Reproductive biologies
Abstract
The placenta is the primary organ for immune regulation, nutrient delivery, gas exchange, protection against environmental toxins, and physiologic perturbations during pregnancy. Placental inflammation and vascular dysfunction during pregnancy are associated with a growing list of prematurity-related complications. The goal of this study was to identify differences in gene expression profiles in fetal monocytes - cells that persist and differentiate postnatally - according to distinct placental histologic domains. Here, by using bulk RNA sequencing (RNA-Seq), we report that placental lesions are associated with gene expression changes in fetal monocyte subsets. Specifically, we found that fetal monocytes exposed to acute placental inflammation upregulate biological processes related to monocyte activation, monocyte chemotaxis, and platelet function while monocytes exposed to maternal vascular malperfusion lesions downregulate these processes. Additionally, we show that intermediate monocytes might be a source of mitogens, such as HBEGF, NRG1, and VEGFA, implicated in different outcomes related to prematurity. This is the first study to show that placental lesions are associated with unique changes in fetal monocytes and monocyte subsets. As fetal monocytes persist and differentiate into various phagocytic cells following birth, our study may provide insight into morbidity related to prematurity and ultimately potential therapeutic targets.
Authors
Abhineet M. Sharma, Robert Birkett, Erika T. Lin, Linda M. Ernst, William A. Grobman, Suchitra Swaminathan, Hiam Abdala-Valencia, Alexander V. Misharin, Elizabeth T. Bartom, Karen K. Mestan
Abstract
Parturition is a well-orchestrated process characterized by increased uterine contractility, cervical ripening, and activation of the chorioamniotic membranes; yet, the transition from a quiescent to a contractile myometrium heralds the onset of labor. However, the cellular underpinnings of human parturition in the uterine tissues are still poorly understood. Herein, we performed a comprehensive study of the human myometrium during spontaneous term labor using single-cell RNA sequencing (scRNA-Seq). First, we established a single-cell atlas of the human myometrium and unraveled the cell type–specific transcriptomic activity modulated during labor. Major cell types included distinct subsets of smooth muscle cells, monocytes/macrophages, stromal cells, and endothelial cells, all of which communicated and participated in immune (e.g., inflammation) and nonimmune (e.g., contraction) processes associated with labor. Furthermore, integrating scRNA-Seq and microarray data with deconvolution of bulk gene expression highlighted the contribution of smooth muscle cells to labor-associated contractility and inflammatory processes. Last, myometrium-derived single-cell signatures can be quantified in the maternal whole-blood transcriptome throughout pregnancy and are enriched in women in labor, providing a potential means of noninvasively monitoring pregnancy and its complications. Together, our findings provide insights into the contributions of specific myometrial cell types to the biological processes that take place during term parturition.
Authors
Roger Pique-Regi, Roberto Romero, Valeria Garcia-Flores, Azam Peyvandipour, Adi L. Tarca, Errile Pusod, Jose Galaz, Derek Miller, Gaurav Bhatti, Robert Para, Tomi Kanninen, Ola Hadaya, Carmen Paredes, Kenichiro Motomura, Jeffrey R. Johnson, Eunjung Jung, Chaur-Dong Hsu, Stanley M. Berry, Nardhy Gomez-Lopez
Abstract
Chronic inflammation and localized alterations in immune cell function are suspected to contribute to the progression of endometriosis and its associated symptoms. In particular, the alarmin, Interleukin (IL)-33 is elevated in the plasma, peritoneal fluid, and endometriotic lesions from endometriosis patients; however, the exact role of IL-33 in the pathophysiology of endometriosis is not well understood. In this study, we demonstrate, in both human patients and a murine model, that IL-33 contributes to the expansion of the novel group 2 innate lymphoid cells (ILC2s) and this IL-33 induced ILC2 expansion modulates the endometriosis lesion microenvironment. Importantly, we show that IL-33 drives hallmarks of severe endometriosis including elevated inflammation, lesion proliferation, and fibrosis and that this IL-33 induced aggravation is mediated by ILC2s. Finally, we demonstrate the functionality of IL-33 neutralization as a promising and novel therapeutic avenue for treating the debilitating symptoms of endometriosis.
Authors
Jessica E. Miller, Harshavardhan Lingegowda, Lindsey K. Symons, Olga Bougie, Steven L. Young, Bruce A. Lessey, Madhuri Koti, Chandrakant Tayade
Abstract
Macrophages are commonly thought to contribute to the pathophysiology of preterm labor by amplifying inflammation — but a protective role has not previously been considered to our knowledge. We hypothesized that given their antiinflammatory capability in early pregnancy, macrophages exert essential roles in maintenance of late gestation and that insufficient macrophages may predispose individuals to spontaneous preterm labor and adverse neonatal outcomes. Here, we showed that women with spontaneous preterm birth had reduced CD209+CD206+ expression in alternatively activated CD45+CD14+ICAM3– macrophages and increased TNF expression in proinflammatory CD45+CD14+CD80+HLA-DR+ macrophages in the uterine decidua at the materno-fetal interface. In Cd11bDTR/DTR mice, depletion of maternal CD11b+ myeloid cells caused preterm birth, neonatal death, and postnatal growth impairment, accompanied by uterine cytokine and leukocyte changes indicative of a proinflammatory response, while adoptive transfer of WT macrophages prevented preterm birth and partially rescued neonatal loss. In a model of intra-amniotic inflammation–induced preterm birth, macrophages polarized in vitro to an M2 phenotype showed superior capacity over nonpolarized macrophages to reduce uterine and fetal inflammation, prevent preterm birth, and improve neonatal survival. We conclude that macrophages exert a critical homeostatic regulatory role in late gestation and are implicated as a determinant of susceptibility to spontaneous preterm birth and fetal inflammatory injury.
Authors
Nardhy Gomez-Lopez, Valeria Garcia-Flores, Peck Yin Chin, Holly M. Groome, Melanie T. Bijland, Kerrilyn R. Diener, Roberto Romero, Sarah A. Robertson
Abstract
Retinoic Acid (RA) signaling has long been speculated to regulate embryo implantation, because many enzymes and proteins responsible for maintaining RA homeostasis and transducing RA signals are tightly regulated in the endometrium during this critical period. However, due to lack of genetic data, it was unclear whether RA signaling is truly required for implantation, and which specific RA signaling cascades are at play. Herein we utilize a genetic murine model that expresses a dominant negative form of RA receptor specifically in female reproductive organs to show that functional RA signaling is fundamental to female fertility, particularly implantation and decidualization. Reduction in RA signaling activity severely affects the ability of the uterus to achieve receptive status and decidualize, partially through dampening follistatin expression and downstream activin B/BMP2 signaling. To confirm translational relevance of these findings to humans, human endometrial stromal cells (hESCs) were treated with pan-RAR antagonist to show that in vitro decidualization is impaired. RNAi perturbation of individual RAR transcripts in hESCs revealed that RARα in particular is essential for proper decidualization. These data provide direct functional evidence that uterine RAR-mediated RA signaling is crucial for mammalian embryo implantation, and its disruption leads to failure of uterine receptivity and decidualization resulting in severely compromised fertility.
Authors
Yan Yin, Meade E. Haller, Sangappa B. Chadchan, Ramakrishna Kommagani, Liang Ma
Abstract
The syndrome of spontaneous preterm birth (sPTB) presents a challenge to mechanistic understanding, effective risk stratification, and management. Individual associations between sPTB, ethnicity, vaginal microbiota, metabolome and innate immune response are known, but not fully understood and knowledge has yet to impact clinical practice. Here we use multi-data type integration and composite statistical models to gain insight into sPTB risk by exploring the cervicovaginal environment of an ethnically heterogenous pregnant population (n=346 women; n=60 sPTB <37 weeks’ gestation, including n=27 sPTB <34 weeks). Analysis of cervicovaginal samples (10-15+6 weeks) identified novel interactions between risk of sPTB and microbiota, metabolite, and maternal host defense molecules. Statistical modelling identified a composite of metabolites (leucine, tyrosine, aspartate, lactate, betaine, acetate and Ca2+) associated with risk of sPTB <37 weeks (Area Under the Curve - AUC 0.752). A combination of glucose, aspartate, Ca2+ and Lactobacillus crispatus and L. acidophilus relative abundance, identified risk of early sPTB <34 weeks, (AUC 0.758); improved by ethnicity stratification (AUC 0.835). Increased relative abundance of L. acidophilus appeared protective against sPTB <34 weeks. By using cervicovaginal fluid samples, we demonstrate the potential of multi-datatype integration for developing composite models towards understanding the contribution of the vaginal environment to risk of sPTB.
Authors
Flavia Flaviani, Natasha L. Hezelgrave, Tokuwa Kanno, Erica M. Prosdocimi, Evonne Chin-Smith, Alexandra E. Ridout, Djuna K. von Maydell, Vikash Mistry, William G. Wade, Andrew H. Shennan, Konstantina Dimitrakopoulou, Paul T. Seed, Andrew James Mason, Rachel M. Tribe
Abstract
Exposure to maternal obesity may promote metabolic dysfunction in offspring. We use infant mesenchymal stem cells (MSC) to experimentally examine cellular mechanisms of intergenerational health transmission. Our earlier reports show MSCs collected from infants of mothers with obesity had a dichotomous distribution in metabolic efficiency; they were either efficient (Ef-Ob) or inefficient (In-Ob) with respect to fatty acid oxidation (FAO). Here, we sought to determine if this was due to a primary defect in FAO. Accordingly, we measured FAO in myogenic differentiating MSCs under three conditions: 1) myogenesis alone, 2) excess fatty acid exposure, and 3) excess fatty acid exposure plus a chemical uncoupler to increase metabolic rate. Compared to NW and Ef-Ob MSCs, In-Ob displayed lower FAO in myogenesis alone and after fatty acid plus uncoupler, indicating In-Ob were less metabolically flexible after increasing lipid availability and metabolic rate, demonstrating a primary deficit in FAO. MSC FAO was negatively associated with fasting maternal glucose and insulin, and positively associated with fasting HDL-cholesterol. MSC FAO was negatively associated with infant fat mass. These data indicate a less favorable maternal metabolic milieu, independent of maternal BMI, reduces intrinsic MSC FAO and is linked to higher infant adiposity as early as birth.
Authors
Melissa L. Erickson, Zachary W. Patinkin, Allison M. Duensing, Dana Dabelea, Leanne M. Redman, Kristen E. Boyle
Abstract
Clinical phenotyping of term and preterm labor is imprecise, and disagreement persists on categorization relative to underlying pathobiology, which remains poorly understood. We performed RNA sequencing (RNA-seq) of 31 specimens of human uterine myometrium from 10 term and 21 preterm cesarean deliveries with rich clinical context information. A molecular signature of 4,814 transcripts stratified myometrial samples into quiescent (Q) and non-quiescent (NQ) phenotypes, independent of gestational age and incision site. Similar stratifications were achieved using expressed genes in Ca2+ signaling and TGF-β pathways. For maximal parsimony, we evaluated the expression of just two Ca2+ transporter genes, ATP2B4 (encoding PMCA4) and ATP2A2 (coding for SERCA2), and found that their ratio reliably distinguished NQ and Q specimens in the current study, and also in two publically available RNA-seq datasets (GSE50599 and GSE80172), with an overall AUC of 0.94. Cross-validation of the ATP2B4/ATP2A2 ratio by qPCR in an expanded cohort (by 11 additional specimens) achieved complete separation (AUC=1.00) of NQ vs. Q specimens. While providing additional insight into the associations between clinical features of term and preterm labor and myometrial gene expression, our study also offers a practical algorithm for unbiased classification of myometrial biopsies by their overall contractile program.
Authors
William E. Ackerman IV, Catalin S. Buhimschi, Ali Snedden, Taryn L. Summerfield, Guomao Zhao, Irina A. Buhimschi
Abstract
The microtubule (MT) cytoskeleton plays a critical role in axon growth and guidance. Here, we identify the MT severing enzyme fidgetin-like 2 (FL2) as a negative regulator of axon regeneration and a therapeutic target for promoting nerve regeneration after injury. Genetic knockout of FL2 in cultured adult dorsal root ganglion (DRG) neurons resulted in longer axons and attenuated growth cone retraction in response to inhibitory molecules. Given the axonal growth-promoting effects of FL2 depletion in vitro, we tested whether FL2 could be targeted to promote regeneration in a rodent model of cavernous nerve (CN) injury. The CN are parasympathetic nerves that regulate blood flow to the penis, which are commonly damaged during radical prostatectomy (RP) resulting in erectile dysfunction (ED). Application of FL2-siRNA after CN injury significantly enhanced functional nerve recovery. Remarkably, following bilateral nerve transection, visible and functional nerve regeneration was observed in 7 out of 8 animals treated with FL2-siRNA, while no control treated animals exhibited regeneration. These studies identify FL2 as a promising therapeutic target for enhancing regeneration after peripheral nerve injury and for mitigating neurogenic ED post-RP—a condition for which, at present, only poor treatment options exist.
Authors
Lisa Baker, Moses Tar, Adam H. Kramer, Guillermo A. Villegas, Rabab A. Charafeddine, Olga Vafaeva, Parimala Nacharaju, Joel Friedman, Kelvin P. Davies, David J. Sharp
Abstract
A maternal Western-style diet (WSD) is associated with poor reproductive outcomes, but whether this is from the diet itself or underlying metabolic dysfunction is unknown. Here, we performed a longitudinal study using regularly cycling female rhesus macaques (n = 10) that underwent 2 consecutive in vitro fertilization (IVF) cycles, one while consuming a low-fat diet and another 6–8 months after consuming a high-fat WSD. Metabolic data were collected from the females prior to each IVF cycle. Follicular fluid (FF) and oocytes were assessed for cytokine/steroid levels and IVF potential, respectively. Although transition to a WSD led to weight gain and increased body fat, no difference in insulin levels was observed. A significant decrease in IL-1RA concentration and the ratio of cortisol/cortisone was detected in FF after WSD intake. Despite an increased probability of isolating mature oocytes, a 44% reduction in blastocyst number was observed with WSD consumption, and time-lapse imaging revealed delayed mitotic timing and multipolar divisions. RNA sequencing of blastocysts demonstrated dysregulation of genes involved in RNA binding, protein channel activity, mitochondrial function and pluripotency versus cell differentiation after WSD consumption. Thus, short-term WSD consumption promotes a proinflammatory intrafollicular microenvironment that is associated with impaired preimplantation development in the absence of large-scale metabolic changes.
Authors
Sweta Ravisankar, Alison Y. Ting, Melinda J. Murphy, Nash Redmayne, Dorothy Wang, Carrie A. McArthur, Diana L. Takahashi, Paul Kievit, Shawn L. Chavez, Jon D. Hennebold
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