry unique signatures of miRNAs and long noncoding RNAs (lncRNAs) reflecting their contribution to disease pathophysiology. Mass spectrophotometry–based proteomic analysis of EVs from patient plasma and peritoneal fluid further revealed enrichment of specific pathways, as well as altered immune and metabolic processes. Functional studies in endometriotic epithelial and endothelial cell lines using EVs from patient plasma and controls clearly indicate autocrine uptake and paracrine cell proliferative roles, suggestive of their involvement in endometriosis. Multiplex cytokine analysis of cell supernatants in response to patient and control plasma–derived EVs indicate robust signatures of important inflammatory and angiogenic cytokines known to be involved in disease progression. Collectively, these findings suggest that endometriosis-associated EVs carry unique cargo and contribute to disease pathophysiology by influencing inflammation, angiogenesis, and proliferation within the endometriotic lesion microenvironment.
Kasra Khalaj, Jessica E. Miller, Harshavardhan Lingegowda, Asgerally T. Fazleabas, Steven L. Young, Bruce A. Lessey, Madhuri Koti, Chandrakant Tayade
Many women with hyperandrogenemia suffer from irregular menses and infertility. However, it is unknown whether androgens directly affect reproduction. Since animal models of hyperandrogenemia-induced infertility are associated with obesity, which may impact reproductive function, we have created a lean mouse model of elevated androgen using implantation of low dose dihydrotestosterone (DHT) pellets to separate the effects of elevated androgen from obesity. The hypothalamic-pituitary-gonadal axis controls reproduction. While we have demonstrated that androgen impairs ovarian function, androgen could also disrupt neuroendocrine function at the level of brain and/or pituitary to cause infertility. To understand how elevated androgens might act on pituitary gonadotropes to influence reproductive function, female mice with disruption of the androgen receptor (Ar) gene specifically in pituitary gonadotropes (PitARKO) were produced. DHT treated control mice with intact pituitary Ar (Con-DHT) exhibit disrupted estrous cyclicity and fertility with reduced pituitary responsiveness to GnRH at the level of both calcium signaling and LH secretion. These effects were ameliorated in DHT treated PitARKO mice. Calcium signaling controls GnRH regulation of LH vesicle exotocysis. Our data implicated upregulation of GEM (a voltage-dependent calcium channel inhibitor) in the pituitary as a potential mechanism for androgen’s pathological effects. These results demonstrate that gonadotrope AR, as an extra-ovarian regulator, plays an important role in reproductive pathophysiology.
Zhiqiang Wang, Mingxiao Feng, Olubusayo Awe, Yaping Ma, Mingjie Shen, Ping Xue, Rexford Ahima, Andrew Wolfe, James Segars, Sheng Wu
Hormones produced by the anterior pituitary gland regulate an array of important physiological functions, but pituitary hormone disorders are not fully understood. Herein we report that genetically-engineered mice with deletion of the hedgehog signaling receptor Patched1 by S100a4 promoter-driven Cre recombinase (S100a4-Cre;Ptch1fl/fl mutants) exhibit adult-onset hypogonadotropic hypogonadism and multiple pituitary hormone disorders. During the transition from puberty to adult, S100a4-Cre;Ptch1fl/fl mice of both sexes develop hypogonadism coupled with reduced gonadotropin levels. Their pituitary glands also display severe structural and functional abnormalities, as revealed by transmission electron microscopy and expression of key genes regulating pituitary endocrine functions. S100a4-Cre activity in the anterior pituitary gland is restricted to CD45+ cells of hematopoietic origin, including folliculo-stellate cells and other immune cell types, causing sex-specific changes in the expression of genes regulating the local microenvironment of the anterior pituitary. These findings provide in vivo evidence for the importance of pituitary hematopoietic cells in regulating fertility and endocrine function, in particular during sexual maturation and likely through sexually dimorphic mechanisms. These findings support a previously unrecognized role of hematopoietic cells in causing hypogonadotropic hypogonadism and provide inroads into the molecular and cellular basis for pituitary hormone disorders in humans.
Yi Athena Ren, Teresa Monkkonen, Michael T. Lewis, Daniel J. Bernard, Helen C. Christian, Carolina J. Jorgez, Joshua A. Moore, John D. Landua, Haelee M. Chin, Weiqin Chen, Swarnima Singh, Ik Sun Kim, Xiang H.-F. Zhang, Yan Xia, Kevin J. Phillips, Harry MacKay, Robert A. Waterland, M. Cecilia Ljungberg, Pradip K. Saha, Sean M. Hartig, Tatiana Fiordelisio Coll, JoAnne S. Richards
Human placenta development and a successful pregnancy is incumbent upon precise oxygen-dependent control of trophoblast migration/invasion. Persistent low oxygen leading to failed trophoblast invasion promotes inadequate spiral artery remodeling, a characteristic of preeclampsia. Angiomotin (AMOT) is a multifaceted scaffolding protein involved in cell polarity and migration, yet its upstream regulation and significance in the human placenta remain unknown. Herein, we show that AMOT is primarily expressed in migratory extravillous trophoblast cells (EVTs) of the intermediate and distal anchoring column. Its expression increases after 10 weeks of gestation when oxygen tension rises and EVT migration/invasion peaks. Time-lapse imaging confirmed that the AMOT 80-kDa isoform promotes migration of trophoblastic JEG3 and HTR-8/SVneo cells. In preeclampsia, however, AMOT expression is decreased and its localization to migratory fetomaternal interface EVTs is disrupted. We demonstrate that Jumonji C domain–containing protein 6 (JMJD6), an oxygen sensor, positively regulates AMOT via oxygen-dependent lysyl hydroxylation. Furthermore, in vitro and ex vivo studies show that transforming growth factor-β (TGF-β) regulates AMOT expression, its interaction with polarity protein PAR6, and its subcellular redistribution from tight junctions to cytoskeleton. Our data reveal an oxygen- and TGF-β–driven migratory function for AMOT in the human placenta, and implicate its deficiency in impaired trophoblast migration that plagues preeclampsia.
Abby Farrell, Sruthi Alahari, Leonardo Ermini, Andrea Tagliaferro, Michael Litvack, Martin Post, Isabella Caniggia
Zika virus (ZIKV) infection during pregnancy causes significant adverse sequelae in the developing fetus, and results in long-term structural and neurologic defects. Most preventive and therapeutic efforts have focused on the development of vaccines, antivirals, and antibodies. The placental immunologic response to ZIKV, however, has been largely overlooked as a target for therapeutic intervention. The placental inflammatory response, specifically IL-1β secretion and signaling, is induced by ZIKV infection and represents an environmental factor that is known to increase the risk of perinatal developmental abnormalities. We show in a mouse model that maternally administrated IL-1 receptor antagonist (IRA; Kineret, or anakinra), following ZIKV exposure, can preserve placental function (by improving trophoblast invasion and placental vasculature), increase fetal viability, and reduce neurobehavioral deficits in the offspring. We further demonstrate that while ZIKV RNA is highly detectable in placentas, it is not correlated with fetal viability. Beyond its effects in the placenta, we show that IL-1 blockade may also directly decrease fetal neuroinflammation by mitigating fetal microglial activation in a dose-dependent manner. Our studies distinguish the role of placental inflammation during ZIKV-infected pregnancies, and demonstrate that maternal IRA may attenuate fetal neuroinflammation and improve perinatal outcomes.
Jun Lei, Meghan S. Vermillion, Bei Jia, Han Xie, Li Xie, Michael W. McLane, Jeanne S. Sheffield, Andrew Pekosz, Amanda Brown, Sabra L. Klein, Irina Burd
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