Infections at the maternal-fetal interface can directly harm the fetus and induce complications that adversely impact pregnancy outcomes. Innate immune signaling by both fetal-derived placental trophoblasts and the maternal decidua must provide antimicrobial defenses at this critical interface without compromising its integrity. Here, we developed matched trophoblast (TO) and decidua organoids (DO) from human placentas to define the relative contributions of these cells to antiviral defenses at the maternal-fetal interface. We demonstrate that TO and DO basally secrete distinct immunomodulatory factors, including the constitutive release of the antiviral type III interferon IFN-λ2 from TOs, and differentially respond to viral infections through the induction of organoid-specific factors. Finally, we define the differential susceptibility and innate immune signaling of TO and DO to human cytomegalovirus (HCMV) and develop a co-culture model of TO and DO which showed that trophoblast-derived factors protect decidual cells from HCMV infection. Our findings establish matched TO and DO as ex vivo models to study vertically transmitted infections and highlight differences in innate immune signaling by fetal-derived trophoblasts and the maternal decidua. The maternal-fetal interface is comprised of both the maternal-derived decidua and the fetal-derived placenta, which together support fetal growth, maintain maternal-fetal immunotolerance, and provide immunologic defenses to protect the fetus from infections. The relative contributions of maternal- and fetal-derived cells to these critical functions remain largely undefined given the limitations of models that recapitulate the complex nature of the human maternal-fetal interface.The fetal-derived placenta contains distinct trophoblast subsets throughout gestation. Trophoblast stem cells located in chorionic villi, the branch-like projections that directly contact maternal blood, give rise to cytotrophoblasts (CTBs), the proliferative mononuclear cells of the placenta, the syncytiotrophoblast (STB), a multinucleated contiguous cell layer that covers the villous surfaces, and extravillous trophoblasts (EVTs). EVTs remodel the maternal microvasculature to form spiral arteries, which ultimately deliver maternal blood to the surface of chorionic villi. On the maternal side, decidualization of the endometrium is required for implantation and appropriate placentation. Taken together, the decidua and placenta maintain immunotolerance throughout gestation and provide the fetus with essential hormones and nutrients to support its growth.Toxoplasma gondii, Zika virus (ZIKV), HIV, Rubella, and human cytomegalovirus [HCMV]) can be vertically transmitted to infect the fetus and in some cases, cause disease and/or pregnancy loss. Studies of tissue explants and histopathologic analysis of the placenta suggest that the decidua and chorionic villi have differing roles in the pathogenesis of vertically transmitted infections. The decidua may be a primary site of replication for several microorganisms associated with congenital disease, including HCMV  or rhesus macaque (RhCMV) cytomegaloviruses rely on viruses that are genetically disparate from HCMV and do not fully recapitulate the human maternal-fetal interface . Prior sThe recent development of trophoblast (TO) and decidua organoids (DO) from first trimester placental tissue has greatly expanded the models available to study the human maternal-fetal interface . PreviouTo directly compare antiviral signaling pathways between the human placenta and decidua, we generated TO and DO from matched mid-to-late gestation (26–41 weeks of gestation) human placentas and profiled their release of cytokines, chemokines, and other factors at baseline and under virally infected conditions. These studies revealed that TOs basally secrete IFN-λ2, which does not occur in matched DOs. To determine whether maternal- and fetal-derived cells respond differently to viral infections, we compared the transcriptional profiles and immunological secretomes from organoids treated with a ligand to stimulate toll-like receptor 3 (TLR3) signaling. TO and DO differentially responded to TLR3 activation through the secretion of organoid-specific cytokines and chemokines. Additionally, we defined the differential susceptibility of trophoblast and decidua organoids to HCMV infection and identified key differences in the innate immune responses of these organoids to HCMV infection. Finally, to simulate the communication between trophoblasts and the decidua epithelium in vivo, we established a co-culture system between matched TO and DO, which showed that fetal-derived trophoblasts protect the decidual epithelium from HCMV infection. Taken together, these studies highlight the differential responses of decidual and placental cells at the maternal-fetal interface to viral infections and provide new models to understand innate immune responses against placental infections.To generate matched human TOs and decidual organoids (DOs), we used human placental tissue isolated from the second and third trimesters of human pregnancy (26–41 weeks gestation) and cultured progenitor cells isolated from chorionic villi or uterine glands of the decidua . Given tTrophoblast stem cells and trophoblast-derived organoids derived from the first trimester placental tissue can be differentiated to an EVT-enriched phenotype through a two-step process that includes treatment with Neuregulin-1 (NRG1) . SimilarCollectively, these data show that TOs and DOs are morphologically and functionally distinct and that they recapitulate human placental trophoblasts and decidua glandular epithelial cells.Next, we performed transcriptional profiling of established TO and DO lines using bulk RNASeq. We found that TOs and DOs were transcriptionally distinct based on differential expression analysis using DESeq2 followed by principal component analysis (PCA) . TOs higTo determine if TOs derived from full-term tissue recapitulated the transcriptional signature of placental tissue throughout gestation, we performed comparative transcriptional profiling of TOs and publicly available RNASeq data sets from placental tissue isolated from the first and second trimesters, or from full-term tissue. We performed differential expression analysis of these data sets and compared the expression profiles of key pregnancy hormones and other factors associated with placental structure and function. Hierarchical clustering showed that TOs most closely clustered with samples derived from first trimester placental tissue . In addiCollectively, these data establish matched TOs and DOs as model systems of the maternal-fetal interface and demonstrate that they can be derived from tissue after the first trimester, including from mid-to-late gestation. Furthermore, these data suggest that TOs derived from full-term tissue recapitulate the expression profile of first trimester placental tissue.We have shown previously that human chorionic villous explants release numerous cytokines, chemokines, and growth factors that impact both maternal and fetal antimicrobial defenses . SimilarWe showed previously that PHT cells isolated from full-term placentas and mid-gestation chorionic villi constitutively release type III IFNs, which protect trophoblasts from viral infections . We foun2 fold-change of ±2) of TOs and DOs revealed that TOs upregulated 448 transcripts whereas DOs upregulated 121 (2 fold change 5.9), CX3CL1 (log2 fold change 7.1), and CCL22 (log2 fold change 7.6), which were the most induced cytokine or chemokine transcripts in poly I:C-treated TOs , a synthetic ligand of TLR3, and profiled the transcriptional and secretory changes induced by poly I:C treatment of TOs and DOs. We found that both TOs and DOs robustly responded to poly I:C treatment, which corresponded with significant transcriptional changes as assessed by RNASeq . Compariated 121 . Of thesated 121 . Of the ated TOs . In contated TOs . Transcrated TOs .To confirm the transcriptional changes described above, we performed parallel multianalyte Luminex profiling of 105 cytokines and chemokines in TOs and DOs treated with poly I:C. Both TOs and DOs mounted robust responses to poly I:C treatment that correlated with the release of high levels of select cytokines and chemokines . CytokinHCMV is the most common vertically transmitted pathogen and is associated with high rates of placental dysfunction, pregnancy loss, and congenital disease. However, how HCMV transmits across the placenta and how the maternal decidua and fetal-derived placenta respond to HCMV infection remains poorly defined. To address these questions, we infected TOs and DOs with HCMV strains AD169r  or 173 transcripts (TB40E strain), of which ~38% were shared between both strains of HCMV . By contTo define the innate immune responses to HCMV infection in TOs and DOs, we used Luminex multianalyte profiling of >100 cytokines and chemokines in TOs and DOs infected with HCMV strains AD169r or TB40E. We found that TOs secreted relatively few cytokines in response to HCMV infection, including pentraxin-3 (PTX3), first described as an endothelial factor induced by IL-1β treatment , IL-8, aBecause we observed high levels of basal IFN-λ2 secretion in TOs, which were resistant to HCMV infection, and high induction of IFN-λ2 in DOs infected with HCMV, we next determined whether inhibiting this IFN signaling would alter the sensitivity of DOs or TOs to HCMV infection. To test this, we used the JAK-specific inhibitor ruxolitinib, which inhibits signaling downstream of type III IFN receptor activation. DOs and TOs were treated with ruxolitinib for 1 hr prior to infection with the AD169r strain of HCMV for 48 hr in the presence of ruxolitinib. We found that treatment of DOs with ruxolitinib significantly increased levels of HCMV replication, which correlated with higher levels of GFP fluorescence . In contTo model communication between the maternal-fetal interface and to define this impact of this communication on HCMV infection, we established a co-culture model of TOs and DOs developed from matched placental tissue. To do this, equivalent numbers of TOs and DOs were pre-mixed prior to culturing and co-cultured for ~5 days prior to infection with HCMV. We confirmed that co-cultured organoids contained both TOs and DOs and distinguished between these organoids using SIGLEC-6 immunostaining, with only TOs positive for this marker . FollowiTaken together, these findings highlight the distinct immunoregulatory and transcriptional cascades induced by HCMV infection of maternal-derived decidual versus fetal-derived placental cells and suggest that IFNs restrict HCMV infection in decidual epithelial cells. In addition, our co-culture studies support a model whereby TO-derived factors confer protection of epithelial cells in the decidua from HCMV infection.Our work establishes fetal- and maternal-derived organoids as models for defining innate immune signaling at the maternal-fetal interface. Similar to PHTs  and mid-The previous establishment of trophoblast and decidua-derived organoids has significantly expanded the in vitro systems that can model the complexity of the maternal-fetal interface . These pUnlike many other barrier cell types such as the epithelium, human trophoblasts have the unique capacity to robustly secrete cytokines and other immunological factors without stimulation. PHTs from full-term placentas and mid-gestation chorionic villi secrete cytokines that mediate innate immune defenses against bacteria, parasites, and viruses . ConsistT. gondii infection through the selective release of CCL22  and in accordance with the guidelines of the University of Pittsburgh and Duke University human tissue procurement. Placental tissue used in this study was collected from the second (23rd–27th weeks) and third (35th–41st weeks) trimesters. Placental tissue was excluded for diagnosis of PPROM or maternal infection.oC incubator to pre-polymerize for ~3 min, turned upside down to ensure equal distribution of the isolated cells in domes for another 10 min, then carefully overlaid with 500 µL prewarmed trophoblast organoid medium (TOM) consisting of Advanced DMEM/F12  supplemented with 1× B27 , 1× N2 , 10% fetal bovine serum (FBS) , 2 mM GlutaMAX supplement , 100 µg/mL Primocin , 1.25 mM N-Acetyl-L-cysteine , 500 nM A83-01 , 1.5 µM CHIR99021 , 50 ng/mL recombinant human EGF , 80 ng/mL recombinant human R-spondin 1 , 100 ng/mL recombinant human FGF2 , 50 ng/mL recombinant human HGF , 10 mM nicotinamide , 5 µM Y-27632 , and 2.5 µM prostaglandin E2 . Cultures were maintained in a 37oC humidified incubator with 5% CO2. Medium was renewed every 2–3 days. Small trophoblast organoid spheroids became visible by approximately day 12 post-isolation. Derived TOs were passaged every 5–7 days depending on their size and density. To passage, TOs were released from domes with ice-cold cell recovery solution  and then were digested in prewarmed TrypLE Express  or Stem Pro Accutase  at 37oC for 5–7 min. Further mechanical dissociation was achieved with an Eppendorf Explorer Plus electronic pipettes at its ‘mix’ function setting by pipetting up and down around 400 times through small-bore pipette tips. Dissociated TOs fragment were collected and washed by centrifuge, then resuspended in fresh ice-cold Matrigel and replated as domes at the desired density for continuous culture. For freezing TOs, overlaid TOM was aspirated, TOs with Matrigel were resuspended in CryoStor CS10 stem cell freezing medium  frozen at –80 oC and then transferred to liquid nitrogen for long-term storage. For thawing cryopreserved TOs, organoids were thawed as quickly as possible, diluted with five times volume of basic TOM containing Advanced DMEM/F12, 2 mM GlutaMAX supplement, 10 mM HEPES , 1× Penicillin/Streptomycin  and centrifuged to pellet. Afterward, TOs were resuspended in new ice-cold Matrigel and replated for recovery culture and passaged as described below. Trophoblast organoids used in this study were derived from 15 independent placentas . Detailed protocols for the isolation and passaging of trophoblast organoids derived from full-term placental tissue have been posted and are available for download  was TOM or ExM (described above) that had not been exposed to TOs or DOs, respectively. All experiments were performed with CM from at least three independent TO and DO lines.Human osteosarcoma U2OS (ATCC HTB-96) and Vero (ATCC CCL-81) cells were grown in Dulbecco’s minimal essential medium (DMEM) with 10% FBS and 1% antibiotic. HEK-Blue IFN-λ reporter cells (hkb-ifnl) were purchased from InvivoGen and cultured according to the manufacturer’s instructions. Cell lines were tested for mycoplasma at least every 2–4 weeks using the MycoStrip Mycoplasma Detection Kit . ZIKV  was propagated in Vero cells. Viral titers were determined by fluorescent focus assay as previously described  using reoC overnight. Organoids were then washed with PBS and then incubated for 1–2 hr at room temperature with Alexa Fluor-conjugated secondary antibodies (Invitrogen). Organoids were washed again with PBS and mounted in Vectashield (Vector Laboratories) containing 4′,6-diamidino- 2-phenylindole (DAPI) and were transferred onto microscope slides with large-orifice 200 µL tips . For staining performed in eight-well chamber slides, the same protocol described above was used, but the releasing of organoids from Matrigel was omitted. The following antibodies or reagents were used: cytokeratin-19 , E-cadherin , EpCAM , Mucin 5AC , SIGLEC6 , HLA-G , Ki67 , SDC-1 , J2 double-stranded RNA antibody as described previously  per well without disrupting their 3D architecture, then were fixed in 4% PFA for 30 min at room temperature, followed by 0.5% Triton X-100/phosphate-buffered saline [PBS] to permeabilize for 30 min at 4°C. Organoids were washed and blocked in 5% (v/v) goat serum/0.1% (v/v) Tween-20 in PBS for 15 min at room temperature and then incubated with primary antibodies in the above-described blocking solution at 4eviously , Alexa Feviously . For who20. Antigen retrieval was performed with slides submerged in 10 mM citrate buffer (pH 6.0) and heated in a steamer for 90°C for 20 min. Slides were cooled to room temperature and incubated with 6% H2O2 in methanol for 30 min. Following washing in 0.1% PBS-T , slides were incubated in Avidin blocking solution for 15 min, followed by subsequent blocking in Biotin blocking solution for 15 min . Following washing in 0.1% PBS-T, slides were then incubated with serum-free Protein Block  for 10 min. Sections were incubated with primary antibodies  and Ki67  diluted 1:250 in PBS-T overnight in a humidified chamber at 4°C. Next, slides were washed with PBS-T and incubated with secondary antibody  for 30 min, washed, and then incubated with avidin/biotin-based peroxidase  for an additional 30 min. Following washes in PBT-T, sections were incubated with DAB substrate  for ~5 min. Slides were washed with ddH20 and then counterstained with hematoxylin for 1 min, thoroughly rinsed with H2O, and incubated in 0.1% sodium bicarbonate in H2O for 5 min. Slides were then dehydrated with increasing concentrations of ethanol, cleared with xylene, and mounted with Vectamount Permanent Mounting Medium . Images were captured on an IX83 inverted microscope (Olympus) using a UC90 color CCD camera (Olympus).Tissue sections were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol , then washed with ddHT method normalized to actin. The expression level of C19MC miRNAs hsa-miR-516b-5p, hsa-miR-517a-3p, hsa-miR-525-5p, and reference gene RNU6 was quantified by using the MystiCq MicroRNA Quantitation System (Millipore Sigma) which includes miRNAs Poly(A) tailing, cDNA synthesis (MIRRT), and miRNA qPCR (MIRRM00). MicroRNAs were first polyadenylated in a poly(A) polymerase reaction, then reverse transcribed using an oligo-dT adapter primer according to the manufacturer’s protocol . Individual microRNAs  were quantified in real-time SYBR Green RT-qPCR reactions with the specific MystiCq microRNA qPCR Assay Primer  and the MystiCq Universal PCR Primer (MIRUP). Cq readings were normalized to the RNU6 internal reference by the 2-ΔCq method.Total RNA was extracted with the Sigma GenElute total mammalian RNA miniprep kit following the manufacturer’s instruction and using the supplementary Sigma DNase digestion. RNA quality and concentration were determined using a Nanodrop ND-1000 Spectrophotometer. Total RNA was reverse-transcribed with the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer’s instructions. Quantitative PCR was performed using the iQ SYBR Green Supermix  on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Gene expression was determined based on a ΔC2 ±2. Heatmaps and hierarchical clustering were performed in R using pheatmap package in R and were based on RPKM (reads per kilobase million). Mapping to HCMV genomes were performed in CLC Genomics (v20) using either AD169r (accession FJ527563) or TB40E (accession MW439039) reference sequences. Files associated with RNA-seq studies have been deposited into Sequence Read Archive under accession number SUB11885513. Previous data sets used in this study include those from PHT cells (accession PRJNA305417 and PRJNA388219)  prior to library preparation by the Duke Center for Genomic and Computational Biology (GCB) using the TruSeq stranded total RNA prep kit (Illumina). Sequencing was performed on a NovaSeq 6000 or NextSeq500 by using 150 bp paired-end sequencing. The reads were aligned to the human reference genome (GRCh38) using QIAGEN CLC Genomics (v20). DESeq2  was usedA388219) , first tA388219) . PrincipA388219)  or GraphA388219)  or in GrAll sample processing was performed in duplicate and each experiment was performed with at least three biological replicates. Luminex assays were performed using the following kits according to the manufacturer’s instructions: Bio-Plex Pro Human Inflammation Panel 1, 37-Plex kit  Bio-Plex Human Chemokine Panel, 40-plex , Bio-Plex Pro Human Cytokine Screening Panel, 48-Plex kit , hCG Human ProcartaPlex Simplex Kit , and CA125 (Mucin-16) Human ProcartaPlex Simplex Kit (EPX010-12437-901). Plates were read on MAGPIX (EMD Millipore) or Bio-Plex 200 (Bio-Rad) Luminex machines and analyzed using xPONENT (EMD Millipore) or Bio-Plex (Biorad) software.oC for 24 hr. Supernatants (20 µL) were collected and then transferred into a 96-well plate and activity measured using QUANTI-Blue Solution  after incubating at 37oC for 2 hr using a spectrophotometer at 655 nm. Each CM preparation was run in triplicate.For IFN-λ activity detection from CM, HEK-Blue IFN-λ reporter cells were used according to the manufacture’s protocol. Briefly, IFN-λ reporter cells were plated into a 96-well plate, then CM was added and incubated at 37oC, then were infected with GFP-tagged AD169r in the presence of ruxolitinib for 48 hr.For Poly (I:C) treatment, TOs or DOs were incubated with 10 µg/mL Poly (I:C)  diluted in organoid complete growth media for ~24 hr, then supernatant was collected for Luminex-based multianalyte profiling or RNA isolated as described for qRT-PCR or RNASeq. For ruxolitinib  treatment, TOs or DOs plated as previously described for HCMV infections were pre-treated with 20 µM ruxolitinib in appropriate complete growth media for 1 hr at 37All experiments reported in this study have been reproduced using independent samples (tissues and organoids) from multiple donors. All statistical analyses were performed using Prism software (GraphPad Software). Data are presented as mean ± SD, unless otherwise stated. Statistical significance was determined as described in the figure legends. Parametric tests were applied when data were distributed normally based on D’Agostino-Pearson analyses; otherwise, nonparametric tests were applied. P values <0.05 were considered statistically significant, with specific p values noted in the figure legends. Yang et al., provide a scientifically sound and compelling manuscript characterizing mid-to-late gestation trophoblast and decidual organoids as ex vivo models to study vertically transmitted microbial infections, using human cytomegalovirus as a model pathogen. They demonstrate organoids have tissue-specific immunological responses and susceptibilities to viral infection. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Sara Sawyer as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Rowan Karvas (Reviewer #2).Thank you for submitting your article "Innate immune signaling in trophoblast and decidua organoids defines differential antiviral defenses at the maternal-fetal interface" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.The reviewers agree that this is a strong and impactful manuscript describing the establishment and characterization of mid-to-late gestation trophoblast and decidual organoids and their use in studying viral infection. Several points need to be addressed to support conclusions of which the essential revisions are pointed out below.Essential revisions:(1) There is only a single readout for viral infection with quantification being % infected organoid (which could include organoids with only a single cell or hundreds of cells infected). A more fine-tuned quantification seems necessary given the conclusions of the manuscript.(2) More convincing experimental data that EVTs contained in TOs are not susceptible should be included. The reviewer suggests: "The authors should differentiate their organoids into EVT organoids as described in Sheridan et al., Nature Protocols 2020 and try infection with their fluorophore-tagged hCMV viruses. Spontaneously formed pre-EVT within TOM-treated organoids are not very mature compared to EVTs within properly differentiated EVT organoids. They could try infection and whole mount image, OR dissociate the organoids and run a flow cytometry experiment to quantify the proportion of infected cells within their EVT organoids and TOM (cytotrophoblastic and syncytiotrophoblastic) organoids. “(3) A weakness in this work is that there was no characterization of the resulting organoids proving that they indeed transcriptionally or epigenetically model a second-third trimester timepoint” or really any experimental comparison with first trimester to see how gestation age is reflected/odellin.Reviewer #1 (Recommendations for the authors):– The introduction should include a description of trophoblasts organoid orientation and what the decidual organoids are odelling .– Does the gestational age of TO and DO derivation correlate with the variability of expression of other cytokines? Or on any other readout?– How was CM standardized across organoids ? Could hcg be used to standardize?– For ease of interpretation, for Figure 6, please indicate what antigen is stained for directly under each image with words/colors as done in other figures.– The current quantification of HCMV infection does not give a sense for the number of cells infected in each organoid. Consider an additional quantification that would better capture infection dynamics.– Are these productive infections?– Is the lack of cytokine and transcriptional response to HCMV infection simply due to the Tos not being infected? This should be included in discussion.– Despite commonly indicating TO and DO are matched, the use of this matched specificity is not utilized either experimentally or in data interpretation. Does matched matter?– Does TO CM prevent infection of DO? Does it act in paracrine to relevant cell types as concluded?– Can TO CM prevent HCMV infection? This seems quite relevant given the focus on HCMV infection dynamics and the stark differences between Zika (an emerging RNA virus) and HCMV (DNA virus that has coevolved with host).Reviewer #2 (Recommendations for the authors):Listed below are my questions:nd-3rd trimester Tos compared to first which show abundant STB formation.(1) The Tos shown in Figure 1C seem largely epithelial with few syncytium. Typically there are sporadically formed portions of STB (syncytiotrophoblast) that form internally. This could be due to their mid-late gestation source. I would like to see some staining for SDC1, hCG, etc. Something to indicate the location and abundance of spontaneously formed STB. I think this piece of data would be important to the trophoblast field to show similarities and differences of the structure of 2(2) The TO medium from your methods is very similar to Turco’s TOM medium, but you do state that nicotinomide was added for improved proliferation. That is reasonable, but I also noticed that 10% FBS is also added but there is no rationale for this addition to the medium.Listed below are my critiques:(1) As stated in my public review, I am concerned about the “attracting forces” of cell culture medias and in vitro culture generally. It is stated that first trimester timepoints are not as susceptible to HCMV infection as later stages and it is shown that your Tos are not susceptible to HCMV. The productions of IFN-lambda2 could be responsible for this protection, but it is not proven to me that these organoids, while derived from mid-late gestation, continue to resemble those timepoints in vitro and this requires transcriptional or maybe epigenetic comparisons to other first and second trimester placental datasets.2) Also stated in the public review- I would really like for the authors to differentiate Tos into EVT organoids and repeat HCMV infections to prove that EVTs are or are not susceptible to HCMV infection. Please provide whole staining/confocal analysis OR infect with the fluorescent HCMVs, dissociate, and perform flow cytometry analysis to demonstrate susceptibility of EVT Tos to HCMV compared to Tos in TOM.(3) An additional experiment that would really improve the impact of this paper is to attempt a co-culture model with both Tos and Dos together and repeat HCMV infection. Could Tos protect Dos from infection? Essential revisions:(1) There is only a single readout for viral infection with quantification being % infected organoid (which could include organoids with only a single cell or hundreds of cells infected). A more fine-tuned quantification seems necessary given the conclusions of the manuscript.Titration of HCMV infections is technically challenging in general, but even more so given the low levels of infection in many cell systems, including our organoid models. Nonetheless, to address this concern, we now include bulk RNASeq data mapping HCMV-associated reads to two genes important for HCMV replication in infected Tos and Dos , which supports the low levels of replication in Tos.(2) More convincing experimental data that EVTs contained in Tos are not susceptible should be included. The reviewer suggests: “The authors should differentiate their organoids into EVT organoids as described in Sheridan et al., Nature Protocols 2020 and try infection with their fluorophore-tagged hCMV viruses. Spontaneously formed pre-EVT within TOM-treated organoids are not very mature compared to EVTs within properly differentiated EVT organoids. They could try infection and whole mount image, OR dissociate the organoids and run a flow cytometry experiment to quantify the proportion of infected cells within their EVT organoids and TOM (cytotrophoblastic and syncytiotrophoblastic) organoids. "To address this concern, we applied the EVT differentiation protocol in Sheridan et al. Because this protocol has not previously been applied to TOs from full-term tissue, we also include key data to support the success of this protocol. We now show that TOs isolated from full-term tissue respond to the EVT differentiation protocol as described in Sheridan et al., which correlated with an increase in HLA-G positive cells from ~18% to ~96% in undifferentiated versus differentiated conditions . This differentiation also correlated with a significant increase in MMP-2, which is released at high levels from first trimester EVTs .Because we successfully applied the EVT differentiation to TOs, we compared the levels of HCMV infection between undifferentiated and differentiated TOs. This showed that TOs differentiated to contain >95% HLA-G positive cells remained highly resistant to HCMV infection , supporting our conclusion that EVTs are not the primary targets of HCMV in TOs.3) A weakness in this work is that there was no characterization of the resulting organoids proving that they indeed transcriptionally or epigenetically model a second-third trimester timepoint" or really any experimental comparison with first trimester to see how gestation age is reflected/modeled.To address this concern, we used publicly available bulk RNASeq datasets generated by our own group and others to perform comparative expression analysis between TOs and placental tissue generated from the first or second trimesters, or from full-term tissue. Hierarchical clustering analysis using key hormones and other factors associated with trophoblast structure and function revealed that TOs were most closely related to tissue explants from the first trimester .Reviewer #1 (Recommendations for the authors):– The introduction should include a description of trophoblasts organoid orientation and what the decidual organoids are modeling .Lines 86-87 of the original manuscript read as follows: “Similarly, decidua organoids isolated from uterine glands recapitulate the transcriptional profile of their tissue of origin and respond to hormonal cues..”. However, to make this clearer, we now include the following statement on lines 124-125 “…cultured progenitor cells isolated from chorionic villi or uterine glands of the decidua…”.– Does the gestational age of TO and DO derivation correlate with the variability of expression of other cytokines? Or on any other readout?There were no apparent differences in cytokine levels or other readouts of TOs used in this study, regardless of the gestational stage in which they were isolated. We have included individual datapoints from unique organoid preparation throughout the manuscript to demonstrate the variability that we observe between organoids lines, which is expected given that they are from human tissue. Of note, we now include a comparison of the transcriptional profiles of the TOs used in our study to placental tissue from the first and second trimesters, and from full-term tissue, and these studies indicated that TOs are most closely associated transcriptionally with first trimester tissue .– How was CM standardized across organoids ? Could hcg be used to standardize?We have added a clearer explanation of the quality control measures we take for CM, which are based on hCG levels and overall organoid numbers and morphology. This description can be found on lines 809-815 of the revised manuscript.– For ease of interpretation, for Figure 6, please indicate what antigen is stained for directly under each image with words/colors as done in other figures.This has been added, as requested.– The current quantification of HCMV infection does not give a sense for the number of cells infected in each organoid. Consider an additional quantification that would better capture infection dynamics.We now show infection data as a percent of CMV+ cells over total nuclei taken from two dimensional images, and as now described on the methods.– Are these productive infections?It is unclear whether the reviewer refers to productive infections in DOs or TOs. In the case of DOs, the reporters of both AD159r and TB40E strains of HCMV are fused to transcripts associated with late replication of HCMV, suggesting that these are in fact productive infections. In the case of TOs, the lack of signal for either reporter, coupled with the low levels of production of key virally-encoded transcripts required for HCMV replication , suggests that the infection is not productive.– Is the lack of cytokine and transcriptional response to HCMV infection simply due to the TOs not being infected? This should be included in discussion.Despite the low level of overall productive infection, there are more transcripts induced in TOs infected with HCMV than in DOs (282 transcripts (AD169r strain) or 173 transcripts (TB40E strain) in TOs and 95 (AD169r strain) or 117 (TB40E strain) in DOs). Thus, the differences observed do not seem to result from differences in overall infection levels.– Despite commonly indicating TO and DO are matched, the use of this matched specificity is not utilized either experimentally or in data interpretation. Does matched matter?There does not appear to be a requirement for the use of matched organoid cultures. In fact, we have successfully generated co-cultures of unmatched TOs and DOs, which appears to work equivalently well regardless of whether they were derived from unmatched tissue (it should be noted that the co-cultures used in this study are from matched tissue). We have made this clearer in the text (lines 814-815).– Does TO CM prevent infection of DO? Does it act in paracrine to relevant cell types as concluded?In the revised manuscript, we show that CM isolated from TOs reduces ZIKV infection in DOs .– Can TO CM prevent HCMV infection? This seems quite relevant given the focus on HCMV infection dynamics and the stark differences between Zika (an emerging RNA virus) and HCMV (DNA virus that has coevolved with host).As requested by reviewer 2, and to address this question, we now provide data from co-cultures of TOs and DOs, which shows that the presence of TOs confers protection of DOs from HCMV infection .Reviewer #2 (Recommendations for the authors):Listed below are my questions:(1) The TOs shown in Figure 1C seem largely epithelial with few syncytium. Typically there are sporadically formed portions of STB (syncytiotrophoblast) that form internally. This could be due to their mid-late gestation source. I would like to see some staining for SDC1, hCG, etc. Something to indicate the location and abundance of spontaneously formed STB. I think this piece of data would be important to the trophoblast field to show similarities and differences of the structure of 2nd-3rd trimester TOs compared to first which show abundant STB formation.We now include SDC-1 immunostaining from TOs, which shows that there are large numbers of SDC-1 positive cells in TOs . We also note that TOs express high levels of many STB markers  as assessed by bulk RNASeq .(2) The TO medium from your methods is very similar to Turco's TOM medium, but you do state that nicotinomide was added for improved proliferation. That is reasonable, but I also noticed that 10% FBS is also added but there is no rationale for this addition to the medium.Our optimization revealed that 10% enhanced the growth rate of TOs. This has been added to the methods.Listed below are my critiques:(1) As stated in my public review, I am concerned about the "attracting forces" of cell culture medias and in vitro culture generally. It is stated that first trimester timepoints are not as susceptible to HCMV infection as later stages and it is shown that your TOs are not susceptible to HCMV. The productions of IFN-lambda2 could be responsible for this protection, but it is not proven to me that these organoids, while derived from mid-late gestation, continue to resemble those timepoints in vitro and this requires transcriptional or maybe epigenetic comparisons to other first and second trimester placental datasets.In the revised manuscript, we show that the transcriptional signature of TOs more closely resembles placental tissue isolated from the first trimester than from the second trimester of from full-term tissue. Other groups have also demonstrated the remarkable resistance of first trimester placental tissue to viral infections .(2) Also stated in the public review- I would really like for the authors to differentiate TOs into EVT organoids and repeat HCMV infections to prove that EVTs are or are not susceptible to HCMV infection. Please provide whole staining/confocal analysis OR infect with the fluorescent HCMVs, dissociate, and perform flow cytometry analysis to demonstrate susceptibility of EVT TOs to HCMV compared to TOs in TOM.As indicated above, we have performed EVT differentiation, which we fully characterized  and show that this does not impact the levels of HCMV infections .(3) An additional experiment that would really improve the impact of this paper is to attempt a co-culture model with both TOs and DOs together and repeat HCMV infection. Could TOs protect DOs from infection?As suggested, we established co-cultures of TOs and DOs and found that presence of TOs in these co-cultures significantly decreased the susceptibility of DOs to HCMV infection


MYOD1, MYF5 and MYOG genes in PRGF groups at T1 compared to Saline treated muscles. At ultrastructural level, PRGF groups presented scarce edema and loss of connective tissue structure, as well as higher mitochondrial density adequately associated to the sarcomere unit in contrast to the Saline group. In conclusion, histological, biochemical, and ultrastructural results showed that PRGF treatment improved muscle regeneration process leading to more mature histological aspect in newly formed muscle tissue after a surgically induced grade II muscle injury.The purpose of this study was to perform a histological and biochemical evaluation of the influence of plasma rich in growth factors (PRGF) on muscle regeneration process after a surgically induced grade II muscle laceration. A randomized, single blind, controlled experimental research was conducted including twenty-one adult healthy sheep, randomly divided in three groups (n = 7). A grade II surgical section was performed in the biceps femoris muscle of both hindlimbs. After two days , intralesional infiltration of autologous PRGF or Saline solution was randomly administered in both hindlimbs. Treatment was repeated once a week. Animal groups were euthanized at 1 (T1), 2 (T2) or 4 (T4) weeks. Histological assessment showed that PRGF intralesional injection induced a significant decrease of inflammatory cells density, significant higher centrally nucleated fibers percentage and significantly smaller fibrotic areas compared to Saline-treated muscles at T1, T2 and T4. Also, lower vascular density, with lower capillaries cross-sectional area, in PRGF group compared to Saline was observed. Biochemical analysis revealed a significant higher expression level of The online version contains supplementary material available at 10.1186/s12917-022-03491-2. Muscle injuries are common lesion in athletes and workers that causes pain and disability with significant social impact and economic losses –3. GradeAn inflammatory response occurs when muscle fibers are partially severed, and different inflammatory cells are sequentially attracted to the damaged area , 6. ThisSkeletal muscle tissue presents reparation capacity led by an intrinsic mechanism conducted by a group of undifferentiated reserve cells called satellite cells . In respThe cellular events described during muscle reparation are orchestrated by a pool of growth factors and cytokines, most of which are released by platelets , 3, 10. The purpose of this experimental study is to investigate muscle regeneration properties of intralesional PRGF therapy for surgically induced grade II muscle injury in a sheep model. It was hypothesized that this treatment could modulate inflammation and enhance regeneration properties to restore normal or nearly skeletal muscle tissue.Twenty-one skeletally mature 2- to 3-year-old female sheep, weighing 50–55 kg were included in the study. All sheep used in this study were deemed healthy beforehand and did not suffer from any orthopedic or systemic conditions. Animals were randomly divided into three groups, T1 (n = 7), T2 (n = 7), and T4 (n = 7), according to the time when they were sacrificed along the experiment, at 1 week, 2 weeks, or 4 weeks after treatment, respectively. All procedures were carried out following the guidelines of the European Union (Directive 2010/63/EU) and approved by the Bioethical Committee on Animal Research at Andalucía . In addition, authors affirm that the research was conducted in accordance with the ARRIVE guidelines.Sheep were anaesthetized and both thighs were aseptically prepared for surgery. First, a sonographer performed an ultrasound evaluation to confirm the absence of abnormalities in biceps femoris muscle . In order to standardize the exact point of the surgical lesion, the thickest muscle area to perform the grade II muscle injury was measured mid-point in between the greater trochanter and the proximal aspect of the lateral condyle of the femur. A surgical muscle laceration equivalent to grade IIc injury, consisting of a partial tears of muscle fibers, was performed under ultrasound guidance in the biceps femoris muscle involving 70% of the muscle fibers in all the sheep using a scalpel blade #22  . The magBlood was collected just prior to treatment from the jugular vein of each animal and was divided into four sterile tubes containing 3.8% trisodium citrate. Each 5-mL extraction tube contained 0.5 mL of anticoagulant. Blood was centrifuged at 630 g for 8 min according to the method reported to obtain PRGF  in sheep . The fraSheep were sedated for treatment injection 48 h after muscle injury. First, hematoma was evacuated from the muscle laceration site using a 21-G needle under ultrasound guidance. PRGF or Saline solution  infiltrations were randomly assigned for each thigh. Platelets contained in PRGF (2 mL) were activated just prior to treatment by adding 50 µL of calcium chloride 10% for 1 mL of plasma ratio, and injected directly into the injured site under ultrasound guidance as previously described in Bubnov et al. . The timAll the animals were humanely sacrificed at 1 week (T1 group), 2 weeks (T2 group), or 4 weeks (T4 group) after the first treatment with PRGF or Saline solution, by using a standard protocol with T61™ .After euthanasia, biceps femoris muscles of both thighs were dissected proximal-distally to the damaged area in each animal. Muscle damage area was divided, so one half was used for histochemical evaluation, and the other half was used for ultrastructural and biochemical studies. Samples of the vastus lateralis quadriceps femoris were taken and used as a normal skeletal muscle control (Control group). Samples were oriented keeping muscle fibers along their longitudinal axis.For histochemical examination, muscles samples were fixed in 10% neutral buffered formalin and embedded in paraffin. Four µm-thick sections were stained with Hematoxylin and Eosin (HE) and Masson Trichrome with aniline blue (MT) . Histological images were evaluated using a photomicroscope  with an attached digital camera controller .2), blood vessels density (blood vessels/mm2), blood vessels area (µm2) and percentage of centrally nucleated fibers with respect to total muscle fibers. MT stained histological images were used to assess areas of fibrillar collagen to estimate the percentage of fibrotic areas (FA) development.From HE and MT stained sections, five histological images per slide were randomly taken at 200x magnification and were analyzed with ImageJ software . HE stained histological images were used to study the inflammatory cells density (cells/mmFor transmission electron microscopy (TEM) evaluation, all the muscle samples were fixed in glutaraldehyde 2% for 48 h at 4 ºC and included in Araldite. Ultrathin sections were stained with uranyl acetate and plumb citrate and evaluated using a transmission electronic microscope Jeol-1400 .−ΔΔ Ct method using GAPDH as housekeeping gene.Muscle tissue samples stored at -80 ºC were thawed and processed for RNA extraction with TRI reagent . Total RNA was quantified by spectrophotometry . Afterwards, total RNA treated with DNAse was used to analyze mRNA expression of MYOD1, MYF5, MYOG and Myostatin (MSTN) genes by RT-PCR using SensiFAST SYBR No-ROX One-Step kit  according to the manufacturer instructions. RT-PCR was performed using a Lightcycler 480 equipment . Primer sequences are shown in Table The data were statistically evaluated using SPSS 17.0 software . All variables were analyzed for normality of distribution by the Kolmogorov-Smirnov test. Histochemical variables , and level of expression of MYOD1, MYF5, MYOG and MSTN were expressed as mean ± standard deviation. A mixed general lineal model was performed to analyze each variable with respect to the muscles sample . Furthermore, each variable was analyzed with respect to different timepoints  throughout the study. A one-way ANOVA with post hoc Bonferroni test were used to evaluate differences between groups . Significance level was standardized as p < 0.05.All animals reached the end of the study without incidents or adverse effects.PRGF group, Saline solution group and Control group results are presented in Table Regarding vascularization analysis, Control group showed small capillaries distributed through the muscular tissue with low vascular density. After surgical grade II injury of biceps femoris, both PRGF-treated muscles and those injected with Saline solution suffered an important increase in the vascular area, showing significantly larger size capillaries than normal skeletal muscle. PRGF treated muscles exhibited statistically significant smaller vascular area than in those treated with Saline solution at T1 (p = 0.023) and T2 (p = 0.001). Vascular density was significantly increased in both surgically injured muscles injected with PRGF or Saline solution compared with the intact skeletal muscle. All the muscles showed a significant progressive drop in vascular density along the study. PRGF groups exhibited lower vascular density than Saline solution groups with significant differences at T4 (p = 0.019)CNF were not observed in normal skeletal muscle. After the surgical damage of biceps femoris, a significant progressive increase of the percentage of CNF was registered from T1 to T4 groups in PRGF and Saline solution groups. Comparison between PRGF and Saline treatments revealed that percentage of CNF was significantly higher in muscles injected with PRGF than those treated with Saline solution at T1, T2 and T4. This difference was maintained throughout the experiment (p = 0.001)  . However, on later stage of muscle healing at weeks 2 and 4, no differences were registered between the PRGF and Saline groups. Regarding MSTN, as inhibitor gene of muscle regeneration, no statistical differences were registered in the comparison either between injured muscles and Control group, or between PRGF and Saline groups throughout the experiment.Level of expression of myogenic genes Muscle injuries are one of most frequently reported lesions related to the sports world . AccelerPRGF is an autologous treatment that accelerates tissue regeneration and reduces the recovery period, improving the quality of tissue repaired with the aim of decreasing relapse incidence –30. TherIn the present study, the anatomical references considered and ultrasound guidance used were useful to locate the thickest muscle area to precisely determine the surgical lesions site. Furthermore, the grade II muscle injuries in the biceps femoris muscle was accurately standardized using an ultrasound muscle injury grading scale previously reported . UltrasoWhen muscle fibers are partially severed, inflammatory phenomena occurs and different inflammatory cells are sequentially attracted to the damaged area , 6. SeveThe restoration of vascular supply is prerequisite for subsequent morphological and functional recovery of the injured muscle . In the Muscle regeneration have been evaluated in several experiments using CNF counting as histological criteria , 15. OurDuring muscle repair process, connective tissue occupies the gap created in between the ruptured myofibers . The proThe hypertrophic reaction described in all the surgically damaged muscles is associated to the activation of satellite cells and the beginning of its division, with the aim of regenerating the injured muscle . The ultMYOD1, MYF5 and MYOG in the precocious stage of muscle regeneration at one week after grade II muscle rupture. This finding, together with statistically higher percentage of CNF previously described in the group of muscles treated with PRGF, suggests that PRGF therapy enhances the regeneration stimulus of muscle, accelerating the repair process, and subsequently it may shorten the convalescence period after grade II muscle injury. On the other hand, MSTN level of expression did not show statistical differences with respect to the PRGF of Saline solution therapy along the study. Thereby, muscle regeneration process is not being inhibited despite PRGF o Saline solution injection.At biochemical level, the expression of muscle regulatory factors becomes objective criteria that have been utilized to evaluate muscle regeneration , 18. In Several types of PRP have been used in clinical setting with the goal of accelerating the muscle regeneration process, as well as avoiding fibrotic tissue scar at the end of muscle healing procedure , 46–52. There are several limitations to this study. First, ultrasonographic follow-up was not evaluated to check the muscle repair stage on ultrasound-imaging. The goal of the present study was to evaluate the histological and genomic effect of PRGF on muscle regeneration process. Therefore, ultrasound technique was only used as guidance to intralesional infiltration of PRGF or Saline solution, as previously described . Second,MYOD1, MYF5 and MYOG) was significantly higher in PRGF-treated muscles at the early stage of muscle regeneration. Furthermore, PRGF treatment leaded to newly formed muscle tissue with significant smaller fibrotic areas along the study, in contrast with Saline-treated muscles. These findings suggested an anti-inflammatory effect of PRGF on muscle repair, with an enhanced muscle regeneration stimulus, and improved repair quality after a surgically induced grade II muscle injury.In conclusion, PRGF intralesional treatment produced a significant decrease in the inflammatory cell density, with lower vascular density and blood vessel area, in comparison with the Saline-treated muscles. PRGF stimulated a significant higher percentage of CNF, and also the expression of myogenic genes (Below is the link to the electronic supplementary material.Supplementary Material 1


Proprotein convertase subtilisin kexin type 9 (PCSK9) is a proprotein convertase that increases plasma low-density lipoprotein cholesterol (LDL-C) levels by triggering the degradation of LDL receptors (LDLRs). Beyond the regulation of circulating LDL-C, PCSK9 also has direct atherosclerotic effects on the vascular wall and is associated with coronary plaque inflammation. Interestingly, emerging data show that women have higher circulating PCSK9 concentrations than men, suggesting that the potential roles of PCSK9 may have different impacts according to sex. In this review, we summarize the studies concerning sex difference in circulating levels of PCSK9. In addition, we report on the sex differences in the relations of elevated circulating PCSK9 levels to the severity and prognosis of coronary artery disease, the incidence of type 2 diabetes mellitus, and neurological damage after cardiac arrest and liver injury, as well as inflammatory biomarkers and high-density lipoprotein cholesterol (HDL-C). Moreover, sex difference in the clinical efficacy of PCSK9 inhibitors application are reviewed. Finally, the underlying mechanisms of sex difference in circulating PCSK9 concentrations and the clinical implications are also discussed. Major differences exist between men and women in the epidemiology, pathophysiology, and outcomes of cardiovascular diseases (CVDs) . SignifiSex differences in CVD are due to differences in gene expression and the regulation of sex hormones that lead to differences in various cardiovascular functions, for example, in nitric oxide (NO) signaling, myocardial remodeling under stress, glucose regulation and lipid metabolism . PreclinThis systematic review examined sex difference in circulating PCSK9 levels and the underlying mechanisms and clinical implications of these sex differences. The selected studies included cross-sectional, case-control and prospective studies. There were no language or time restrictions for eligible studies. The PubMed electronic database was used. The following search terms were used: “proprotein convertase subtilisin kexin type 9” OR “PCSK9” AND “sex difference” AND “estrogen” OR “estradiol” AND “coronary artery diseases” OR “type 2 diabetes mellitus” OR “neurological damage after cardiac arrest” OR “liver injury” OR “inflammatory biomarkers” OR “HDL-C” OR “PCSK9 inhibitor”.Increasing evidence shows that PCSK9 is a well-known therapeutic target in the prevention and treatment of atherosclerosis. PCSK9 is an important enzyme in cholesterol metabolism that regulates serum low-density lipoprotein cholesterol (LDL-C) levels through the degradation of LDLRs. PCSK9 inhibitors could lead to significant LDL-C reductions in high-risk patients, and they have shown a favorable safety profile in recent clinical trials . Beyond p < 0.001)  in the IMPROVE cohort study, a large-scale multicenter study encompassing several European countries with the centralized measurement of PCSK9 . The eff< 0.001) . Althoug< 0.001) . Many st< 0.001) . Levensop < 0.05), whereas the two groups of male participants had similar PCSK9 levels   .in vitro fertilization. In vitro fertilization involves 2 treatment phases: Extreme suppression and strong stimulation of the endogenous estrogen levels. A comparison of PCSK9 levels in these two phases shows that PCSK9 levels are significantly reduced by 14% after the stimulation of estrogen synthesis, which indicates that high levels of endogenous estrogens reduce circulating PCSK9 levels (The regulating effects of estrogen on PCSK9 expression noted in recent studies are as follows: 1) PCSK9 levels increase in women after menopause, along with the sharp reduction in estrogen that occurs during this time period; however, PCSK9 levels do not increase in men during this time period . 2) PCSK9 levels .In vitro studies have also found that estrogens could affect the PCSK9 functional state through a posttranscriptional mechanism. One of the possible mechanisms for the posttranscriptional inhibiting effect is that estradiol could block PCSK9 internalization in hepatic cells. The activation of GPER in the membrane by estradiol rapidly initiates the phosphorylation of phospholipase C-γ (PLC-γ), which in turn alters clathrin distribution or influences the clathrin trafficking pathway to affect the internalization of the PCSK9-LDLR complex in HepG2 cells , neurological damage after cardiac arrest, liver injury as well as inflammatory biomarkers and HDL-C are complex; moreover, the clinical implications of sex difference in PCSK9 levels need to be clarified.It is clear that PCSK9 contributes to every step of the molecular pathway of atherosclerosis. Recent studies have revealed that PCSK9 has various effects on the progression of atherosclerosis, including inflammation, foam cell formation, endothelial cell apoptosis, smooth muscle cell phenotypic switching, and platelet activation . Many clInvasive and pathological evidence has suggested sex-specific differences in the pattern of compositional plaque progression: Men are more likely to have plaque rupture and occlusion that is associated with sudden onset symptoms than women, and women can present with plaque rupture as well as plaque erosion and an indolent course of anginal symptoms . Women sIn addition to the associations between PCSK9 levels and coronary artery lesion severity in women, knowledge of the relation of PCSK9 to CAD outcomes may also require further, more specific exploration. Although sex differences in CAD outcomes are not consistent among all reports, many studies have shown that female patients do not have more favorable outcomes than male patients; moreover, several studies have demonstrated an even worse prognosis in female patients than male patients. Women with stable angina and nonobstructive CAD were found to be 3 times more likely to experience a cardiac event within the first year of cardiac catheterization than men . Comparep = 0.01) (The circulating PCSK9 level is independently predictive of major adverse cardiovascular events (MACEs) in patients with stable CAD . PCSK9 w = 0.01) . A prosp = 0.01) . The sam = 0.01) . Patient = 0.01) . The sexThe findings from the available clinical study on PCSK9 and T2DM suggest a trend toward a positive association between circulating PCSK9 levels and the incidence of T2DM in renal transplant recipients . MoreoveWe suggest that gonadal hormones may be an important confounding factor for the relationship between circulating PCSK9 levels and the incidence of T2DM. Circulating PCSK9 levels change in women depending on their reproductive stage of life, and serum estrogen is inversely correlated with circulating PCSK9 levels, while in men, serum testosterone is not correlated with circulating PCSK9 levels . In womeCardiac arrest causes significant morbidity and mortality, and women have been found to have worse outcomes despite improvements in prehospital and hospital care. Women were associated with a lower likelihood of good neurological outcomes at discharge and the 6-month follow-up in a multinational retrospective registry of patients who suffered out-of-hospital cardiac arrest . HoweverAn extensive investigation of PCSK9 revealed its novel potential functions, including the regulation of neuronal development, apoptosis and differentiation, but the precise role of PCSK9 in brain physiology remained unclear . MoreovePCSK9 has recently been shown to influence inflammatory responses in the liver. Circulating PCSK9 levels were associated with steatosis severity in patients who underwent liver biopsy for suspected nonalcoholic steatohepatitis . The meaPCSK9 inhibition seems to exert a protective effect against hepatic damage in NAFLD. A PCSK9 loss-of-function variant in humans was protective against liver steatosis and fibrosis in individuals with NAFLD . The genThe difference in vascular inflammation between men and women has recently become the focus of many studies . The proHDL-C is inversely associated with CVD across a wide range of concentrations. Indeed, different HDL subpopulations may have different functional properties since HDL particles are heterogeneous in size and biochemical composition. Numerous studies have shown that small- and medium-sized HDL particles are inversely related to cardiovascular risk . On averDespite the overwhelming evidence of cardiovascular benefits from trials with lipid-lowering medications, there is evidence that women are often undertreated in clinical practice . MoreoveOne concern related to PCSK9 inhibitors is whether they may increase the risk of impaired glucose metabolism and the development of new-onset diabetes; however, recent clinical trial data for PCSK9 monoclonal antibodies suggest that the application of alirocumab and evolocumab in the treatment of atherosclerotic disease does not increase the risk of new-onset diabetes or worsen glycemia. In addition, the inhibition of PCSK9 by polydatin, a natural antidiabetic product, could modify glucose metabolism disorders and thereby ameliorate diabetic complications by increasing the expression of liver glucokinase, a key enzyme in glucose metabolism . NonetheIn the FOURIER trial, the LDL-C reduction with evolocumab at 4 weeks was nominally greater in men than women, but the relative risk reductions in endpoint analysis were similar in women and men. The large size of the FOURIER trial population provides a robust evidence base that the absolute risk reductions of adverse events with evolocumab application were similar in both men and women, and no important safety issues were observed in either sex . ConsistThe administration of PCSK9 monoclonal antibodies to patients with hyperlipidemia and CAD led to a 50–70% reduction in LDL-C levels . Small-mAlthough the overall mortality in patients with CAD has dramatically declined over recent decades as a result of preventive strategies, the decline has been less significant for women . The phy


IV cisplatin in randomized studies in patients undergoing optimal surgical debulking at diagnosis. In the past two decades, with the introduction of novel anticancer interventions , advantages of IP drug delivery are less clear and concerns are raised on cisplatin's therapeutic index. The discovery of BRCA genes and their key role in DNA repair, on the other hand, have strengthened the rationale for IP drug delivery: high grade serous cancers arising in the Mullerian epithelium in association with hereditary or somatic BRCA function inactivation are linked to peritoneal spread of cells that - while initially sensitive - are prone to emergence of platinum resistance. Therefore, selection of patients based on genomic features and focusing on the better tolerated IP carboplatin are ongoing. Recent examples of leveraging the peritoneal route include (1) targeting the cell membrane copper transport receptor - that is shared by platinums - by the combination of the proteasome inhibitor bortezomib and IP carboplatin; and (2) enhancing IP 5-fluoro-2-deoxyuridine cytotoxicity when coupled with PARP inhibition.Intraperitoneal (IP) delivery of cisplatin was developed in the 1970s based on a strong pharmacologic rationale and rodent models. Its advantage over intravenous (IV) administration was supported initially by observational studies in treating recurrent ovarian cancer and eventually by better outcomes from IP  Early in its clinical development, cisplatin showed unprecedented activity against ovarian cancer - a disease that is usually diagnosed at advanced stages, with surgery most often playing an ancillary role in its treatment. Although cisplatin - and the subsequent platinum analogs introduced in order to attenuate some of its toxicities - can be relied to induce tumor regressions in up to 90% of late presentations, most women’s cancers eventually recur. In its most common subtype, the high-grade serous adenocarcinoma, tumor progression occurs predominantly in the peritoneal cavity a few months to years after treatment with surgery and the initial “platinum-doublet”. These regimens usually combine a platinum with paclitaxel, with the latter occasionally replaced by other drugs with similar anti-tumor activity as single agent taxanes. Recurrences are commonly classified as “platinum resistant” or “platinum sensitive” based on the timing of when these relapses are documented: if after completing 5 or 6 cycles of platinum-containing regimen, the designation “platinum resistant” is given,3. This arbitrary “operational definition” recognizes that cancer reappearance within 6 months of completing 5-6 platinum cycles usually yields at best short-lasting benefits when treated again with a platinum as single agent or as part of a doublet. In fact, even “platinum sensitive” recurrences may reflect some degree of platinum resistance, as suggested by retrospective analysis of 176 women receiving two or more platinum-based regimens for recurrent ovarian cancer, with “the length of a prior response (to the preceding regimen) highly predictive of the upper limit of the duration of response achieved to a subsequent platinum program”.Platinum resistance is the central issue in the treatment of most ovarian cancers. The development of cisplatin as an anticancer agent was reviewed by us at the 50th anniversary of the discovery of its potential anticancer properties,6, their role in homologous recombination,8, the sub-classifications of ovarian cancer based on pathology and genomic characteristics, and treatment strategies supplementing chemotherapy are in full development. These advances have further emphasized why optimal utilization of the platinum drugs coupled with knowledge of homologous recombination deficiency (HRD) is central to the treatment of the most common subset of ovarian cancer . Typically, such cancers are initially not only highly “platinum sensitive”, presumably from germline or somatic deleterious BRCA mutations with HRD, but also characterized by their early peritoneal dissemination leading to peritoneal surface implants. These features, bolstered by clinical experience to be cited in this overview suggest a possible role for intraperitoneal (IP) therapy in controlling these molecular subtypes. After reviewing the historical background for cisplatin administration, we shall comment on the possible integration of IP drug administration in strategies designed to minimize the emergence of platinum resistance. As trialists persuaded by improved IP therapy outcomes - in spite of some justified skepticism - we shall focus on how optimizing platinum pharmacodynamics in selected patients manifesting early spread is the appropriate target for IP platinum administration.Future investigations need to probe deeper into these definitions and how to deal with consolidation drugs applied after completion of first-line treatment cycles. Insights resulting from the cloning of BRCA genes-14 led to routine use of these drugs as systemic monotherapy. Cyclophosphamide’s “platelet-sparing” effects led to testing it in drug combinations that eventually claimed to improve median times to progression by a few months over the non-specific DNA damaging alkylating agents. At the unusual early stages of ovarian cancer, IP radioactive colloidal chromic phosphate P32 was tested vs. melphalan as consolidation in the absence of residual disease after surgery; subsequently, in a randomized trial vs. systemic cisplatin + cyclophosphamide with a non-significant advantage in survival for the latter (GOG95).In 1964 as a Hematology fellow at Columbia’s Francis Delafield Cancer Hospital , FM recalls treating ovarian cancer patients suffering with massive ascites by injecting ThioTepa directly into their distended abdomens, sometimes repeatedly. Severe thrombocytopenia was dose-limiting, and any improvements observed were short-lived. Such treatment interventions were promptly discarded but remissions after oral melphalan or cyclophosphamide,17. Medicine Branch trainees  became involved in this area of therapeutics and the first two started extensive interactions with extramural gynecologists who since 1970 with NCI support began clinical trials under the umbrella of the Gynecologic Oncology Group (GOG)-paralleling actions created under NCI Clinical Cooperative Groups seeking to integrate clinical drug development with surgery and radiation. GOG became the centerpiece for the future testing of cisplatin and paclitaxel - the most successful anticancer drugs emerging from studies fully coordinated by the NCI.By the 1970s, the IP route for drug administration had become the focus of pharmacologic and clinical studies at the National Cancer Institute (NCI), National Institutes of Health (NIH). With Vincent T DeVita Jr, assuming the Directorship of the Division of Cancer Treatment in 1974 to succeed C Gordon Zubrod - who in 1954 was recruited to run the Chemotherapy Program of NCI emphasizing anticancer drug development for hematologic malignancies - its Medicine Branch turned its attention to curative strategies for breast and ovarian cancers. Laparoscopic staging and drug combinations were the initial focus of ovarian cancer clinical protocols. Out of these studies, led by Robert C Young established benefits from the regimen HexaCAF , and soon thereafter a number of phase I/II and pharmacokinetics studies with methotrexate, 5-fluoruracil, and the novel antitumor antibiotic doxorubicin were launched at the Medicine and Surgery Branches under DeVita’s prompting-21. Initially, the clinical focus was on drug peritoneal/plasma concentration ratios that were tolerable, but drug diffusivity (penetrance) into tissues and intrinsic tumor sensitivity to a drug was later emphasized as being most relevant to eventual clinical results. In fact, Stephen Howell at University of California, San Diego, initiated IP cisplatin studies after the 1979 approval for systemic treatment of ovarian cancer, and reported the most noteworthy clinical results. These were compelling enough for Baltimore NCI unit-trained David Alberts in 1985 to a phase III study of cisplatin IP vs. IV (+ IV cyclophosphamide in both arms) within the Ovarian Committee of the Southwest Oncology Group (SWOG). This study completed its lagging accrual with GOG participation (GOG104): its results presented at the American Society of Clinical Oncology plenary session and published in 1996 fostered enthusiasm for further exploration of IP cisplatin. Accordingly, it was followed by GOG114: this study adopted the new standard IV cisplatin + 24h IV paclitaxel as a comparator to the IP cisplatin arm. However, this experimental IP arm was modified to be preceded by IV carboplatin AUC 9 × 2 cycles as “chemical debulking” before IP cisplatin + 24h IV paclitaxel; this resulted in excessive hematologic toxicity. Therefore, even with an improved overall median survival, because of its greater toxicity it could not be “recommend for routine use”. Such a conclusion led to the third GOG IV vs. IP comparison in GOG172 that included yet another major change in the experimental IP regimen, - now adding a day 8 IP paclitaxel to the IP cisplatin. The median survival results exceeding 5 years first disclosed at the GOG meeting were striking for the IP arm, even though associated with considerably more toxicity and lesser completion rates. An NCI Clinical Announcement followed in January 2006 urging IP treatments was provided for optimally cytoreduced women with stage III ovarian cancer. With each of the IP arms differing among the 3 GOG studies and problematic toxicities blunted adoption of IP regimens outside the US. The late Martin Gore articulated the European position over several years with insightful comments: e.g, “even if IP superiority is confirmed by a subsequent trial, tell me how I should use it”, and after the brilliant results of GOG172, his overall critique was “Lack of robustness of the data lies in the design of the main IP trials, not their conduct or analysis”. Introduction of IP paclitaxel in GOG172, in particular, was problematic for testing the original hypothesis of IP vs. IV cisplatin and it raised new local and systemic toxicity issues . Additionally, critics of IP approaches objected that the comparator in the IV arm was cisplatin and not the by now current standard IV carboplatin .The pharmacologic basis for IP drug delivery was developed by Robert L Dedrick at the NIH2 given on day 2 (coupled with IV paclitaxel on day 1) were further compounded by an additional paclitaxel IP on day 8. Therefore, the GOG embarked on piloting several modified IP regimens with cisplatin at 75 mg/m2 (but still retaining the IP paclitaxel on day 8). After 3 years of IP pilot studies, other concepts gained traction . These innovations led to GOG252: a 3-way comparison enrolling 1,560 women randomized to (1) a reduced cisplatin dose-modified GOG172 IP regimen; (2) 80 mg/m2 IV paclitaxel d1, 8, 15 (acquired from the Japanese GOG study) given together with IP carboplatin at target AUC dosing of 6 or 3) the same two drugs and respective doses with both given IV. Bevacizumab every 3 weeks was added to each arm. The final analysis of this study was published in 2019 - three years after initial analysis of progression free survival (PFS) had been presented showing no significant differences between the 3 arms but with quality of life less affected in the IP or IV carboplatin arms. As anticipated, the mature overall survival (OS) analysis was similar in all three arms, exceeding 6 years in all three regimens. These results were definitive enough to reverse any impact on clinical practice including IP cisplatin administration that the 2006 Clinical Announcement had recommended. The brisk enrollment into this trial, however, reflects the ease gynecologic oncologists have adopted IP interventions, not impeding entry into such administration into studies. The overall lack of advantage in outcome, however, has essentially nullified IP interventions as a standard option relative to IV regimens that uniformly rely on carboplatin doublets. Whether differences were blurred by introducing weekly paclitaxel in two of the arms  and by bevacizumab improving PFS in all three arms, or by diminished efficacy of the lower IP cisplatin dose (75 mg/m2 per cycle rather 100 g/m2) is a currently unanswerable question. While trialists consider future directions of IP interventions, the Dutch group randomized patients to hyperthermic IP chemotherapy (HIPEC) utilizing cisplatin × 1 HIPEC following 3 cycles of carboplatin/paclitaxel and surgical cytoreduction vs. the same surgical intervention without HIPEC. Both arms went on to receive additional 3 cycles of carboplatin/paclitaxel. This study provided a statistically significant survival advantage for the HIPEC arm. HIPEC has been applied at different time points of treatment of advanced epithelial ovarian carcinoma, namely up-front, interval, and recurrent setting. However, the use of HIPEC following neo-adjuvant chemotherapy (NACT) at the time of interval debulking surgery is the most promising since it is based on a higher level of evidence and perhaps should be able to reverse the platinum-resistance induced by neo-adjuvant chemotherapy. Furthermore, the observation that hyperthermia delayed the repair of DNA damage induced by cisplatin or doxorubicin acting upstream of different repair pathways to block histone polyADP-ribosylation (PARylation), as efficiently as pharmacologic inhibitors of PARP (PARPi), producing comparable delay in DNA repair, induction of double-strand breaks, and cell cytotoxicity after chemotherapy could suggest a potential synergistic effect of HIPEC and PARP1 inhibitor, to be evaluated in clinical trials. In the post-NACT and optimal surgical debulking setting the Gynecologic Cancer Intergroup performed a two-stage adaptively designed randomized phase II study of intraperitoneal vs. intravenous chemotherapy. Patients were randomized to one of 3 arms: (1) i.v. carboplatin/paclitaxel control arm; (2) i.p. cisplatin plus i.v./i.p. paclitaxel; or (3) i.p. carboplatin plus i.v./i.p. paclitaxel with the primary endpoint being 9-month progressive disease (PD9) rate before going on to an anticipated two-arm phase III comparison of the intravenous standard vs. one of the intraperitoneal arms. The IP cisplatin arm did not progress beyond the first stage of the study after failing to meet the PD9 pre-set superiority rule the final analysis compared i.v. carboplatin/paclitaxel (n = 101) while i.p carboplatin, i.v./i.p. paclitaxel (n = 102) did show a lower PD9 rate compared with the i.v. carboplatin/paclitaxel control arm: 24.5% (95%CI: 16.2%-32.9%) vs. 38.6% (95%CI: 29.1%-48.1%) (P = 0.065). Regrettably, the planned phase III trial was aborted due to insufficient funding.In the GOG172 IP regimen the long-lasting neurotoxicity and other concerns associated with cisplatin for up to 6 cycles at the excessive doses (for women) of 100 mg/m-37. Early NCI studies on IP therapy concentrated on ovarian cancer because one could attain a pharmacologic advantage with a number of chemotherapy agents. Subsequently, the IP route would be deemed ideal following “early diagnosis” or “optimally debulked” in a disease initially confined to the peritoneal cavity -for example, we have since learned from BRCA mutation-related serous cancers discovered incidentally at risk-reducing salpingo-oophorectomies in hereditary mutation carriers. The studies conducted by UCSD and the GOG reviewed in detail had signaled improvements in outcome for IP cisplatin administration and reinforced by the initial 3 GOG studies utilizing cisplatin at 100 mg/m2 per cycle, but varied in some aspects of patient eligibility  as well as accompanying added treatments in the IP cisplatin arm. In the last GOG252, all differences became blurred with superior overall outcome results suggesting a contribution of bevacizumab to IV and both IP arms. Rather than discarding the prior undoubtedly positive signals following IP drug administration, we suggest that one should consider the following steps: (1) refining eligibility to concentrate on HRD deficient disease and where most favorable low-volume disease distribution might be expected; (2) testing out other potentiators of IP platinums; and (3) reinforcing potential practical advantages of IP administered drugs.Most positive signals from IP drug administration have been obtained in epithelial ovarian cancer with high-grade serous cancers and early peritoneal spread - many originate in the fimbria of the Fallopian tubes. If an advantage from IP administration over IV is going to emerge, the patient selection should initially be confined to those with demonstrable genetic mutations of BRCA and/or other germline or somatic alterations resulting in HRD. Retrospective studies have shown an improved prognosis, higher response rates to platinum-containing regimens, and longer treatment-free intervals between relapses in patients with BRCA 1 and BRCA 2 (BRCA1/2)-mutated ovarian cancer compared with patients who are not carriers of this mutation. Regarding the role of IP therapy in BRCA-mutated ovarian cancer, an ancillary study of a subset of GOG 172 patients revealed that those with reduced BRCA 1 expression greatly benefitted from IP rather than IV chemotherapy: in tumors with aberrant BRCA1 expression the median OS was 84 months vs. 47 months in the IP vs. IV group, respectively (P = 0.0002). Moreover, in a retrospective study of OS among 62 patients entering in IP platinum doublet phase II trials performed at NYU, a subset of 10 patients later documented to have germline BRCA mutations experienced a very long median survivals (10+ years).As a reference point, the NCI The Cancer Genome Atlas on ovarian cancer provided a comprehensive analysis of genomic alteration in high grade serous cancersvs. IV comparisons, one should consider using the same platinum agent in IV and IP arms, that is, carboplatin. In this era of pretreatment genomic analysis, one could include additional accompanying biomarker studies of outcome, and detection of early recurrence by ctDNA, as an example. In addition to attempting to confirm an IP vs. IV advantage in this selected population, the IP delivery lends itself to potentiating the action of platinums by this route. The next section will describe studies that provided positive leads but unfortunately have not followed up to date, in part because clinical investigations without pharmaceutical industry sponsorship are challenging to get underway more than ever.Beyond BRCA mutations and other HRD features, patients should be optimally cytoreduced up front and include only stages II or III. Anyone undergoing additional resections of bowel and visceral organs might also need to be excluded. To avoid additional issues not related to IP ,42. The GOG set out to prove this hypothesis by conducting a clinical trial in patients with peritoneal recurrence mimicking Howell’s preclinical data. In the study by Jandial et al., 33 heavily pretreated women meeting eligibility criteria were enrolled in the phase I/pharmacokinetic study to evaluate the safety of IP carboplatin AUC 5 and bortezomib 0.5 mg/m2. Twenty-one of these patients had measurable disease with one complete and 3 partial responses  were noted. With the clinical experience of IP placement over several decades the leads from this phase I study should be pursued among other hypotheses that need to be tested about overcoming resistance, especially since this study demonstrated clinical benefit with a satisfactory therapeutic index. Moreover, it exemplifies a path of going from animal models into human study that has not been exploited.Data from Howell’s laboratory targeting the cell membrane copper transport receptor (CTR) shared by platinums suggested that cisplatin resistance was overcome by the combination of the proteasome inhibitor bortezomib and cisplatin and this arm was one of the two selected of a SWOG randomized phase 2 study. In this trial its progression free survival vastly exceeded that of IP mitoxantrone that had been selected based on anecdotal reports of activity. Subsequently, we went on to combine FUDR with platinums for further development as a promising combination but it was displaced by the emerging interest of IP paclitaxel (that was later included in GOG172 and in the modified arm of GOG252). While further development stalled, Scott Kaufmann’s laboratory studies in Mayo Clinic had established that IP FUDR but not 5-fluorouracil potentiated the effect of PARP inhibitors,47. This led to an institutional Phase I clinical study of IP FUDR coupled with oral veliparib  that completed accrual in 2019. These are examples of how leveraging PARP inhibition and IP drug combinations overcome relative resistance to the platinum drugs that may be less achievable by the systemic route. All our experience with peritoneal ports may allow monitoring for ctDNA in peritoneal washings. In fact, in our unpublished experience based on monitoring peritoneal cytology every 6 weeks × 4 after completion of IP treatment does lead to a 10% early detection of peritoneal recurrences not otherwise detectable by imaging or CA125 increase . Removing the port at 6 months was chosen as a compromise of having to deal with the port a few more months after completion of treatment  and detecting a “platinum-resistant” peritoneal relapse that might be appropriate for experimental IP therapies.Series of trials utilizing the IP route in patients with ovarian cancer recurrences to assess potential drug activity alone and in combination with platinums were carried out by the GOG102 in the early 1990s (terminated when joining the phase III SWOG study as GOG104). We had studied IP fluoropyrimidines, and specifically 5-fluoro-2-deoxyuridine (FUDR) as single agent-52 is reported in  and by the Cancer Care Ontario. The Cochrane concluded that women receiving primary treatment for ovarian cancer were less likely to die if they received an IP component to chemotherapy  and that there was greater serious toxicity with regard to gastrointestinal effects, pain, fever and infection but less ototoxicity with the IP than the IV route. Platinum-resistance is a key element of the challenging relapses that follows all approaches to high-grade serous ovarian cancers. We have reviewed how intraperitoneal therapy was developed and its pharmacologic basis led to phase III trials that have provided mostly positive signals, but also associated with increasing toxicities. A summary of published phase III trials comparing IP with IV administrations of platinum-containing regimens, including studies performed before the introduction of taxanes


Ttr, Folr1, and Prlr. We used the Ttr gene as a marker to query the Gene Expression Omnibus database for transcriptome studies of brain tissue and identified at least some level of likely choroid contamination in numerous studies that could have potentially confounded data analysis and interpretation. We also analyzed transcriptomic datasets from human samples from Allen Brain Atlas and the Genotype-Tissue Expression (GTEx) database and found abundant choroid contamination, with regions in closer proximity to choroid more likely to be impacted such as hippocampus, cervical spinal cord, substantia nigra, hypothalamus, and amygdala. In addition, analysis of both the Allen Brain Atlas and GTEx datasets for differentially expressed genes between likely “high contamination” and “low contamination” groups revealed a clear enrichment of choroid plexus marker genes and gene ontology pathways characteristic of these ciliated choroid cells. Inclusion of these contaminated samples could result in biological misinterpretation or simply add to the statistical noise and mask true effects. We cannot assert that Ttr or other genes/proteins queried in targeted assays are artifacts from choroid contamination as some of these differentials may be due to true biological effects. However, for studies that have an unequal distribution of choroid contamination among groups, investigators may wish to remove contaminated samples from analyses or incorporate choroid marker gene expression into their statistical modeling. In addition, we suggest that a simple RT-qPCR or western blot for choroid markers would mitigate unintended choroid contamination for any experiment, but particularly for samples intended for more costly omic profiling. This study highlights an unexpected problem for neuroscientists, but it is also quite possible that unintended contamination of adjacent structures occurs during dissections for other tissues but has not been widely recognized.The choroid plexus, a tissue responsible for producing cerebrospinal fluid, is found predominantly in the lateral and fourth ventricles of the brain. This highly vascularized and ciliated tissue is made up of specialized epithelial cells and capillary networks surrounded by connective tissue. Given the complex structure of the choroid plexus, this can potentially result in contamination during routine tissue dissection. Bulk and single-cell RNA sequencing studies, as well as genome-wide in situ hybridization experiments (Allen Brain Atlas), have identified several canonical markers of choroid plexus such as  Ttr) gene between groups that we could not validate, nor did it make biological sense. We decided to examine the expression of transthyretin (Ttr) and other similar genes more comprehensively. Ttr showed almost exclusive expression in the choroid plexus of the mouse in the Allen Brain Atlas, a unique and powerful resource to examine mRNA levels of virtually every transcript across the brain and visualize the expression from in situ hybridization [Ttr was the canonical marker gene that defined choroid plexus cell clusters, with extremely low expression in other cell types. In addition, a comprehensive query of deposited microarray and RNAseq datasets from Gene Expression Omnibus (GEO) profiles [TTR samples yielded high enrichment of choroid plexus marker genes. These findings provide a cautionary tale for neuroscientists and indicate that determining the level of choroid contamination is necessary for proper biological interpretation, especially when factoring in the considerable costs of most omics studies. Examination of other tissues may also reveal other unintended dissection artifacts that are worth reexamination.The mammalian brain is tightly packed with subregions that have unique gene expression profiles. In order to understand these regional differences in brain tissue, scientists often dissect freshly obtained brains into separate regions  that are then used to purify RNA, proteins, lipids, or metabolites for further analyses. Often these inquiries utilize targeted methods such as western blotting, RT-qPCR, etc., but frequently investigators choose to perform unbiased “omic” profiling using microarrays, RNA sequencing (RNAseq), proteomics, lipidomics, metabolomics, etc. When analyzing our unpublished datasets, we noticed an occasional differential expression of the transthyretin  expression in brain tissue datasets from Homo sapiens and Mus musculus. Our query searched for “Ttr AND ” in the central nervous system (CNS) of Homo sapiens and Mus musculus. This yielded 586 GEO Profiles. Each sample within each profile has a value and rank for TTR/Ttr expression. Values stem from the original user-supplied data that may be, but not limited to, raw counts, log-transformed counts, or fold change [TTR/Ttr value for each sample is displayed with a red bar. Ranks are determined by placing all gene expression values per sample in bins from 0 to 100 and then assigning a percentile to each gene. Ranks are denoted with blue squares [The GEO database  was usedHomo sapiens and 135 are Mus musculus. After compiling the data from GEO profiles into dictionaries of profiles and their corresponding ranks, each profile was assigned 1 of 5 priority numbers to categorize the level of TTR/Ttr contamination , studies on cultured cells, predominantly tumor profiling, and studies listed more than once in the query results . GEO profiles that contained faded-out bars or squares to signify absent calls with Affymetrix microarrays were included. Post-filtering yielded 158 GEO profiles , as well as de-identified clinical information for all donors within the study [TTR for each sample in each brain region. Then, to determine if potential choroid plexus contamination could contribute to biological misinterpretation, we ran a differential expression analysis using the Allen Brain Atlas hippocampus samples as this region is in close proximity to the choroid plexus.We investigated how potential in Atlas . The humin Atlas , 2. Speche study , 2. Ther2 (TPM) of TTR and defined samples with a log2(TTR) of less than 3.32 as having low choroid plexus contamination and samples with a log2(TTR) greater than 5.32 as potentially having high choroid plexus contamination. Samples with greater than 3.32 log2(TTR) expression, but less than 5.32 log2(TTR) expression were not assigned to either the low or high contamination groups. There are 58 samples in the low contamination group and 10 samples in the potentially high contaminated group, and 26 samples in neither low nor high contamination groups. The TPM value data for all genes were then converted to counts per million (CPM), and then filtered to keep genes with greater than 2 CPM in at least three samples. Post-filtering resulted in 21,959 genes. The counts data for each sample were then normalized for gene expression distributions by calculating the trimmed mean of M-values with edgeR [2(TTR) expression distributions among the age bins for Allen Brain Atlas [p value is <0.05 and absolute log2 fold change >1 (Supplementary Table FOLR1) (Supplementary Table 2(TPM) of FOLR1 and defined samples with a log2(FOLR1) of less than −1 as having low choroid plexus contamination and samples with a log2(FOLR1) greater than 1 as potentially having high choroid plexus contamination. There are 40 samples in the low contamination group and 3 samples in the potentially high contaminated group. The remaining 51 samples were not assigned to a group and were not included in the FOLR1 differential expression analysis.Examining the 94 hippocampus samples from the Allen Brain Atlas data, we assigned each sample as either having little choroid plexus contamination or as potentially having a lot of choroid plexus contamination. To assign the samples to a group, we plotted the logth edgeR . The Allhttps://gtexportal.org/home/datasets). There are 12 brain regions in the GTEx data (Supplementary Table 2(TTR) expression distributions among the age bins. Differential expression analyses between the lowly and highly contaminated groups were then carried out by linear modeling as implemented in the R package limma [p value is <0.05 and absolute log2 fold change >1 .Data processing pipeline available on GitHub  , a small mammal in the primate family with a gyrencephalic brain, from GEO dataset GDS4128 [Ttr was expressed at several orders of magnitude higher only in a subset of samples that is best explained by choroid contamination rather than biological variation , which could potentially bias the biological interpretation   Fig. . Thus, oTTR/Ttr was also found to be highly expressed but with no clear group-level differences in an additional 27.2% of these 143 GEO profiles  and the frontal cortex, at a mean percentile rank of 62.70 and 65.11, respectively  is 32, while the highest within that tissue is 100. Unlike the Allen Brain Atlas data, there are no samples with a TTR percentile rank below 32 in GTEx, whereas some Allen Brain Atlas samples show TTR in the zero percentile rank. Regions nearest the choroid plexus show the highest mean percentile rank of TTR, and there are wide ranges in TTR percentile rank expression among samples within tissues  expression of 3.32 to define lowly contaminated samples and log2(TTR) of 5.32 to define potentially highly contaminated samples. These values retained enough samples in each group for each dataset. Using an arbitrary delineation of “low” (blue shading) vs “high” (red shading) TTR expression, we found several differentially expressed genes between these groups for both the Allen Brain Atlas dataset  expression distributions among the age bins and found no statistically significant difference between TTR expression by different age bins for the Allen Brain Atlas data  or for the GTEx data   < 1  in both the Allen Brain Atlas dataset  < −1 vs “high” (red shading) of log2(FOLR1) > 1 expression, we found several differentially expressed genes between these groups for both the Allen Brain Atlas dataset  are probably better suited to infer contamination since TTR is a highly secreted protein.It is possible that choroid plexus contamination may not be limited to bulk transcriptome data. Single-cell RNAseq is becoming a widely popular tool for profiling thousands of individual cell types within a system for characterizing cell function, regulation, and interactions among cells , 15. Sevar genes , 19. ChoTTR, FOLR1, and PRLR to determine unintended group-level differences or unlikely high levels of expression for TTR, FOLR1, and PRLR before inferring biological interpretations when utilizing these data sets.Recent publications have shown that the choroid plexus is a crucial target for COVID-19  as well To avoid costly analyses that may lead to misinterpretation, we suggest that researchers might prescreen samples via RT-qPCR or western blot for choroid markers before sequencing or other omics studies. If utilizing already sequenced data, researchers should percentile rank choroid plexus markers for each sample and determine if there are unintended group-level differences, or biologically unreasonably high expression levels of choroid plexus markers. To avoid contaminated omics profiling and unintended missed biological conclusions, researchers should carefully consider the experimental design to prevent contamination and the biology of the sample to identify potentially contaminated samples. We highlight here an example of tissue profiling that results in unwanted contamination of the target region within the brain, but other tissues with poorly discerned tissue boundaries might also suffer from unintentional introduction of an adjacent structure that could cause similar problems and thus caveat emptor.Supplementary information fileSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5