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Regular article Biomarkers, genomics, proteomics, and gene regulation| Volume 188, ISSUE 6, P1345-1353, June 2018

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Alterations in Placental Gene Expression of Pregnant Women with Chronic Chagas Disease

Open ArchivePublished:March 12, 2018DOI:https://doi.org/10.1016/j.ajpath.2018.02.011
      Trypanosoma cruzi infection in women of reproductive age is associated with congenital transmission and adverse pregnancy outcomes. The placenta is a key barrier to infection. Gene expression profiles of term placental environment from T. cruzi–seropositive (SP) and -seronegative (SN) mothers were characterized by RNA-Seq. Nine pools of placental RNA paired samples were used: three from SN and six from SP tissues. Each pool consisted of female/male newborns and vaginal/cesarean delivery binomials. No newborn was congenitally infected. T. cruzi satellite DNA quantitative PCR in placental tissues and maternal and neonatal blood, and parasite 18S quantitative RT-PCR from placental RNA were negative, except in three SP women's bloodstream. To identify pathways associated with maternal T. cruzi infection, a gene-set association analysis was implemented: SP placental samples showed overexpression of inflammatory response and lymphocytic activation, whereas numerous biosynthetic processes were down-regulated. About 42 genes showed a significant fold-change between SP and SN groups. KISS1 and CGB5 were down-regulated, whereas KIF12, HLA-G, PRG2, TAC3, FN1, and ATXN3L were up-regulated. Several expressed genes in SP placentas encode proteins associated with preeclampsia and miscarriage. This first transcriptomics study in human term placental environment shows a placental response that may affect the fetus while protecting it from parasite infection; this host response could be responsible for the low rate of congenital transmission in chronic Chagas disease.
      About 1.125 million women of fertile age are infected with Trypanosoma cruzi worldwide.
      • WHO
      Integrating Neglected Tropical Diseases into Global Health and Development: Fourth WHO Report on Neglected Tropical Diseases.
      The incidence of congenital transmission from a chronically T. cruzi–infected pregnant woman to her fetus is relatively low, making the passage of the parasite across the placental barrier an infrequent event. Several mechanisms exerted by the placenta to protect the fetus from the infection have been reported, such as epithelial trophoblast turnover,
      • Liempi A.
      • Castillo C.
      • Carrillo I.
      • Muñoz L.
      • Droguett D.
      • Galanti N.
      • Maya J.D.
      • Kemmerling U.
      A local innate immune response against Trypanosoma cruzi in the human placenta: the epithelial turnover of the trophoblast.
      fetal production of proinflammatory cytokines,
      • Cuna W.R.
      • Choque A.G.
      • Passera R.
      • Rodriguez C.
      Pro-inflammatory cytokine production in chagasic mothers and their uninfected newborns.
      as well as an important role of fetal natural killer response.
      • Hermann E.
      • Alonso-Vega C.
      • Berthe A.
      • Truyens C.
      • Flores A.
      • Cordova M.
      • Moretta L.
      • Torrico F.
      • Braud V.
      • Carlier Y.
      Human congenital infection with Trypanosoma cruzi induces phenotypic and functional modifications of cord blood NK cells.
      Different strategies for studying the mechanisms of placental resistance to T. cruzi infection have been implemented, namely, in vitro monolayer cell culture models, ex vivo infection of human chorionic villi explants, and mammal experimental models.
      • Fretes R.E.
      • Kemmerling U.
      Mechanism of Trypanosoma cruzi placenta invasion and infection: the use of human chorionic villi explants.
      However, they all have disadvantages: cell cultures cannot reflect physiological conditions; in chorionic villi explants, the immune system, essential in this process, does not participate; and placentas of experimental models are dissimilar to human placentas.
      • Fretes R.E.
      • Kemmerling U.
      Mechanism of Trypanosoma cruzi placenta invasion and infection: the use of human chorionic villi explants.
      In search of a better understanding of host–pathogen interaction in the pregnancy context, we aimed to characterize the gene expression profile of the term placental environment from T. cruzi–infected mothers in comparison with that of healthy noninfected mothers.

      Materials and Methods

      Ethics Statement

      The research protocols followed the tenets of the Declaration of Helsinki and Guidelines according to Resolution N°1480/11 of the Ministerio de Salud from Argentina and were approved by the local medical ethics committees named Comité Provincial de Bioética de Jujuy and Comité de Bioética Dr. Vicente Federico del Giúdice – Hospital Nacional Prof. Alejandro Posadas. All mothers provided informed consent on their behalf before sample collection and after the purposes of the study were explained.

      Subjects and Samples

      Serodiagnosis of pregnant women was done by means of conventional serological methods and performed by the respective health centers based on routine assays. In maternal and umbilical cord blood samples, as well as placental tissues, presence of T. cruzi was tested using multiplex real-time PCR as previously described.
      • Duffy T.
      • Cura C.I.
      • Ramirez J.C.
      • Abate T.
      • Cayo N.M.
      • Parrado R.
      • Bello Z.D.
      • Velazquez E.
      • Muñoz-Calderon A.
      • Juiz N.A.
      • Basile J.
      • Garcia L.
      • Riarte A.
      • Nasser J.R.
      • Ocampo S.B.
      • Yadon Z.E.
      • Torrico F.
      • de Noya B.A.
      • Ribeiro I.
      • Schijman A.G.
      Analytical performance of a multiplex real-time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples.
      Maternal infection with other pathogens that produce congenital transmission and adverse pregnancy outcome, as well as missing data or incorrect sampling, were considered as exclusion criteria.
      Fresh normal placentas were obtained after labor from vaginal or cesarean deliveries and placed within 24 hours at 4°C. Each placenta was dissected, and the middle section
      • Sood R.
      • Zehnder J.L.
      • Druzin M.L.
      • Brown P.O.
      Gene expression patterns in human placenta.
      at 2 cm distance from the umbilical cord was isolated and placed into RNAlater solution (Applied Biosystems, Foster City, CA). Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) and stored at −80°C until used.

      Transcriptomic Studies

      A RNA-Seq experiment was performed in nine pools of two different placental RNA samples each: three pools (C1, C2, and C3) belonging to placentas from seronegative mothers (SN) and six pools (TC4 to TC9) from seropositive mothers (SP). Each pool consisted of a binomial of a female/male newborn and a vaginal/cesarean delivery. The cDNA libraries were prepared according to Illumina's TruSeq Stranded Total RNA with Ribo-Zero Gold for Human (Illumina, San Diego, CA), and a Hiseq 2.500 Illumina platform with 100-bp paired-end reads was used for sequencing. The RNA-Seq data have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo; accession number GSE107376).

      RNA-Seq Data Analysis

      Raw reads were subjected to a quality test with FastQC software version 0.11.5 (Babraham Bioinformatics, Cambridge, UK; http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Sequences were mapped implementing STAR software version 2.5,
      • Dobin A.
      • Gingeras T.R.
      Mapping RNA-seq reads with STAR.
      and Picard software version 2.9.4 (Broad Institute, Cambridge, MA) tools were applied to calculate the gene expression level. Finally, the differentially expressed genes (DEGs) were identified using edgeR software version 3.6,
      • Robinson M.D.
      • McCarthy D.J.
      • Smyth G.K.
      edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.
      and principal component analysis was calculated in R software version 3.4.3 (R Foundation for Statistical Computing, Vienna, Austria). Gene Ontology (GO) functional enrichment analysis was performed by the web-based application GOrilla (MULTI-KNOWLEDGE Project; http://cbl-gorilla.cs.technion.ac.il) and gene-set association analysis for RNA-Seq with gene permutation GSAASeqGP software version 2.0.
      • Xiong Q.
      • Mukherjee S.
      • Furey T.S.
      GSAASeqSP: a toolset for gene set association analysis of RNA-Seq data.
      Gene interaction networks were analyzed by GeneMANIA software version 3.4.0 (University of Toronto, Toronto, ON, Canada; http://www.genemania.org) and visualized by the corresponding Cytoscape software version 3.0.2 plugins.
      • Merico D.
      • Isserlin R.
      • Stueker O.
      • Emili A.
      • Bader G.D.
      Enrichment map: a network-based method for gene-set enrichment visualization and interpretation.

      RNA-Seq Validation

      To validate the RNA-Seq results, total RNA pools used in transcriptomics and 12 independent samples (four from SN and eight from SP women) were analyzed. The cDNA was obtained implementing TaqMan Reverse Transcription Reagents (Applied Biosystems). Quantitative RT-PCR (RT-qPCR) of selected DEGs were performed using 1X FastStart Universal SYBR Green Master (Roche Diagnostics Corp., Indianapolis, IN) using the following cycling conditions: 95°C 10 minutes; 5 cycles of 95°C for 15 seconds and 64°C 1 minute, 40 cycles of 95°C for 15 seconds, and annealing temperature as listed in Table 1, for 1 minute. The expression levels were normalized to GAPDH with primers GAPDH 5′-GGTCTCCTCTGACTTCAACA-3′ (forward) and GAPDH 5′-GTGAGGGTCTCTCTCTTCCT-3′ (reverse).
      Table 1Oligonucleotides Used in RNA-Seq Validation
      GeneForwardReverseTa, °CAmplicon length, bp
      HLA-G5′-CTGGTTGTCCTTGCAGCTGTAG-3′5′-CCTTTTCAATCTGAGCTCTTCTTTCT-3′5780
      FN15′-CAAACACTAATGTTAATTGCCCA-3′5′-GGTCTGCTAACATCACTCCA-3′56156
      ATXN3L5′-AAGATGAACATCTCCGCAGT-3′5′-GGCCTTTCGTGTAGGTATGA-3′56214
      TAC35′-GGATCATGCTGCTATTCACAG-3′5′-ATGCATGTCACGTTTCTCGG-3′56245
      CGB55′-CAGCATCCTACAACCTCCTG-3′5′-CGCGGGTCATGGTGG-3′67277
      KISS15′-GGACCTGCCTCTTCTCACCA-3′5′-ATTCTAGCTGCTGGCCTGTG-3′54140
      PRG25′-TGGATTGGAGGCAGGATCA-3′5′-TGCCGTCAACCCACTGAAA-3′5461
      Primer sequences of selected DEGs, annealing temperatures of RT-qPCR procedures (Ta) and amplicons length.
      A RT-qPCR assay targeting T. cruzi 18S RNA was also performed in these samples as described previously.
      • Juiz N.A.
      • Solana M.E.
      • Acevedo G.R.
      • Benatar A.F.
      • Ramirez J.C.
      • da Costa P.A.
      • Macedo A.M.
      • Longhi S.A.
      • Schijman A.G.
      Different genotypes of Trypanosoma cruzi produce distinctive placental environment genetic response in chronic experimental infection.
      Primer sequences of selected DEGs are listed in Table 1. The relative expression of each sample was calculated using the 2−ΔΔCT method with the control group as calibrator. Statistical analyses were performed using GraphPad Prism software version 6.01 (GraphPad Software, La Jolla, CA).

      Results

      Mother/Newborn Binomials Characterization

      Sixty-five pregnant women (age range 18 to 41 years) were consecutively enrolled at Posadas Hospital, Buenos Aires, from January 2014 to November 2015 and at Jujuy Maternal Hospital from January to September 2016 (Figure 1). Sixteen were SN and 49 SP, but 24 of these binomials were omitted because of exclusion criteria. Numbers of vaginal and cesarean deliveries and also numbers of female/male newborns in each case are detailed in Figure 1. All newborn umbilical cord and peripheral blood samples were collected during the first hour of life and had nondetectable T. cruzi satellite DNA by PCR, in agreement with microhematocrit findings. Placental tissues were also real-time quantitative PCR negative (Table 2).
      Figure thumbnail gr1
      Figure 1Flowchart of enrolled mother/newborn binomials in the prospective study.
      Table 2Characteristics of Mother/Newborn Binomials under Study
      PoolSample IDMotherNewbornPlacentaDeliveryGestation, weeks
      Age, yearsSerologyqPCRqPCRSexRT-qPCR and qPCR
      C1C326NDNDMNDC39
      C5229NDNDFNDV40
      C2C432NDNDMNDC38.4
      C718NDNDFNDV41
      C3C633NDNDMNDV39.2
      C828NDNDFNDC39.5
      TC4TC122+15p/mLNDMNDV37
      TC1118+D/NQNDFNDC39.5
      TC5TC335+43p/mLNDMNDC39.4
      TC3441+D/NQNDFNDV40.3
      TC6TC4528+D/NQNDFNDV39
      TC3340+D/NQNDMNDC38
      TC7TC426+D/NQNDFNDC39.4
      TC4022+NDNDMNDV38.5
      TC8TC4220+NDNDMNDV41
      TC4424+NDNDFNDC37
      TC9TC531+NDNDFNDC39.2
      TC2228+NDNDMNDV40.5
      Maternal age and serological diagnosis, newborn sex and results of T. cruzi satellite DNA qPCR expressed as parasites per milliliter (p/mL) in maternal and neonatal blood, as well as T. cruzi satellite DNA qPCR and 18S RNA–based RT-qPCR in placental tissues. It is also indicated delivery mode and time of gestation.
      F, female; M, male; C, cesarean; D/NQ, detectable but nonquantifiable (parasitic load below 1.5 p/mL; [6]); ND, nondetectable; RT-qPCR, quantitative RT-PCR; qPCR, real-time quantitative PCR; V, vaginal.
      For RNA-Seq studies, only term pregnancies with times of gestation above 37 weeks (mean = 39.22 ± 1.15 weeks) were selected. Of 29 placental samples from SP, 12 were selected, grouped into six pools, and of 12 samples from SN, 6 were selected, grouped into three pools. The construction of pools took into account a balance (1:1) of newborn sex and mode of delivery variables, because they could become confounding factors (Table 2). These samples belonged to mothers with a mean age of 27.92 ± 7.55 and 27.67 ± 7.55 years, in the SP and SN groups, respectively (P > 0.05). No significant differences were found in time of gestation between the SP and SN groups (Table 2) (P = 0.597).
      Of SP binomials, three pools were prepared with samples from women with positive bloodstream PCR results (TC4, TC5, and TC6) and another three with nondetectable parasite DNA in maternal peripheral blood after admission to maternity service (TC7, TC8, and TC9). Moreover, none of the placental tissues were positive for the determination of 18s RNA for T. cruzi (Table 2).

      Relatedness among Gene Expression Patterns of Pools

      First, principal component analysis was performed for a preliminary clustering of samples on the basis of complete gene expression data without prior statistical filtering (Figure 2). The first two principal components, which explain 86% (PC1 61% and PC2 25%) of the variance among samples, separate pools of SN (C1, C2, and C3) from those of SP (TC4 to TC9) mothers. However, the gene expression observed in the SP group could not differentiate between mothers with detectable or nondetectable T. cruzi DNA in peripheral blood close to delivery.
      Figure thumbnail gr2
      Figure 2Principal component analysis (PCA). PCA plot of human placental transcriptome from pooled samples of seronegative (SN) and seropositive (SP) mothers with detectable (PCR+) or nondetectable (PCR-) T. cruzi DNA in peripheral blood at the time of delivery. Circles cluster SN and SP samples. The x and y axes correspond to the first (PC1) and second (PC2) principal components, respectively, with their explained variances.

      Differentially Expressed Genes

      Only 42 genes were differentially expressed with a significance level <0.05 and a fold change |FC| >1.5 and 26 with |FC| >2, comparing SN and SP placentas (Table 3). The most represented transcript in SP group placentas was KIF12, a kinesin superfamily member with a role in microtubule cytoskeleton. Another interesting gene that turned out to be more expressed in the SP group was HLA-G. It belongs to the HLA class I that is expressed on fetal-derived placental cells and is related to immune tolerance in pregnancy. In addition, PRG2, a cytotoxin involved in antiparasitic defense mechanisms, MUC4, a mucin that provides protection to epithelial cells, and TAC3, the protein of which is expressed in the syncytiotrophoblast, had also a greater transcription level in the SP group.
      Table 3List of Differentially Expressed Genes Detected by RNA-Seq Analysis
      Up-regulated DEGsDown-regulated DEGs
      Locus
      Loci are available from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov).
      GeneFCpadjLocusGeneFCpadj
      NM_138424KIF126.360.040NR_106781MIR6723−4.800.000
      NR_027278FAM230C6.120.035NR_104646LOC101927592−4.630.043
      NR_046087GS15.800.040NR_033557LOC100422737−3.710.033
      NM_001135995ATXN3L5.770.028NM_020672S100A14−3.160.003
      NR_108087LINC010873.720.038NM_058173MUCL1−2.820.004
      NR_122046LOC2849303.620.000NM_002256KISS1−2.720.000
      NM_013251TAC33.560.000NM_001260512MGST1−2.510.000
      NM_002127HLA-G3.290.000NM_033043CGB5−2.370.033
      NM_022073EGLN33.170.003NM_173161IL1F10−2.300.011
      NM_002728PRG23.070.000NM_001191321GABRB3−2.270.000
      NM_001306094NPIPB152.880.003NM_000771CYP2C9−1.990.037
      NM_018406MUC42.480.021NM_014400LYPD3−1.950.000
      NR_126062CELF2-AS12.260.011NR_015411MIR9-3HG−1.870.000
      NM_212482FN12.180.003NM_001875CPS1−1.690.010
      NM_177551HCAR22.170.015NM_182767SLC6A15−1.630.007
      NM_001012302ANO92.080.043NM_022659EBF2−1.580.033
      NR_026758FAR2P11.770.033NM_024807TREML2−1.530.033
      NR_033968GUSBP91.760.033NM_018643TREM1−1.510.001
      NM_152666PLD51.750.039
      NM_001447FAT21.660.010
      NR_003263SDHAP31.640.034
      NM_003670BHLHE401.610.039
      NM_000887ITGAX1.590.010
      NM_001083538POTEE1.580.010
      Genes with fold change |FC| >1.5 and adjusted P < 0.05, detected after comparing placental samples from SN and SP pregnant women.
      Loci are available from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov).
      There was also a down-regulation of immunomodulatory activity genes in the SP group, such as IL1F10 and KISS1, inhibitors of chemotaxis and invasion, with a putative role in cell-matrix adhesion and that stimulate the secretion of gonadotropin. Related to the latter, the gene encoding the beta subunit of chorionic gonadotropin, CGB5, was also down-regulated, whereas fibronectine 1 (FN1), presents in cell surface and the extracellular matrix, was overexpressed in placentas from SP mothers. Finally, the most down-regulated gene was MIR6723, a microRNA that may be involved in post-transcriptional regulation.

      Functional Pathway Analysis

      To identify pathways significantly associated with the SP group in the placental environment, we first used the GOrilla tool for identifying enriched GO terms, considering genes with an adjusted P < 0.05 (Supplemental Table S1). Extracellular matrix organization emerged from positive FC genes, whereas pathways related to exocytosis, neutrophil degranulation, and glutamine and glutathione biosynthetic processes emerged from the list of negative FC genes.
      A gene-set association analysis was also performed by GSAA-Seq, a cutoff-free strategy of genome-wide patterns in the measured gene expression. In this approach, choosing arbitrary cutoffs is avoided, and accumulative subtle changes in gene expression within the same gene set are identified. The resulting gene sets significantly associated with a false discovery rate q value < 25% and nominal P < 5% were represented in an enrichment map (Figure 3). Several pathways were overexpressed in the SP compared with SN placentas; some of them were related to immune response, cytoskeleton and calcium transport, cell cycle, DNA synthesis, and chromatin modifications. On the other hand, processes such as biosynthesis and respiratory chain, vesicle transport, and metabolism of lipids and hormones were down-regulated, as were networks associated with protein metabolism (synthesis and proteolysis by proteasome).
      Figure thumbnail gr3
      Figure 3Gene-set association analysis. Representation of biological pathways with false discovery rate q value < 25% and nominal P < 5%. Nodes correspond to gene sets, where red nodes correspond to up-regulated gene-sets and nodes in blue correspond to down-regulated ones, and links correspond to overlap of member genes. To improve layout, the networks were manually rearranged. Major clusters were manually labeled.
      Within the top 10 GO pathways of the ranked list of over-represented genes from SP, which is based on the false discovery rate q values and on the Gene Set Normalized Association Score, which reflects how much a pathway is over- or under-represented, five pathways were related to calcium transport, and three to immune response (Supplemental Table S2). Interestingly, the fourth in this list was the GO Positive Regulation of Acute Inflammatory Response with 16 (57%) genes contributing to the enrichment score. In the eighth and tenth positions, there were T-cell Selection and Positive Regulation of Neutrophil Migration networks.
      Regarding gene sets that were under-represented in placentas of SP mothers, several pathways related to respiratory chain were on the top of the list (Supplemental Table S2): Mitochondrial Respiratory Chain Complex I Biogenesis and Energy Derivation By Oxidation of Organic Compounds. Moreover, translation processes were also represented in the first positions: Translational Initiation and Translational Termination. Lastly, processes of vesicle transport were also affected by maternal infection, for example, Establishment of Protein Localization to Endoplasmic Reticulum (Supplemental Table S2).

      RNA-Seq Validation

      To validate the RNA-Seq data using RT-qPCR, seven DEGs were selected on the basis of their high FCs and low P values: FN1, ATXN3L, TAC3, CGB5 and HLA-G, PRG2, and KISS1. The expression patterns of these genes are shown in Figure 4. The results showed that the expression patterns as determined by RT-qPCR were consistent with those obtained by RNA-Seq (P > 0.05), confirming the accuracy of the RNA-Seq results in this study.
      Figure thumbnail gr4
      Figure 4RT-qPCR validation analysis of gene expression. Bars represent the mean gene expression levels in seropositive (SP) relative to seronegative (SN) samples, and vertical lines show SD. Data are expressed as means ± SD.

      Discussion

      Congenital T. cruzi infection remains a global health problem with an estimation of 8700 cases per year in endemic countries.
      • WHO
      Integrating Neglected Tropical Diseases into Global Health and Development: Fourth WHO Report on Neglected Tropical Diseases.
      Maternal parasitic loads and strain placental tropism have been implicated in the likelihood of congenital transmission, which is an infrequent event in chronic Chagas disease pregnancy.
      • Bua J.
      • Volta B.J.
      • Velazquez E.B.
      • Ruiz A.M.
      • De Rissio A.M.
      • Cardoni R.L.
      Vertical transmission of Trypanosoma cruzi infection: quantification of parasite burden in mothers and their children by parasite DNA amplification.
      • Bisio M.
      • Seidenstein M.E.
      • Burgos J.M.
      • Ballering G.
      • Risso M.
      • Pontoriero R.
      • Moreau M.
      • Altcheh J.
      • Leguizamón M.S.
      • Freilij H.
      • Marceillac M.
      • Schijman A.G.
      Urbanization of congenital transmission of Trypanosoma cruzi: prospective polymerase chain reaction study in pregnancy.
      Indeed, trypanocidal treatment of women at reproductive age reduces the rate of vertical transmission.
      • Messenger L.A.
      • Gilman R.H.
      • Verastegui M.
      • Galdos-Cardenas G.
      • Sanchez G.
      • Valencia E.
      • Sanchez L.
      • Malaga E.
      • Rendell V.R.
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      • Shah V.
      • Santos N.
      • Abastoflor M.D.C.
      • LaFuente C.
      • Colanzi R.
      • Bozo R.
      • Bern C.
      Working Group on Chagas Disease in Bolivia and Peru
      Toward improving early diagnosis of congenital Chagas disease in an endemic setting.
      Family clustering and congenital transmission in second generations in nonendemic areas contribute to perpetuate Chagas disease.
      • Bisio M.
      • Seidenstein M.E.
      • Burgos J.M.
      • Ballering G.
      • Risso M.
      • Pontoriero R.
      • Moreau M.
      • Altcheh J.
      • Leguizamón M.S.
      • Freilij H.
      • Marceillac M.
      • Schijman A.G.
      Urbanization of congenital transmission of Trypanosoma cruzi: prospective polymerase chain reaction study in pregnancy.
      • Sánchez Negrette O.
      • Mora M.C.
      • Basombrío M.A.
      High prevalence of congenital Trypanosoma cruzi infection and family clustering in Salta, Argentina.
      A case of a triplet born to a Chagas disease mother from a triamniotic dichorionic delivery was described by Burgos et al,
      • Burgos J.M.
      • Altcheh J.
      • Petrucelli N.
      • Bisio M.
      • Levin M.J.
      • Freilij H.
      • Schijman A.G.
      Molecular diagnosis and treatment monitoring of congenital transmission of Trypanosoma cruzi to twins of a triplet delivery.
      who reported the occurrence of congenital infection in both sisters sharing one placenta and no infection in the third one, indicative of the protective role of the placenta. Human genomic polymorphism in placental genes has been also associated to the occurrence of transmission.
      • Juiz N.A.
      • Cayo N.M.
      • Burgos M.
      • Salvo M.E.
      • Nasser J.R.
      • Búa J.
      • Longhi S.A.
      • Schijman A.G.
      Human polymorphisms in placentally expressed genes and their association with susceptibility to congenital Trypanosoma cruzi infection.
      Anti–T. cruzi seropositivity in mothers was associated with a history of adverse reproductive outcomes.
      • Gamboa-León R.
      • Ramirez-Gonzalez C.
      • Pacheco-Tucuch F.S.
      • O'Shea M.
      • Rosecrans K.
      • Pippitt J.
      • Dumonteil E.
      • Buekens P.
      Seroprevalence of Trypanosoma cruzi among mothers and children in rural Mayan communities and associated reproductive outcomes.
      Nevertheless, there is still a knowledge gap regarding the effect of the parasite infection in pregnancy outcome.

      Placental Gene Expression Profile in SP Women

      In the present study, even though a few genes (N = 42) had a significant FC between SP and SN groups, the deeper gene-set association analysis of the RNA-Seq data suggests the existence of a gene expression profile in the chorionic villi region of placentas from T. cruzi–seropositive mothers that differs from that of placentas from uninfected women, as revealed by principal component analysis.
      This first study in human placental environment is consistent with our previous findings in placental gene expression from mice infected with T. cruzi compared with noninfected dams.
      • Juiz N.A.
      • Solana M.E.
      • Acevedo G.R.
      • Benatar A.F.
      • Ramirez J.C.
      • da Costa P.A.
      • Macedo A.M.
      • Longhi S.A.
      • Schijman A.G.
      Different genotypes of Trypanosoma cruzi produce distinctive placental environment genetic response in chronic experimental infection.
      Indeed, the analysis showed several biosynthetic processes down-regulated in both murine and human placental studies, including lipid and protein metabolisms, whereas immunological gene sets were enriched in infected dams and mothers. In fact, in both studies, the inflammatory response and lymphocytic activation were overexpressed in placental tissues from infected groups. It has been already reported that newborns of T. cruzi–infected mothers are prone to produce higher levels of proinflammatory cytokines in comparison with those born to noninfected mothers, and there is increasing evidence suggesting that maternal infection during pregnancy affects the developing immune system of fetuses independent of the vertical transmission of pathogens.
      • Dauby N.
      • Goetghebuer T.
      • Kollmann T.R.
      • Levy J.
      • Marchant A.
      Uninfected but not unaffected: chronic maternal infections during pregnancy, fetal immunity, and susceptibility to postnatal infections.
      Nevertheless, the placenta could reduce the damage produced by the inflammatory response by decreasing the exocytosis process, including neutrophil degranulation, because we have observed a significantly lower expression in related genes in the SP group. These results are also consistent with those from mice placenta, where genes associated with the secretory granule were down-regulated.
      • Juiz N.A.
      • Solana M.E.
      • Acevedo G.R.
      • Benatar A.F.
      • Ramirez J.C.
      • da Costa P.A.
      • Macedo A.M.
      • Longhi S.A.
      • Schijman A.G.
      Different genotypes of Trypanosoma cruzi produce distinctive placental environment genetic response in chronic experimental infection.
      Moreover, vesicle transport processes also decreased in placentas from SP women and infected mice.
      Because inflammatory activity leads to extracellular matrix damage, it is not surprising that we have found an activation of extracellular matrix organization–related genes. Previous studies had reported the importance of the extracellular matrix remodeling and that fibronectin was not affected at a protein level
      • Duaso J.
      • Yanez E.
      • Castillo C.
      • Galanti N.
      • Cabrera G.
      • Corral G.
      • Maya J.D.
      • Zulantay I.
      • Apt W.
      • Kemmerling U.
      Reorganization of extracellular matrix in placentas from women with asymptomatic Chagas disease: mechanism of parasite invasion or local placental defense?.
      ; however, our findings suggest that the placenta produces de novo synthesis of this protein, as well as collagen, during T. cruzi infection.
      This differential gene expression in SP placentas was not associated with the presence of parasitic DNA or RNA in the tissues subjected to RNA-Seq analysis. Nevertheless, real-time quantitative PCR and RT-qPCR analysis in in-term placentas do not reflect the dynamics of placental parasite burden during pregnancy. Indeed, our transcriptomic analysis represents a placental terminal stage.

      Differentially Expressed Genes in Placentas from SP Women

      Infection with T. cruzi during pregnancy could result in low birth weight, still birth, spontaneous abortion, growth restriction, intrauterine fetal death, and fetal abnormalities.
      • Nourollahpour Shiadeh M.
      • Behboodi Moghadam Z.
      • Adam I.
      • Saber V.
      • Bagheri M.
      • Rostami A.
      Human infectious diseases and risk of preeclampsia: an updated review of the literature.
      There are some studies reporting similar or slightly higher prevalence of abortions and stillbirths in T. cruzi–seropositive mothers than in uninfected mothers, and there are also some contradictions about the consequences on low birth weight.
      • Torrico F.
      • Alonso-Vega C.
      • Suarez E.
      • Rodriguez P.
      • Torrico M.C.
      • Dramaix M.
      • Truyens C.
      • Carlier Y.
      Maternal Trypanosoma cruzi infection, pregnancy outcome, morbidity, and mortality of congenitally infected and non-infected newborns in Bolivia.
      In addition, if that effect really exists it, is not clear whether it is a result of congenital infection or placental dysfunction.
      • Carlier Y.
      • Truyens C.
      Congenital Chagas disease as an ecological model of interactions between Trypanosoma cruzi parasites, pregnant women, placenta and fetuses.
      It is worth noting that in this study, SP human placentas showed a down-regulation of the KISS1 gene, which encodes kisspeptin, low serum levels of which have been related to preeclampsia and intrauterine growth restriction.
      • Armstrong R.A.
      • Reynolds R.M.
      • Leask R.
      • Shearing C.H.
      • Calder A.A.
      • Riley S.C.
      Decreased serum levels of kisspeptin in early pregnancy are associated with intra-uterine growth restriction and pre-eclampsia.
      Moreover, KISS1 low expression levels have been associated with recurrent pregnancy loss,
      • Park D.W.
      • Lee S.K.
      • Hong S.R.
      • Han A.R.
      • Kwak-Kim J.
      • Yang K.M.
      Expression of Kisspeptin and its receptor GPR54 in the first trimester trophoblast of women with recurrent pregnancy loss.
      and low serum levels of its protein, as well as of human chorionic gonadotropin (hCG), have been proposed as markers of miscarriage.
      • Jayasena C.N.
      • Abbara A.
      • Izzi-Engbeaya C.
      • Comninos A.N.
      • Harvey R.A.
      • Gonzalez Maffe J.
      • Sarang Z.
      • Ganiyu-Dada Z.
      • Padilha A.I.
      • Dhanjal M.
      • Williamson C.
      • Regan L.
      • Ghatei M.A.
      • Bloom S.R.
      • Dhillo W.S.
      Reduced levels of plasma kisspeptin during the antenatal booking visit are associated with increased risk of miscarriage.
      Interestingly, the gene CGB5 that encodes the beta 5 subunit of hCG was down-regulated in SP human placentas.
      Moreover, the DEG TAC3 had a higher transcription activity in SP placentas. Its coding protein, neurokinin B (NKB), is expressed by syncytiotrophoblasts, and its overexpression has been also associated with preeclampsia.
      • Page N.M.
      • Woods R.J.
      • Gardiner S.M.
      • Lomthaisong K.
      • Gladwell R.T.
      • Butlin D.J.
      • Manyonda I.T.
      • Lowry P.J.
      Excessive placental secretion of neurokinin B during the third trimester causes pre-eclampsia.
      Both, kisspeptin and NKB play a key role in the placental expression of hCG in response to estradiol.
      • Oride A.
      • Kanasaki H.
      • Mijiddorj T.
      • Sukhbaatar U.
      • Ishihara T.
      • Kyo S.
      Regulation of kisspeptin and gonadotropin-releasing hormone expression in rat placenta: study using primary cultures of rat placental cells.
      Furthermore, PRG2 gene coding protein was also up-regulated in SP placentas. It encodes the preform of eosinophil major basic protein (proMBP), the predominant constituent of eosinophil granules, which have been associated with T. cruzi clearance through extracellular killing in the absence of antibodies.
      • Nakhle M.C.
      • de Menezes Mda C.
      • Irulegui I.
      Eosinophil levels in the acute phase of experimental Chagas' disease.
      MBP is present in placenta inhibiting the pregnancy-associated plasma protein A (PAPPA).
      • Weyer K.
      • Glerup S.
      Placental regulation of peptide hormone and growth factor activity by proMBP.
      Low levels of PAPPA are related to intrauterine growth restriction,
      • Albu A.
      • Anca A.
      • Horhoianu V.
      • Horhoianu I.
      Predictive factors for intrauterine growth restriction.
      so the higher transcription of its inhibitor observed in SP placentas might play a role in pregnancy outcome. Both PAPPA and hCG low levels have been proposed as biomarkers of preeclampsia.
      • Paredes V.
      • Espinoza-Caicedo J.A.
      • Salazar-Pousada D.
      • Escobar G.S.
      • Pérez-López F.R.
      • Chedraui P.
      Lower placental growth factor and higher free β-hCG and PAPP-A levels in the fetal circulation of near-term pregnancies complicated with severe preeclampsia.
      The previously mentioned inflammatory response due to maternal infection has also been associated with preeclampsia.
      • Rustveld L.O.
      • Kelsey S.F.
      • Sharma R.
      Association between maternal infections and preeclampsia: a systematic review of epidemiologic studies.
      Finally, GOrilla analysis allowed detection of altered glutamine and glutathione biosynthetic processes, consistently with a recent proteomics analysis of human placenta, which showed that, not only inflammatory response, but also glutathione metabolism dysfunction may contribute to preeclampsia.
      • Jin X.
      • Xu Z.
      • Cao J.
      • Shao P.
      • Zhou M.
      • Qin Z.
      • Liu Y.
      • Yu F.
      • Zhou X.
      • Ji W.
      • Cai W.
      • Ma Y.
      • Wang C.
      • Shan N.
      • Yang N.
      • Chen X.
      • Li Y.
      Proteomics analysis of human placenta reveals glutathione metabolism dysfunction as the underlying pathogenesis for preeclampsia.
      Taken together, it is tempting to speculate that mothers with Chagas disease could be more prone to manifest preeclampsia than noninfected ones, which can be worth studying. This would be of relevance in chronic Chagas disease, because preeclampsia is associated with an increase of future heart failure and cardiovascular diseases.
      • Wu P.
      • Haththotuwa R.
      • Kwok C.S.
      • Babu A.
      • Kotronias R.A.
      • Rushton C.
      • Zaman A.
      • Fryer A.A.
      • Kadam U.
      • Chew-Graham C.A.
      • Mamas M.A.
      Preeclampsia and future cardiovascular health: a systematic review and meta-analysis.
      However, there are no studies exploring the role of T. cruzi infection in development of preeclampsia, such as has been observed in placental malaria
      • Gueneuc A.
      • Deloron P.
      • Bertin G.I.
      Usefulness of a biomarker to identify placental dysfunction in the context of malaria.
      and other parasitic infections.
      • Alvarado-Esquivel C.
      • Vázquez-Alaníz F.
      • Sandoval-Carrillo A.A.
      • Salas-Pacheco J.M.
      • Hernández-Tinoco J.
      • Sánchez-Anguiano L.F.
      • Liesenfeld O.
      Lack of association between Toxoplasma gondii infection and hypertensive disorders in pregnancy: a case–control study in a Northern Mexican population.
      • Soydinc H.E.
      • Kan I.
      • Dal T.
      • Evsen M.S.
      • Sak M.E.
      • Ozler A.
      • Turgut A.
      • Yildiz I.
      Evaluation of the relationship between preeclampsia and seropositivity of infectious disease in maternal plasma.
      Another DEG was HLA-G with a positive FC, an immunomodulatory molecule involved in generating tolerance during pregnancy. A study of malaria showed that higher levels of HLA-G in infected mothers correlated with higher HLA-G levels in their newborns and infants; this phenomenon was related to a higher susceptibility of Plasmodium infection in children belonging to binomials with high HLA-G serum levels.
      • Sadissou I.
      • d'Almeida T.
      • Cottrell G.
      • Luty A.
      • Krawice-Radanne I.
      • Massougbodji A.
      • Moreau P.
      • Moutairou K.
      • Garcia A.
      • Favier B.
      • Rouas-Freiss N.
      • Courtin D.
      High plasma levels of HLA-G are associated with low birth weight and with an increased risk of malaria in infancy.
      It would be of interest to study this phenomenon in the context of T. cruzi infection.
      This is the first placental transcriptomics study in natural human infection, which may contribute to the understanding of placental mechanisms induced by maternal T. cruzi infection. Our results suggest that although most fetuses from infected mothers remain uninfected, they may be affected by the placental response that protects them from the parasite.

      Acknowledgments

      We thank Paula Beati (INGEBI-CONICET), Leandro Simonetti (INGEBI-CONICET), and Carlos David Bruque (ANLIS-MALBRAN) for their bioinformatics contributions.

      Supplemental Data

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