Differential expression during placentation in pregnancies conceived with different fertility treatments compared with spontaneous pregnancies

Bora Lee, Ph.D.,a Alex F. Koeppel, Ph.D.,b Erica T. Wang, M.D., M.A.S.,a,c Tania L. Gonzalez, Ph.D.,a Tianyanxin Sun, Ph.D.,a Lindsay Kroener, M.D.,c Yayu Lin, M.S.,a Nikhil V. Joshi, M.D.,a,c Tejal Ghadiali, B.S.,a Stephen D. Turner, Ph.D.,b Stephen S. Rich, Ph.D.,b Charles R. Farber, Ph.D.,b Jerome I. Rotter, M.D.,d Yii-Der Ida Chen, Ph.D.,d Mark O. Goodarzi, M.D., Ph.D.,e Seth Guller, Ph.D.,f Bryna Harwood, M.D.,g Tania B. Serna, M.D.,g John Williams III, M.D.,c,h and Margareta D. Pisarska, M.D.a,c a Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, Los Angeles, California; b Center for Public Health Genomics, University of Virginia, Charlottesville, Virginia; c Department of Obstetrics and Gynecology, University of California, Los Angeles (UCLA) David Geffen School of Medicine, Los Angeles, California; d LABiomed/Harbor-UCLA Medical Center, Torrance, California; e Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California; f Department of Obstetrics/Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut; g Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, Los Angeles, California; and h Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, Los Angeles, California

Objective: To identify differences in the transcriptomic profiles during placentation from pregnancies conceived spontaneously vs. those with infertility using non-in vitro fertilization (IVF) fertility treatment (NIFT) or IVF. Design: Cohort study. Setting: Academic medical center. Patient(s): Women undergoing chorionic villus sampling at gestational age 11–13 weeks (n ¼ 141), with pregnancies that were conceived spontaneously (n ¼ 74), with NIFT (n ¼ 33), or with IVF (n ¼ 34), resulting in the delivery of viable offspring. Intervention(s): Collection of chorionic villus samples from women who conceived spontaneously, with NIFT, or with IVF for analysis using RNA sequencing. Main Outcome Measure(s): Baseline maternal, paternal, and fetal demographics, maternal medical conditions, pregnancy complica- tions, and outcomes. Differential gene expression of first-trimester placenta. Result(s): There were few differences in the transcriptome of first-trimester placenta from NIFT, IVF, and spontaneous pregnancies. There was one -coding differentially expressed gene (DEG) between the spontaneous and infertility groups, CACNA1I, one protein-coding DEG between the spontaneous and IVF groups, CACNA1I, and five protein-coding DEGs between the NIFT and IVF groups, SLC18A2, CCL21, FXYD2, PAEP, and DNER. Conclusion(s): This is the first and largest study looking at transcriptomic profiles of first-trimester placenta demonstrating similar transcriptomic profiles in pregnancies conceived using NIFT or IVF and spontaneous conceptions. Gene expression differences found to be highest in the NIFT group suggest that the underlying infertility, in addition to treatment-related factors, may

Received May 30, 2018; revised November 3, 2018; accepted November 5, 2018; published online January 2, 2019. J.I.R. reports grants from the National Institutes of Health, during the conduct of the study. J.W. reports personal fees from Natera, Inc. outside the sub- mitted work. B.L. has nothing to disclose. A.F.K. has nothing to disclose. E.T.W. has nothing to disclose. T.L.G. has nothing to disclose. T.S. has nothing to disclose. L.K. has nothing to disclose. Y.L. has nothing to disclose. N.V.J. has nothing to disclose. T.G. has nothing to disclose. S.D.T. has nothing to disclose. S.S.R. has nothing to disclose. C.R.F. has nothing to disclose. Y.-D.I.C. has nothing to disclose. M.O.G. has nothing to disclose. S.G. has nothing to disclose. B.H. has nothing to disclose. T.B.S. has nothing to disclose. M.D.P. has nothing to disclose. Supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award number R01HD074368. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National In- stitutes of Health. Presented at the 65th Annual Meeting of the Society for Reproductive Investigation, San Diego, CA, March 6–10, 2018. Reprint requests: Margareta D. Pisarska, M.D., Cedars-Sinai Medical Center, Department of Obstetrics and Gynecology, Division of Reproductive Endocri- nology and Infertility, Los Angeles, California 90048 (E-mail: [email protected]).

Fertility and Sterility® Vol. 111, No. 3, March 2019 0015-0282/$36.00 Copyright ©2018 American Society for Reproductive Medicine, Published by Elsevier Inc. https://doi.org/10.1016/j.fertnstert.2018.11.005

VOL. 111 NO. 3 / MARCH 2019 535 ORIGINAL ARTICLE: GENETICS contribute to the observed gene expression profiles. (Fertil SterilÒ 2019;111:535–46. Ó2018 by American Society for Reproductive Medicine.) El resumen está disponible en Español al final del artículo. Key Words: Non-IVF fertility treatment (NIFT), in vitro fertilization (IVF), placentation, RNA sequencing, transcriptome Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertility- and-sterility/posts/40658-26402

nfertility affects approximately 6.1 million people in the couples with infertility—that are in the late first trimester of United States, equivalent to 10% of the reproductive-age pop- pregnancy at the time of chorionic villus sampling (CVS) I ulation (1). The use of assisted reproductive technology (ART), and followed until delivery. Non-IVF fertility treatment was including in vitro fertilization (IVF), contributes to 1.5% of live defined as treatment using either medications for ovulation births in the United States, and other non-IVF fertility treatments induction or controlled ovarian stimulation and intrauterine (NIFTs) contribute to 4.6% (2, 3). Adverse pregnancy outcomes, insemination (IUI). IVF pregnancies included fresh or frozen including low birth weight and small for gestational age babies, embryo transfers using either cleavage-stage or blastocyst- pre-eclampsia, retained placenta, placental abruption, placenta stage embryos. Our SMAART Study cohort consisted of 409 previa, preterm labor and delivery, and birth defects, have been singleton pregnancies, of which 208 were spontaneous con- associated with ART compared with pregnancies conceived spon- ceptions and 201 pregnancies conceived with a history of taneously (2, 4–11).However,itisunclearwhethertheseadverse infertility. Of the infertility group, 90 were conceived with outcomes are the result of the ART procedures, such as IVF, or the NIFT and 111 were conceived with IVF. All pregnancies had underlying infertility, because pregnancies conceived by couples a normal karyotype and delivered (20). utilizing other types of fertility treatments, NIFT, are also at Transcriptomic profiling was performed on a subset of increased risk of adverse outcomes, including placental 141 subjects who had chorionic villi available from the first abruption, fetal loss, and gestational diabetes (12).Furthermore, trimester. The SMAART Transcriptome cohort consisted of pregnancies conceived in couples with infertility regardless of 74 spontaneous conceptions and 67 pregnancies conceived treatment are at increased risk of adverse outcomes, including with a history of infertility. Of the infertility group, 33 were placenta accreta (13), earlier gestational age at delivery, late conceived with NIFT and 34 were conceived with IVF. preterm birth, and greater neonatal intensive care unit – admissions (11, 14), as well as birth defects (15 17). Demographics Statistical Analysis Because many of these adverse outcomes are related to t placentation, a better understanding of placental function A -test and analysis of variance were used for the baseline in pregnancies conceived in couples with infertility, utilizing and pregnancy outcome demographics. fertility treatments, may uncover the underlying pathology leading to adverse outcomes. CVS Collection Previous studies examining the transcriptome of human Chorionic villus samples were collected at 11–13 weeks' placenta have been limited to term placentas with specific gestation at the Cedars-Sinai Prenatal Diagnostic Center, as placenta pathologies, predominantly pre-eclampsia (18). Yet previously described (21, 22). Leftover tissue after clinical it is during the first trimester of a pregnancy, during placenta- genetic testing was collected from consenting patients per tion, when trophoblast proliferation, differentiation, and inva- institutional review board–approved protocol and placed in sion, as well as angiogenesis and vasculogenesis, crucial for RNAlater RNA Stabilization Reagent (Qiagen) and stored at laying the groundwork for successful placental function, are 80C in the Cedars-Sinai Prenatal Biorepository until taking place; this is a necessary time point to study placental processing. development (19). It is also the closest time point to conception, either spontaneously or through fertility treatments, that can RNA Extraction for RNA-Sequencing be studied to minimize placental changes due to placental pa- thology and not the mode of conception. This is the first and Chorionic villus sampling tissues were thawed on ice in 1% b- largest study looking at the first-trimester placenta (chorionic mercaptoethanol-containing lysis buffer using the AllPrep villi) transcriptome in our SMAART (Spontaneously/Medically DNA/RNA Extraction Mini (Qiagen). Cells were homoge- Assisted/ART) study cohort consisting of pregnancies that nized by repeatedly passing through single-use needles with delivered from couples with infertility, utilizing either NIFT decreasing gauge (22G, 25G, then 27G) attached to a 1-mL or IVF, and pregnancies conceived spontaneously. sterile, RNase-free syringe before being loaded onto AllPrep spin columns (21, 22). Extraction of DNA/RNA was performed according to the manufacturer's instructions and MATERIALS AND METHODS as previously described. Purity of the extracted total RNA Patient Population was evaluated using the NanoDrop 2000 spectrophotometer Our SMAART Study cohort consisted of pregnancies— (Thermo Scientific) and the concentration of the total RNA conceived spontaneously or through either NIFT or IVF in measured using the Qubit Fluorometer (Thermo Fisher

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Scientific). Integrity of RNA was assessed using the Fragment and pregnancy-related diabetes in the infertility group Analyzer (Advanced Analytical Technologies), which (Table 1). When birth outcomes were examined, there was analyzes the integrity of the total RNA by measuring the no difference in gestational age at delivery but a significant ratio between the 18S and 28S ribosomal peaks. difference in birth weight (3,430 590 g for spontaneous and 3,192 623 g for infertility). When the infertility group RNA Sequencing and Statistical Analysis was divided into pregnancies conceived with NIFT or IVF, the significant difference in maternal BMI was found to be be- The RNA-seq libraries were constructed from 200 ng of total tween spontaneous and NIFT pregnancies (22 3.2 vs. RNA using Illumina TruSeq Stranded Total RNA with Ribo- 25 6.3 kg/m2, respectively), rather than between sponta- Zero Gold sample prep kits (Illumina). Constructed libraries neous and IVF pregnancies. There was a higher rate of cesarean contained RNAs of >200 nt and were depleted of cytoplasmic section in the NIFT and IVF pregnancies, as was observed in the and mitochondrial ribosomal RNAs. The RNA-seq reads were SMAART cohort. The difference in birth weight was found to assessed for quality using FastQC (23). Transcript abundances be between spontaneous and IVF pregnancies (3,430 590 g were then quantified against the human reference genome in spontaneous vs. 3,082 586 g in IVF) (Table 1). (Ensembl build GRCh38) using Kallisto (24) and read into the R statistical computing environment (25) as gene-level counts using the tximport package (26). We then used the DE- Transcriptome Seq2 Bioconductor package (27, 28) to normalize for Two-dimensional principal component analysis, which pro- differences in sequencing depth between samples (using the jects the RNA-seq dataset onto two dimensions to identify default median-of-ratios method), estimate dispersion, and principal causes of variation in the dataset as represented fit a negative binomial model for each gene. Datasets were by clustering of samples as data points (30), revealed that adjusted for fetal sex and sequencing runs. The Benjamini- RNA sequencing samples clustered together with similar Hochberg false discovery rate (FDR) procedure (29) was then data points. This clustering was observed regardless of pre- used to re-estimate the adjusted P values. pregnancy maternal health conditions and medications, preg- nancy complications, or in cases of IVF pregnancies, whether Pathway Analysis ET was performed with fresh vs. frozen embryos or at cleavage To investigate biological significance of expressed tran- vs. blastocyst stages (data not shown) and therefore they did scripts, Ingenuity Pathway Analysis (IPA) software (Qiagen; not contribute to differences in outcomes. Because there was http://www.qiagen.com/ingenuity) was used to analyze en- clustering based on fetal sex, also demonstrated by other riched pathways. Differentially expressed were first studies showing differences in the first-trimester placenta filtered as significant at P<.05 and uploaded to the IPA plat- due to sex (31), we corrected the dataset for fetal sex. In addi- form to perform core analyses, which pathways repre- tion, there was clustering due to fetal race, so we selected to sented by the dataset and upstream regulators according to use our largest populations that clustered together, which P values calculated using Fisher's exact test. included Caucasian and Caucasian–Asian fetuses. All samples had at least 10 106 uniquely mapped reads, with an average of 20.8 106 uniquely mapped reads per RESULTS sample. All samples had at least 67% reads uniquely mapped, Clinical Characteristics of the SMAART with an average of 82% reads uniquely mapped per sample. Transcriptome Cohort Overall, there were small differences in the transcriptome From the SMAART cohort, which consisted of 409 singleton of first-trimester placenta from spontaneous, NIFT, and IVF pregnancies, all subjects underwent CVS for diagnostic pregnancies (Fig. 1), as the principal component analysis plots testing. All subjects had a fetus with normal karyotype anal- of spontaneous vs. infertility (Fig. 1A) and spontaneous vs. ysis results and birth outcomes (20). Extra chorionic villi from NIFT vs. IVF pregnancies (Fig. 1B) illustrate. Samples clus- the first trimester were available after tissue was removed for tered according to sample subset (shown on the PC1 axis) genetic testing for 141 subjects who were available for tran- and fetal sex (shown on the PC2 axis) (Fig. 1), both of which scriptome profiling and analysis. This SMAART Transcrip- were corrected in our analysis. When the transcriptome of tome cohort, which was matched for maternal age, race, spontaneous pregnancies was compared with that of preg- ethnicity, and fetal sex, consisted of 74 spontaneous concep- nancies conceived with infertility treatment, of the 61,799 tions and 67 pregnancies conceived with a history of infer- genes examined, after adjustment for multiple comparisons, tility. Of the infertility group, 33 were conceived with NIFT, one protein-coding gene, CACNA1I was significantly differ- and 34 were conceived with IVF. When comparing pregnan- entially expressed at an FDR of 5%, with 6.82-fold higher cies conceived spontaneously vs. those from couples with expression in the spontaneous group than in the infertility infertility, there was no significant difference in maternal group (Table 2). and paternal age and race. There was no significant difference When we compared the transcriptome profiles among in fetal sex distribution or fetal race. There was no difference spontaneous, NIFT, and IVF conceptions, CACNA1I was in maternal underlying conditions but a significant found to be the only significantly differentially expressed difference in maternal body mass index (BMI) (22 3.2 and gene (DEG) between spontaneous and IVF pregnancies again, 24 5.5 kg/m2, respectively). When pregnancy outcomes with its expression 9.85-fold higher in the spontaneous group were evaluated, there was a higher rate of cesarean section (Table 2). There were no protein-coding genes significantly

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TABLE 1

Baseline demographics and pregnancy outcomes for spontaneous vs. infertility, and spontaneous vs. NIFT vs. IVF SMAART Transcriptome cohort. P-value P-value, P-value, differences SPONT INFERT SPONT vs. NIFT IVF SPONT vs. among Variable (n [ 74) (n [ 67) INFERT (n [ 33) (n [ 34) NIFT vs. IVF groups Baseline demographics Maternal age (y) 39 2.2 40 3.0 .2483 40 2.7 40 3.2 .4985 Paternal age (y) 41 4.9 41 6.9 .6589 40 6.3 43 7.1 .1092 Maternal race .275 .326 Caucasian 71 (96) 60 (90) 30 (91) 30 (88) Asian 3 (4.1) 6 (9.0) 3 (9.1) 3 (8.8) African American –– –– Biracial 0 1 (1.5) 0 1 (2.9) (Caucasian/Asian) Biracial (Other) Paternal race .308 .546 Sex of fetus .455 .416 Male 40 (54) 32 (48) 18 (55) 14 (41) Female 34 (46) 35 (52) 15 (45) 20 (59) Fetal race .130 .181 Caucasian 68 (92) 56 (84) 29 (88) 27 (79) Asian –– –– African American –– –– Biracial 6 (8.1) 11 (16) 4 (12) 7 (21) (Caucasian/Asian) Biracial (Other) –– –– Multiracial –– –– Maternal BMI (kg/m2)22 3.2 24 5.5 .0078a 25 6.3 23 4.5 .0108a .009a (SPONT vs. NIFT) Hypertension 2 (2.7) 2 (3.0) .92 1 (3.0) 1 (2.9) .995 Diabetes 0 3 (4.5) .066 1 (3.0) 2 (5.9) .133 Thyroid disease 16 (22) 14 (21) .916 7 (21) 7 (21) .993 Other medical conditions 1 (1.4) 2 (3.0) .502 1 (3.0) 1 (2.9) .798 Gestational age at CVS (d) 83 6.1 82 6.4 .4572 82 6.6 82 6.4 .7324 Crown rump length (mm) 56 11 53 11 .1701 54 11 53 12 .3504 Pregnancy outcomes Gestational age at 273 13 270 14 .2004 272 13 268 15 .1399 delivery (d) Birth weight (g) 3,430 590 3,192 623 .0317a 3,294 647 3,082 586 .04a .035a (SPONT vs. IVF) Mode of delivery .007a Vaginal, spontaneous 44 (64) 23 (37) 12 (39) 11 (34) Cesarean section 25 (36) 40 (63) .002a 19 (61) 21 (66) Pregnancy complications 10 (14) 18 (27) .047a 7 (21) 11 (32) .073 Hypertension 3 (4.1) 4 (6.0) .601 1 (3.0) 3 (8.8) .481 Diabetes 5 (6.8) 13 (19) .025a 6 (18) 7 (21) .077 Coagulation disorders 0 2 (3.0) .134 1 (3.0) 1 (2.9) .326 Placenta previa 2 (2.7) 2 (3.0) .920 0 2 (5.9) .348 Placental abruption 1 (1.4) 0 .340 0 0 .634 Placenta other 0 0 Note: Values are mean standard deviation or number (percentage). INFERT ¼ infertility; SPON ¼ spontaneous. a P-value < .05. Lee. Placental transcriptome in infertility. Fertil Steril 2018. differentially expressed between spontaneous and NIFT preg- of SLC18A2, CCL21, FXYD2, PAEP, and DNER, with IVF nancies at an FDR of 5%. Comparing the transcriptome of pregnancies clustering with spontaneous pregnancies NIFT vs. IVF pregnancies, five protein-coding genes (Fig. 2C). (SLC18A2, CCL21, FXYD2, PAEP, and DNER) were signifi- cantly up-regulated in NIFT pregnancies compared with IVF pregnancies, with fold changes ranging from 3.39 to 6.41 Pathway Analysis (Table 2). When expression levels of these DEGs represented Because of the small number of differentially expressed by fragments per kilobase transcript per million, a normalized genes, studies were not possible for the DEGs version of RNA expression that accounts for gene length and that were identified after adjustment for multiple compari- sequencing depth (32), among the three groups were plotted sons. However, to identify enriched pathways, differentially and examined, the NIFT group had the highest expression expressed genes with a P value of <.05 were analyzed with

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FIGURE 1

Principal component analysis plots of (A) spontaneous vs. infertility pregnancies, and (B) spontaneous vs. NIFT vs. IVF pregnancies. Samples clustered according to sample subset (shown on the PC1 axis) and fetal sex (shown on the PC2 axis). Green ¼ spontaneous; purple ¼ infertility; blue ¼ NIFT; pink ¼ IVF. INFERT ¼ infertility; SPON ¼ spontaneous. Lee. Placental transcriptome in infertility. Fertil Steril 2018.

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ways were immune system-related, but spontaneous vs. TABLE 2 NIFT pregnancies were represented by a more diverse set of canonical pathways, which included Crosstalk between Den- Differentially expressed genes in spontaneous vs. infertility, spontaneous vs. IVF, and NIFT vs. IVF pregnancies. dritic Cells and Natural Killer Cells and Production of Nitric Fold P-value, Oxide and Reactive Oxygen Species in Macrophages Symbol Description change adjusted (Supplemental Table 1). Spontaneous vs. infertility Upstream analysis was performed to identify master CACNA1I Calcium voltage-gated 6.82 .000779 regulators of DEGs with a P value of <.05. In spontaneous channel subunit alpha1 vs. IVF pregnancies, cytokines were abundantly represented Spontaneous vs. IVF fi CACNA1I among the most signi cant upstream regulators of DEGs, Calcium voltage-gated 9.85 .000568 a channel subunit alpha1 including interleukins such as IL27, various interferon- NIFT vs. IVF subtypes, chemokine ligands such as CCL11, and other SLC18A2 Solute carrier family 18 6.41 .000283 cytokine families (Supplemental Table 2). Notably, cyto- member A2 kines were rarely found among upstream regulators of CCL21 C-C motif chemokine ligand 21 6.02 .0118 FXYD2 FXYD domain containing 3.39 .0235 spontaneous vs. NIFT pregnancy DEGs (Supplemental ion transport regulator 2 Table 3). PAEP Progestagen associated 3.94 .0137 endometrial protein DNER Delta/notch like EGF 3.73 .0291 repeat containing Lee. Placental transcriptome in infertility. Fertil Steril 2018. DISCUSSION Transcriptomic profiling of the first-trimester placenta (chori- onic villi) from spontaneous, NIFT, and IVF pregnancies IPA. When we used the P value cutoff of .05, there were 966 demonstrated little differences in global gene expression genes that were significantly different between spontaneous among the three groups. One protein-coding gene (CACNA1I) and infertility, 729 genes between spontaneous and NIFT, was significantly differentially expressed in infertility preg- 893 genes between spontaneous and IVF, and 482 between nancies when compared with spontaneous pregnancies, and NIFT and IVF pregnancies. The IPA of these DEGs revealed this differential expression of CACNA1I was significant be- similar canonical pathways in all comparisons with IVF tween spontaneous and IVF pregnancies as well. There were cohort, including spontaneous vs. infertility, spontaneous no DEGs between spontaneous vs. NIFT pregnancies. Five vs. IVF, and NIFT vs. IVF pregnancies (Supplemental protein-coding genes (SLC18A2, CCL21, FXYD2, PAEP, Table 1, available online). Pathogenesis of Multiple Sclerosis and DNER) were significantly differentially expressed in was the most represented in those comparison datasets NIFT vs. IVF pregnancies. (Supplemental Table 1), but this pathway was not represented A number of DEGs had higher expression levels in NIFT in spontaneous vs. NIFT pregnancies. Cytokines and cytokine pregnancies, with IVF pregnancies segregating more with receptors involved in Pathogenesis of Multiple Sclerosis were spontaneous pregnancies (Fig. 2C). This result complements down-regulated in infertility and IVF pregnancies compared our previous finding in a pilot study demonstrating differen- with spontaneous pregnancies; CCR5, CXCL9, and CXCL10 tial methylation between NIFT and IVF pregnancies at select were down-regulated in infertility compared with sponta- loci, with global methylation remaining unchanged among neous pregnancies; CCR5, CXCL9, CXCL10, and CXCL11 spontaneous, NIFT and IVF pregnancies (33). In addition, it were down-regulated in IVF compared with spontaneous may reflect the differences in the underlying cause of infer- pregnancies; and CXCL9, CXCL10, and CXCL11 were tility, and not the IVF itself, where fertilization occurs in the down-regulated in IVF compared with NIFT pregnancies. laboratory and has been a source implicated in adverse out- None of these genes were differentially expressed in sponta- comes. This is also consistent with characteristics of our neous vs. NIFT (P values ranged from .305 to .928). Another cohort, where the NIFT group had a higher BMI, which may enriched canonical pathway, Neuroinflammatory Signaling indicate a different etiology for infertility, including polycy- Pathway, was significantly enriched in all three comparisons stic ovary syndrome. Because adverse outcomes exist, some with spontaneous pregnancies (Supplemental Table 1) but not likely due to an underlying placental pathology, it also re- significantly enriched in NIFT vs. IVF (P¼.11). Neuroinflam- mains to be determined whether adverse outcomes are due matory Signaling Pathway genes were more represented in to the underlying genetics of infertility or the intrauterine spontaneous vs. IVF pregnancies than other comparisons, environment throughout gestation, given that differences in and nine genes overlapped in all comparisons with sponta- methylation status in placenta at delivery have been reported neous pregnancies: five consistently down-regulated in spon- in pregnancies conceived spontaneously vs. those conceived taneous pregnancies (CALB1, GABRR1, HLA-A, IKBKG, and using ART (33–37). TNF) and four consistently up-regulated (GABRA2, GABRG3, Because race did play a potential role in our transcriptome HLA-A, PIK3CA, and PPP3CB). Antigen Presentation study, we opted to look at the Caucasian and Caucasian– Pathway was significantly enriched in spontaneous vs. IVF Asian cohort; this group had the most similar global tran- (P¼5.85 10 4) and NIFT vs. IVF (P¼1.07 10 3) scriptomic profile and would limit variability based on race, (Supplemental Table 1) and less significant in spontaneous because race has been reported to play a role in reproductive vs. NIFT (P¼.0309). In general, all enriched canonical path- and birth outcomes (38, 39).

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FIGURE 2

A Spontaneous vs Inferlity CACNA1I

0.005 S

0.004

0.003

(FPKM) 0.002 Spontaneous IF Inferlity 0.001

0

B Spontaneous vs IVF CACNA1I

0.005 S

0.004

0.003 Spont

(FPKM) N NIFT 0.002 IVF 0.001 I

0 C NIFT vs IVF SLC18A2 CCL21 PAEP FXYD2 DNER

0.07 0.6 12 0.06 0.003 N N N 0.06 N 0.5 10 0.05 N 0.0025 0.05 0.4 8 0.04 0.002 0.04 Spont 0.3 0.03 (FPKM) 6 0.0015 0.03 S NIFT 0.2 4 0.02 0.001 0.02 S S IVF S I I 0.01 0.1 I 2 0.01 0.0005 I I 0 0 0 0 0 Gene expression levels of differentially expressed genes. (A) CACNA1I is differentially expressed in spontaneous vs. infertility pregnancies, (B) CACNA1I is differentially expressed in spontaneous vs. IVF, and (C) SLC18A2, CCL21, FXYD2, PAEP, and DNER are significantly up-regulated in NIFT pregnancies compared with IVF pregnancies, with spontaneous pregnancies segregating with IVF pregnancies. Green ¼ spontaneous; purple ¼ infertility; blue ¼ NIFT; pink ¼ IVF. FPKM ¼ fragments per kilobase of transcript per million mapped reads; I ¼ IVF; IF ¼ infertility; N ¼NIFT; S ¼ spontaneous. Lee. Placental transcriptome in infertility. Fertil Steril 2018.

Although global transcriptomic profiles were not found lower birth weight in the IVF group among our transcriptomic to differ, differences in specific gene expression and their po- cohort (Table 1). tential roles in placental development may provide insight The one gene found to be differentially expressed be- into long-term regulation of placental function, because we tween pregnancies conceived spontaneously and those did find differences in fetal growth with birth weight, with a conceived with infertility treatment, specifically with IVF,

VOL. 111 NO. 3 / MARCH 2019 541 ORIGINAL ARTICLE: GENETICS was CACNA1I. CACNA1I encodes the pore-forming alpha chemokine- profiles. For example, it has been shown subunit of a voltage-gated calcium channel (VGCC) that CXCL12 secreted by human first-trimester trophoblast belonging to the low voltage-activated, T-type calcium chan- cells stimulates the decidualization process through increased nel subfamily. The VGCCs mediate calcium (Ca2þ) signaling production of CXCR4 (72). In other cell types, the CCL21/ upon depolarization of membrane potential created by the CCR7 pair has been shown to promote growth and metastasis Ca2þ gradient, and activate a variety of downstream events, in tumors by stimulating angiogenesis and lymphangiogene- including muscle contraction, excitation of neurons, regula- sis (73, 74). Thus, CCL21 may be acting as a chemokine tion of gene expression, and release of hormones or neuro- promoting decidualization at the maternal–fetal interface transmitters (40–42). In the placenta, Ca2þ signaling has by stimulating angiogenesis/lymphangiogenesis necessary been reported to be primarily mediated by the high voltage- for proper placentation and placental function throughout activated, L-type VGCCs and transient receptor potential gestation as it remains expressed in term placental mesen- channels (43, 44). chymal stem cells (75). Although the role of T-type VGCC in placentation remains FXYD2 encodes the sodium/potassium (Naþ/Kþ)-trans- to be elucidated, the presence of other calcium channels porting ATPase subunit gamma of the FXYD family of trans- including L-type VGCCs has been identified in syncytiotropho- membrane protein. In the placenta, Naþ/Kþ ATPases are blasts (43, 44),andCa2þ plays a role in hormone secretion found in both the microvillous and basal membranes of syn- regulation, including human chorionic gonadotropin (hCG) cytiotrophoblast (76) and play an important role in transport and human placental lactogen, which is important in fetal of nutrients. Inhibition of Naþ/Kþ ATPase impairs first- growth and development (45–52). Calcium channel-mediated trimester cytotrophoblast cell proliferation, migration, and Ca2þ influx regulates vasoconstriction of uterine radial arteries invasion (77), and reductions in Naþ/Kþ ATPase activity through L- and T-type VGCCs (53). Remodeling of uterine were seen in syncytiotrophoblast plasma membranes from in- vasculature is critical for development of a healthy placenta trauterine growth restricted placentas (76). Thus, misregula- to ensure normal fetal development. Impaired vascular remod- tion of FXYD2 may affect function of Naþ/Kþ transporters, eling has been associated with spontaneous abortion, intra- impairing nutrient transport to the fetus and resulting in uterine growth restriction, pre-eclampsia, and lower birth growth restricted phenotypes. weights (54–59). Thus, decreased expression of CACNA1I in Glycodelin-A, the protein encoded by PAEP is a glycopro- IVF pregnancies may be impacting regulation of Ca2þ tein found abundantly in the endometrium, decidua, and am- homeostasis in the placenta and leading to impaired vascular niotic fluid and has been identified as a marker of endometrial remodeling and altered hormone secretion necessary for receptivity (78). It plays an important role in regulation of im- normal placentation and fetal development. mune responses at the maternal–fetal interface to help prevent Among the genes significantly differentially expressed be- immune rejection of the fetus. Specifically, glycodelin-A facil- tween NIFT vs. IVF pregnancies are SLC18A2, CCL21, FXYD2, itates the type 1 T helper cell (Th1)-type 2 T helper cell (Th2) and PAEP, which may be involved in regulation of immune shift of T lymphocytes in the decidua during pregnancy to in- responses and transport of signaling molecules at the crease Th2 and decrease Th1, balancing activity and cytokine maternal–fetal interface. SLC18A2 encodes a vesicular mono- secretion by the different T lymphocyte populations. Addition- amine transporter that acts to accumulate cytosolic monoamines, ally, glycodelin-A regulates cytokine production by natural such as norepinephrine, serotonin, dopamine, and histamine, killer cells and dendritic cells to induce a tolerogenic phenotype into synaptic vesicles. It has been reported that histamine plays (78–80). Glycodelin-A has also been suggested to induce dif- a role in decidualization and implantation, as well as immune ferentiation of cytotrophoblast, as evidenced by an increase and blood flow regulation (60–63). There are many transporter in the extravillous trophoblast lineage population and stimula- expressed in the placenta that normally keep tion of hCG production (81, 82). Thus, altered expression of extracellular monoamine concentrations low (64, 65).During PAEP may lead to impaired immune regulation at the decidualization, however, the accumulated histamine in maternal–fetal interface as well as a disrupted trophoblast dif- intracellular vesicles is released and stimulates initial decidual ferentiation, resulting in placental dysfunction. formation (66, 67), because monoamines are potent vasoactive DNER encodes a transmembrane protein that binds agents. In fact, an elevated concentration of monoamines was Notch1 (83). In early pregnancy, the Notch signaling pathway reported in pre-eclamptic pregnancies (68), possibly through functions through cell–cell interactions to regulate stem cell monoamine transporters. Thus, SLC18A2 may be important in renewal and differentiation, decidualization, implantation, regulating monoamines necessary for regulation of uterine and placentation, and angiogenesis (84). Notch1 protein expres- placental blood flow at the maternal–fetal interface. sion is higher in the mid-secretory (receptive phase) endome- CCL21 is a small cytokine belonging to the CC chemokine trium of fertile women, compared with infertile women (85). family and is a ligand for chemokine receptor 7 (CCR7). Thus, DNER may be important for maternal embryonic/fetal Because their function is to recruit immune cells in response communication, leading to appropriate placentation, and to inflammation or homeostatic conditions, cytokines play down-regulation of DNER in IVF pregnancies may impact an active role at the maternal–fetal interface by modulating this communication. Further functional studies are necessary interactions between the decidua and trophoblasts through to understand differences at the maternal–embryonic/fetal its selective recruitment of leukocytes (69–71). Trophoblasts interface. have been shown to actively recruit immune cells through Enrichment analysis of top canonical pathways suggests chemokine production and expression of different that differences in fertility are largely linked to differences in

542 VOL. 111 NO. 3 / MARCH 2019 Fertility and Sterility® inflammatory pathways during the late first trimester. Specif- 3. Sunderam S, Kissin DM, Crawford SB, Folger SG, Boulet SL, Warner L, et al. ically, the C-X-C motif chemokine ligand family (including Assisted reproductive technology surveillance - United States, 2015. MMWR – CXCL9, CXCL10, CXCL11) associated with autoimmunity Surveill Summ 2018;67:1 28. 4. Jackson RA, Gibson KA, Wu YW, Croughan MS. Perinatal outcomes in sin- (e.g. Multiple Sclerosis) seems to be lower in IVF pregnancies, gletons following in vitro fertilization: a meta-analysis. Obstet Gynecol 2004; driving differences in the infertility group. These C-X-C motif 103:551–63. cytokines are important in immune cell migration at the 5. Hansen M, Kurinczuk JJ, Bower C, Webb S. The risk of major birth defects maternal–fetal interface and increase implantation success after intracytoplasmic sperm injection and in vitro fertilization. N Engl J (86, 87). Further, in upstream regulator analysis, multiple Med 2002;346:725–30. cytokine families were among the top upstream regulators 6. Verlaenen H, Cammu H, Derde MP, Amy JJ. Singleton pregnancy after that predicted transcript differences in spontaneous vs. IVF in vitro fertilization: expectations and outcome. Obstet Gynecol 1995;86: 906–10. pregnancies, but not spontaneous vs. NIFT pregnancies. 7. Klemetti R, Gissler M, Sevon T, Koivurova S, Ritvanen A, Hemminki E. Chil- These results suggest that IVF pregnancies may have slightly dren born after assisted fertilization have an increased rate of major congen- altered regulation of cytokine signaling pathways in late first ital anomalies. Fertil Steril 2005;84:1300–7. trimester, compared with other pregnancies, which may be 8. Rimm AA, Katayama AC, Diaz M, Katayama KP. A meta-analysis of altering normal placentation. In contrast, spontaneous vs. controlled studies comparing major malformation rates in IVF and ICSI infants NIFT pregnancies had more diverse top canonical pathways with naturally conceived children. J Assist Reprod Genet 2004;21:437–43. and upstream regulators, which may reflect different 9. Stromberg B, Dahlquist G, Ericson A, Finnstrom O, Koster M, Stjernqvist K. Neurological sequelae in children born after in-vitro fertilisation: a etiology of infertility between NIFT and IVF pregnancies. population-based study. Lancet 2002;359:461–5. Limitations in our study include the racial distribution of 10. Jackson S, Hong C, Wang ET, Alexander C, Gregory KD, Pisarska MD. Preg- our study population because the majority of our cohort was nancy outcomes in very advanced maternal age pregnancies: the impact of Caucasian. Future studies looking at the impact of race on assisted reproductive technology. Fertil Steril 2015;103:76–80. transcriptomic differences of placenta will be important. 11. Wang ET, Ramos L, Vyas N, Bhasin G, Simmons CF, Pisarska MD. Maternal Additionally, because CVS is more commonly performed in and neonatal outcomes associated with infertility. J Matern Fetal Neonatal Med 2018:1–4. women of advanced reproductive age, we were limited by 12. Shevell T, Malone FD, Vidaver J, Porter TF, Luthy DA, Comstock CH, et al. advanced maternal age in our cohort. However, multiple Assisted reproductive technology and pregnancy outcome. Obstet Gynecol studies, including our own, have identified increased 2005;106:1039–45. maternal age with infertility (10, 11, 15). Although maternal 13. Sundheimer LW, Chan JL, Buttle R, DiPentino R, Muramoto O, Castellano K, age in our infertility cohort was greater, it was only slightly et al. Mode of conception does not affect fetal or placental growth param- increased, which may not be clinically significant, and the eters or ratios in early gestation or at delivery. J Assist Reprod Genet 2018; – spontaneous conceptions of advanced maternal age 35:1039 46. 14. Wang ET, Sundheimer LW, Spades C, Quant C, Simmons CF, Pisarska MD. minimize differences that may be due to maternal age. Fertility treatment is associated with stay in the neonatal intensive care unit Another limitation was that chorionic villi consist of and respiratory support in late preterm infants. J Pediatr 2017;187:309–12. multiple cell types. Future studies looking at specific cell 15. Davies MJ, Moore VM, Willson KJ, Van Essen P, Priest K, Scott H, et al. Repro- types within the chorionic villus tree will be necessary to ductive technologies and the risk of birth defects. N Engl J Med 2012;366: determine their functional activity in overall fetal outcomes 1803–13. and how they relate to the underlying infertility or 16. Merritt TA, Goldstein M, Philips R, Peverini R, Iwakoshi J, Rodriguez A, et al. treatments utilized. Impact of ART on pregnancies in California: an analysis of maternity out- fi comes and insights into the added burden of neonatal intensive care. J Peri- In conclusion, this is the rst and largest study to demon- natol 2014;34:345–50. strate that the transcriptomic profiles of first-trimester 17. Boulet SL, Kirby RS, Reefhuis J, Zhang Y, Sunderam S, Cohen B, et al. Assis- placenta, during placentation, in pregnancies conceived ted reproductive technology and birth defects among liveborn infants in with infertility using NIFT or IVF and spontaneous concep- Florida, Massachusetts, and Michigan, 2000-2010. JAMA Pediatr 2016; tions are similar. It is reassuring that overall pregnancies 170:e154934. ~ conceived as a result of infertility treatments are similar to 18. Sober S, Reiman M, Kikas T, Rull K, Inno R, Vaas P, et al. Extensive shift in placental transcriptome profile in preeclampsia and placental origin of spontaneous conceptions. 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Expresion genica diferencial durante la placentacion de embarazos concebidos con diferentes tratamientos de fertilidad en compara- cion con embarazos espontaneos Objetivo: identificar las diferencias en los perfiles transcriptomicos durante la placentacion de embarazos concebidos es- pontaneamente frente a aquellos con infertilidad que utilizan el tratamiento de fertilidad (FIV) sin fertilizacion in vitro (NIFT) o FIV. Diseno:~ Estudio de cohorte. Entorno: Centro medico academico. Paciente (s): mujeres sometidas a muestreo de vellosidades corionicas en edad gestacional de 11–13 semanas (n ¼ 141), con embarazos que fueron concebidos espontaneamente (n ¼ 74), con NIFT (n ¼ 33), o con FIV (n ¼ 34), resultantes en partos con descendencias viables. Intervencion (es): coleccion de muestras de vellosidades corionicas de mujeres que concibieron espontaneamente, con NIFT o con FIV para el analisis de la expresion genica mediante secuenciacion de ARN. Principales medidas de resultado: datos demograficos maternos, paternos y fetales de referencia, estado medico materno, complica- ciones del embarazo y resultados. Expresion genica diferencial de la placenta del primer trimestre. Resultado (s): Hubo pocas diferencias en el transcriptoma de la placenta del primer trimestre de NIFT, IVF y embarazos espontaneos. Hubo expresion diferencial de un gen codificador de proteínas (DEG) entre los grupos espontaneos y de infertilidad, CACNA1I, un co- dificador DEG de proteínas entre los grupos espontaneos y de FIV, CACNA1I, y cinco codificadores DEG de proteínas entre los grupos NIFT e IVF, SLC18A2, CCL21, FXYD2, PAEP y DNER. Conclusion (es): Este es el primer estudio y el mas grande que analiza los perfiles transcriptomicos de la placenta del primer trimestre y que demuestra perfiles transcriptomicos similares en embarazos concebidos con NIFT o FIV y concepciones espontaneas.

546 VOL. 111 NO. 3 / MARCH 2019 S upp.ement a1 T a bl e 1 Genes from Compare Top Canonical Pathways P-value Pathway SPONTvs Toll-like Receptor Signaling 1.23E-03 10/76 INFERT Neuroinflammation Signaling Pathway 3.11E-03 24/311 Pathogenesis of Multiple Sclerosis 5.09E-03 3/9 NF -KB Signaling 5.50E-03 16/187 Role of PKR in Interferon Induction and Antiviral Response 6.82E-03 6/41 SPONTvs Crosstalk between Dendritic Cells and Natural Killer Cells 4.99E-04 10/89 NIFT Production ofNitric Oxide and Reactive Oxygen Species in Macrophages 1.32E-03 151194 Dopamine-DARPP32 Feedback in cAMP Signaling 2.18E-03 13/164 Neuroinflammation Signaling Pathway 2.23E-03 20/311 Sonic Hedgehog Signaling 2.29E-03 5/30 SPONTvs Pathogenesis of Multiple Sclerosis 2.47E-04 4/9 IVF Calcium Signaling 4.70E-04 19/206 Neuroinflammation Signaling Pathway 5.41E-04 25/311 Antigen Presentation Pathway 5.85E-04 7/38 GABA Receptor Signaling 1.17E-03 11195 NIFT Pathogenesis of Multiple Sclerosis 6.78E-04 3/9 vs Antigen Presentation Pathway 1.07E-03 5/38 IVF Granulocyte Adhesion and Diapedesis 1.45E-03 11 /181 Agranulocyte Adhesion and Diapedesis 2.40E-03 11/193 Proline Biosynthesis I 2.50E-03 2/4 Supplemental Table 2: Upstream regulators of differentially expressed genes, spontaneous versus IVF pregnancies. Input genes: D"1f flerent1a . 11 y expresse d genes w1t . h P -vaI u e<0 .05 m. RNA -seq. Rank Upstream Regulator Molecule Type P-value Target molecules in dataset 1 SH3RF1 other 0.0000786 EOMES,MAP3K10,TBX21 ,TNF 2 muromonab-CD3 biologic drug 0.000145 GZMB,ICOS,TBX21 ,TFRC,TNF CD74,CXCL1 O,CXCL11 ,CXCL9,EOMES,GZMB,HLA -A,HLA-B,HLA-DP A 1, HLA- 3 IL27 cytokine 0.00105 DQA1 ,ICOS,TBX21 ,TNF,TNFSF18 4 C4A/C4B peptidase 0.00114 C4A/C4B,CXCL1 O,TNF 5 formoterol chemical drug 0.00133 CXCL10,CXCR1 ,FOSL1 ,UBE2B 6 IKK (complex) complex 0.00147 BCL2,CXCL1 O,EOMES,TBX21 ,TNF 7 MARCH2 enzyme 0.00158 TFRC,TNFRSF10A 8 SMYD5 other 0.00158 CXCL10,TNF 9 AP1M1 transporter 0.00158 ATP7 A,HLA-A 10 mir-370 m1croma 0.00158 GAD2,PTPRN 11 GGA2 transporter 0.00158 GGA1 ,GGA3 12 IFNA10 cytokine 0.0018 CXCL10,CXCL11 ,CXCL9,STAT4 13 IFNA21 cytokine 0.0018 CXCL10,CXCL11 ,CXCL9,STAT4 14 IFNA5 cytokine 0.0018 CXCL10,CXCL11,CXCL9,STAT4 15 IFNA17 cytokine 0.0018 CXCL10,CXCL11,CXCL9,STAT4 16 IFNA7 cytokine 0.0018 CXCL10,CXCL11,CXCL9,STAT4 17 IFNA14 cytokine 0.0018 CXCL10,CXCL11,CXCL9,STAT4 18 IFNA6 cytokine 0.0018 CXCL10,CXCL11,CXCL9,STAT4 BCL2,CXCL11 ,CXCL5,CXCL9,CXCR1 ,FGF5,PDGFA 19 CCL11 cytokine 0.00198 ,TNF CXCL10,CXCL11 ,CXCL9,GBP5,JAML,PMEPA1 ,STA 20 IFNA4 cytokine 0.00211 T4 chemical - endogenous 21 isopentenyl diphosphate mammalian 0.00238 CCR5,FCGR3A/FCGR3B,TNF,XCL1 22 IFNA8 cytokine 0.00238 CXCL10,CXCL11 ,CXCL9,STAT4 23 IFNA16 cytokine 0.00238 CXCL10,CXCL11 ,CXCL9,STAT4 24 PTF1A transcription regulator 0.00274 CDX2,CEL,CPA2,GAD2,GRIK1 ,GRM5,SEZ6,TF AP2B 25 DEPTOR other 0.00301 CXCL10,CXCL11 ,CXCL9 26 OCLN other 0.00301 CLDN1,MMP1 ,SIRT1 27 PAX9 transcription regulator 0.00301 CD19,NKX3-2,SOX9 CD74,HLA-A,HLA-B,HLA-DPA 1,HLA - 28 CIITA transcription regulator 0.00313 DQA1,0SCAR salmonella minnesota R595 chemical - endogenous ADAM9,CCNL2,CD38,CXCL10,CXCL5,CXCL9,FOS 29 non-mammalian 0.00318 L 1, HLA-DP A1 ,HLA-DPB 1, HLA-DQA 1, TFEC,TNF ADAM9,ADAMDEC 1, CD74,CFB,CHI3L1 ,CSF2RA,C XCL10,CXCL5,CXCL9,GBP5,HLA-DPA1 ,HLA- 30 fluticasone propionate chemical drug 0.00387 DPB1 ,HLA-DQA1 ,HTRA1 ,SERPINA1 31 asbestos chemical toxicant 0.00438 FOSL1 ,PDGFA,TNF 32 sethoxydim chemical toxicant 0.0046 BCL2,HDAC4 33 Raet1b other 0.0046 HLA-A,TNF miR-370-3p (and other miRNAs w/seed 34 CCUGCUG) mature microma 0.0046 GAD2,PTPRN 35 H60a other 0.0046 HLA-A,TNF 36 YWHAE . other 0.0046 FOSL1 ,MMP1 37 ODN1411 chemical reagent 0.0046 CXCL10,TNF chemical - kinase 38 ( Ala9)-autocamtide-2 inhibitor 0.0046 CRH,MMP1 ACACB,ACADL,AXIN2,BRCA1,BTK,CD19,CHPT1 ,E VI2A,EVL,HLA-B,HLA- G,IL17RE,KIF1B,LIG4,MMP1 ,NKX3- 1,NTN1 ,PDGFA,PIPOX,PPFIBP2,PPM1E,RAPGEF4,S 39 EP300 transcription regulator 0.00465 IRT1 ,SLC27A1 ,STAT4,TLE6,TNF,TRPM1 ,ZFP36L2 40 IL17R complex 0.00485 CXCL10,CXCL11 ,CXCL5,MMP1 41 IFNW1 cytokine 0.00485 CXCL10,CXCL11 ,CXCL9,STAT4 CD74,CNTNAP2,CXCL10,CXCL11,HLA-DPA1 ,HLA- 42 SAFB other 0.00531 DPB1,UBE2B 43 PIM1 kinase 0.00588 BCL2,EPHA2,FOSL1 ,NKX3-1 ,0SCAR,STAT4,TBX21 44 CD2 transmembrane receptor 0.00597 BCL2,HLA-DPA1 ,ITGAL,PTPRC,STAT4,TNF 45 ADCY7 . enzyme 0.00608 IRAK2, TNF ,TRAF6 46 ANG enzyme 0.00608 BCL2,CXCL 1O,CXCL9 47 PDE4B enzyme 0.00608 CXCLlO,CXCLll,TNF 48 PIM3 kinase 0.00608 STAT4,TBX21,TNF 49 SLC6Al transporter 0.00608 BCL2,TBX21 , TNF 50 IL17D cytokine 0.00608 CD38,CXCL10,TBX21 51 PRKCQ kinase 0.0061 FOSL 1, ITGAL,MMPl, TBX21 ,TNF 52 etanercept biologic drug 0.0061 CXCL10,CXCL9,GZMB,PTGES,TNF 53 CARD9 other 0.0061 CCR5,CLEC4E,CXCL10,FCGR1A,TNF ENKUR,GABRQ,HTRA 1, NTNl ,SATB 1,SLC 17 A8,SL 54 FEV transcription regulator 0.00708 C 18A2,SOX9, TCF7L2 55 HRG other 0.00721 CD74,FCGR1A,FCGR3A/FCGR3B,HLA-DQA1 56 ETVl transcription regulator 0.00721 ASCL 1,BRCA 1,MMP 1,SOX9 g-protein coupled 57 CXCR2 receptor 0.00721 CXCLl O,ITGAL,SOX9,TNF ASPSCRl,ATG16Ll,BCL2,ClQTNF12,C4A/C4B,CAP Nl O,CHST8,CTXN1 ,CYLD,DCAF4,DNAJC2,FAH ,FE S,FOSL1,GABRA3,GNB5,HLA- A,IL4Il,IN080E,KCNK4,KIAA0556,MOB3A,NAV1 ,N PTX2,PDGF A,PPTC7 ,PRKG2,PSMC3IP ,RANBP3L,R chemical - endogenous BL2,SATB 1,SEZ6,SLC22A 18,SLC29 A3 ,SLC3 8A2,SO 58 L-dopa mammalian 0.00779 X9,SRPK3,ST8SIA3,WDR17,ZNF385B 59 vismodegib chemical drug 0.00812 BCL2,CXCL9,SOX9 60 TXK kinase 0.00812 EOMES,TBX21 ,TNF chemical - endogenous 61 oligomycin non-mammalian 0.00812 BCL2,CXCL10,TNF 62 albuterol chemical drug 0.00812 CCR5,GRK2,TNF ACACB,ACADL,ANGPTL3,APOC2,BCL2,CCR5,CD5 63 Nrlh Igroup 0.00812 L,CLEC4E,CXCL10,FLT4,ITGAL,PTGES,TNF g-protein coupled 64 CXCR3 receptor 0.00863 BCL2,CXCL 1O,MAP 1LC3B ,MMP 1 65 RNASE I enzyme 0.00863 CXCLl O,CXCL5 ,CXCL9,TNF 66 NLRC5 transcription regulator 0.00863 HLA-A,HLA-B,HLA-G,TNF 67 Lymphotoxin complex 0.00863 CCL19,CXCL10,MMP1 ,TNF 68 Klra7 (includes others) transmembrane receptor 0.00863 ABCD3,TBX21 ,TNF,XCL1 BCL2,BRCA 1, HLA- 69 FOXP3 transcription regulator 0.00896 A,ICOS,PAXIP1 ,SATB1 ,TBX21 ,TNF,TRAF6,USP21 70 Il12 receptor complex 0.00897 TBX21,TNF 71 IGF2BP2 translation regulator 0.00897 LIMS2,TRIM54 72 DHCR24 enzyme 0.00897 GGA3,TNF 73 DACT3 other 0.00897 AXIN2,DVL2 74 FLII other 0.00897 CLDN1 ,TNF 75 CACNB4 ion channel 0.00897 CACNA1B,CACNB3 miR-149-5p (miRNAs 76 w/seed CUGGCUC) mature microrna 0.00897 RAP 1A,RAP 1B 77 CELF2 other 0.00897 BCL2,SIRT1 78 TRPV5 ion channel 0.00897 CALB 1, TRPV 6 79 GABRA1 ion channel 0.00897 GABRA2,GABRA3 80 chlorothiazide chemical drug 0.00897 CALB 1, TRPV 6 81 bupropion chemical drug 0.00897 TNF ,TNFRSF1 OA 82 galanthamine chemical drug 0.00897 BCL2,TNF chemical - endogenous 83 acetate mammalian 0.00897 SIRT1 ,TNF 84 IL15RA transmembrane receptor 0.0102 BCL2,GAD2,PTPRC, TNF 85 JUN/JUNB/JUND group 0.0102 COL7A1,FOSL1,ICOS,MMP1 86 CISH other 0.0102 CXCL 1O ,EOMES, TBX21 ,TNF ABCD3,ADCY5,ADORA2B,ARAP2,ASCL 1, BCL2,C4 A/C4B,CALB 1,CCL19,CCR5,CD38,CD5L,CD74, 2,CFB,CHRNE,CHST8,CLEC10A,CLEC4E,CXCL10,C XCL11 ,CXCL5,CXCL9,CYLD,FAH,FCGR1A,FCGR3 A/FCGR3B,FGG,FLT4,GAD2,GBP5,GGA1 ,GNB5,GP R146,GRK2,GZMB,HLA-A,HLA-B,HLA-DQA1 ,HLA- G,HTRA1 ,1L13RA2,IL411 ,1RAK2,ITGAL,MMP1 ,MUC 4,NMI,NTN1 ,P AX6,PDGFA ,PRKCA,PTGES,PTPRN ,S BF1 ,SERPINA1 ,SERPINA5,SIX1 ,SLC28A2,STAT4,TB C 1D 1OA , TBX21 , TCF7L2,TFRC,TNF,TNFRSF1 OA,TR 87 IFNG cytokine 0.0105 AF6,UBE2D1 ,XCL1 ,ZKSCAN1 highly active antiretroviral 88 therapy chemical drug 0.0105 BCL2,CCR5,TNF 89 LILRB4 other 0.0105 CXCL10,CXCL11 ,TNF 90 JUP other 0.0105 AXIN2,BCL2,SATB 1 chemical - endogenous 91 D-sphingosine mammalian 0.0105 BCL2,CLDN1 ,TNF EOMES,HLA-A,HLA-B,HLA-DP A1 ,HLA- 92 EBB cytokine 0.0114 DQA1 ,TBX21 93 oxymatrine chemical drug 0.012 BCL2,CLDN 1,PTPRC, TNF 94 IFNK cytokine 0.012 CXCL10,CXCL11,CXCL9,STAT4 AHRR,BCL2,BLK,CCL19,CFB,CHI3L1 ,CSF2RA,CXC L10,CXCL11,CXCL9,FGG,IKBKG,NMI,PDGFA,POL 95 NFKB1 transcription regulator 0.0127 B,PXN,SOX9,TBX21 ,TNF chemical - endogenous 96 D-galactose mammalian 0.0133 BCL2,CRYBA1,TNF 97 GNB3 enzyme 0.0133 GNB5,PRKCA,TRPM1 98 B4GALNT1 enzyme 0.0133 C4A/C4B,MAG,TNF 99 DOK1 kinase 0.0133 STAT4,TBX21 ,TNF clasto-lactacystin beta- chemical - protease 100 lactone inhibitor 0.0133 MAPK6,PRKCA,TP53BP2 chemical- endogenous 101 lpalmitoleic acid mammalian 0.0133 CLEC 1OA,RICTOR , TNF 102 TBX21 transcription regulator 0.0133 CCR5,CD38,EOMES,GZMB,ICOS,STAT4,TBX21 103 IFNE cytokine 0.0133 ANGPTL3,CXCL10,CXCL11 ,CXCL9,STAT4 BCL2,CCR5,CXCL1 O,CXCL9,EOMES,EVI2A,EVL,G FI1B,GRIN3B,HLA-B,HLA- G,IL17RE,KIF1B,LIG4,NTN1,PIPOX,PPFIBP2,PPM1E ,RAPGEF4,SLC18A2,STAT4,TLE6,TNF,TRPM1,ZFP3 104 CREBBP transcription regulator 0.0137 6L2 105 Ikb group 0.0139 BCL2,CFB,MMP1,TNF 106 CDK12 kinase 0.0146 BRCA1,SMARCC2 107 FOXIl transcription regulator 0.0146 SLC12A3,SLC4A9 108 RIPK4 kinase 0.0146 GRHL3,MAD1L1 109 WNK4 kinase 0.0146 SLC12A3,WNK4 110 ARG2 enzyme 0.0146 BCL2,TNF 111 ADAMS peptidase 0.0146 CXCL10,TNF 112 ARL5B enzyme 0.0146 CXCL10,TNF 113 KRT10 other 0.0146 IKBKG,TNF 114 Mucl transmembrane receptor 0.0146 PDGFA,TNF 115 MARCHI enzyme 0.0146 TNF,TNFRSF10A 116 RFXANK transcription regulator 0.0146 HLA-A,HLA-B 117 butylphen chemical toxicant 0.0146 ADORA2B,TNFRSF10A chemical - endogenous 118 azelaic acid mammalian 0.0146 BCL2,TNF chemical - endogenous 119 N -arachidonoyl-dopamine mammalian 0.0146 PTGES,TNF 120 !phloroglucinol chemical drug 0.0146 BCL2,MMP1 chemical - endogenous 121 starch mammalian 0.0146 CALB 1, TRPV 6 AXIN2,BCL2,BRCA1 ,CCR5,CDX2,CX:CL10,CXCL11 , CYB5R2,EOMES,EPC1 ,FGF5,GABRA2,GRIK5,MAP K15,mir- 154,P AX6,PCDHB5,PTGES,SATB 1, SERPINA1 ,SIRT1 122 EZH2 transcription regulator 0.0148 ,TBX21,TNF CDX2,DMBT1 ,NKX3- 123 SOX9 transcription regulator 0.0155 2,PRKCA,SOX9,TCF7L2, WWP2 chemical - kinase 124 JAK inhibitor I inhibitor 0.0161 CD38,CXCL10,CXCL11 ,CXCL9 125 CSF3R transmembrane receptor 0.0164 FCGR1A,LTF,L YZ 126 CCL19 cytokine 0.0164 CCL19,CXCL10,TNF 127 picryl chloride chemical toxicant 0.0164 CXCL10,HLA-DQA1 ,TNF 128 temozolomide chemical drug_ 0.0166 BCL2,CXCL10,CXCL9,RAP1B,TNFRSF10A 129 POU4F2 transcription regulator 0.0166 BCL2,BRCA1,CALB1,PAX6,ZFHX4 ACADL,BCL2,DDX55,GPR146,ICOS,IQCE,MMP1 ,SO 130 SOD2 enzy}lle 0.017 RT1 ,TNF 131 ITK kinase 0.0172 EHD4,EOMES,HLA- A,SLAMF7,STAT4,TBX21 ,TNF,XCL1 BCL2,CXCL 1O ,CXCL5,DMBT1 ,HLA- 132 IL22 cytokine 0.0172 B,MMP1,MUC4,PDGFA,RBL2,TNF 133 MMP1 I peptidase 0.0181 BCL2,BRCA1 ,MMP1,PDGFA,PTGES,TNF 134 CEBPE transcription regulator 0.0181 BCL2,CLEC 1OA,L TF ,L YZ,MAD 1L 1,TNF 135 chloroquine chemical drug 0.0181 CXCL10,MAP1LC3B,STAT4,TNF,TRAF6,ULBP2 136 JAG2 !growth factor 0.0184 CXCL5,CXCL9,CXCR1 ,MMP1 ,TNF chemical - endogenous 137 acetylcholine mammalian 0.0184 CRH,MMP1 ,PNMT,TNF 138 ADAMTS12 Ipeptidase 0.0184 CHI3L 1, CLEC4E,CXCL 1O ,SOX9 139 Tcf7 transcription regulator 0.0184 BCL2,EOMES,TBX21 ,TNF CALB 1,CAMTA2 ,FOSL 1, GABRG3,HDAC4,PPM1E,P 140 HDAC4 transcription regulator 0.0192 RKCA,RTN1 ,TNF ,TRPVl ACADL,CCL19,CCR5,CXCL10,CXCL11 ,EOMES,FLT 4,GZMB,ICOS,ITGAL,STAT4,TBX21 ,TFRC,TNF,XC 141 IL12 (complex) complex 0.0193 142 RELB transcription regulator 0.0195 BCL2,CCL19,CHI3Ll,CRH,STAT4,TBX21,TNF ACACB,ATP7 A,BCL2,BRCA1 ,CDX2,FOSL 1,MAFB , 143 MYOD1 transcription regulator 0.0198 MYH3,PDGFA ,PMEP A 1, PNMT,SIX1 , TNNil, TNNT2 ABCD3,ADORA2B,BCL2,CALB1,CD74,CDX2,CFB,C LEC4E,CXCL10,CXCL11,CXCL5,CXCL9,CYLD,DM BT1,EOMES,FCGR1A,FGF5,FGG,GAD2,HDAC6,HL A- A,ICOS,IL 13RA2,ITGAL,MMP 1, P ASK,PTGES,PXN ,S ENP2,STAT4,STK1 0, TCF12,TFEC,TFRC,TNF, TNFRS 144 NFkB (complex) complex 0.0198 FlOC 145 MAZ transcription regulator 0.0199 FGG,MMP1 ,PNMT 146 Cux1 transcription regulator 0.0199 CXCL10,DKK2,HTRA1 147 histone deacetylase complex 0.0204 BCL2,CRH,CSF2RA,TNF,TNFRSF10A 148 ETS2 transcription regulator 0.0209 BRCA1,CDX2,CHRNE,L YZ,MMP1,TFEC,TNF ACADL,CLEC4E,CXCL1 O,PTGES,RALGDS,SLC27 A 149 RIPK2 kinase 0.0209 1,TNF 150 alitretinoin chemical drug 0.021 ACACB,ANGPTL3,BCL2,CALB1,CD38,CD5L,CXCL 1O,CXCL11 ,HOT AIRM1 ,L TF ,MMP 1,SLC27 A1 chemical - endogenous 151 lpsychosine mammalian 0.0213 FOSL1,TNF 152 Stat1 dimer complex 0.0213 FCGR1A,TBX21 153 LRRK2 kinase 0.0213 MAP1LC3B,TNF 154 HDAC11 transcription regulator 0.0213 MGAT5B,TNF 155 PHLPP2 enzyme 0.0213 PRKCA,TNF chemical - endogenous 156 vaccenic acid mammalian 0.0213 PLIN4,TNF 157 ERAP1 peptidase 0.0213 HLA-A,HLA-G 158 CXCL5 cytokine 0.0213 ITGAL,TNF 159 RBX1 enzyme 0.0213 BCL2,BRCA1 160 LRIG1 other 0.0213 BCL2,TNF 161 HTRA1 peptidase 0.0213 HTRA1,MMP1 162 TNFRSF14 transmembrane receptor 0.0213 MMP1,TNF 163 Muc4 other 0.0213 LYZ,TNF 2,3 -dioxo-6-nitro-7- sulfamoy1 benzo( f)quinoxalin 164 e chemical reagent 0.0213 GRIK4,GRIK5 chemical - endogenous 165 xestospongin C non-mammalian 0.0213 CRH,TNF ACCS,ADCY5,CRLF2,FAH,GABRG3,HDX,IGSF1,NK 166 KMT2D transcription regulator 0.0218 APL,PCDH7,REEP6,SLC38A6,TNNT2,WDR17 BCL2,BTK,CXCL10,FCGR1A,HTR7,ICOS,IL4I1 ,ITG . . 167 wnomycm chemical reagent 0.0218 AL,SIRT1 ,SLC18A2,TBX21 ,TNF,XCL1 168 NFKB2 transcription regulator 0.0224 BCL2,CCL19,CRH,HLA-DQA1,mir-154,PXN,TNF AOAH,CCR5,FCGR1A,GBP5,GZMB,HLA- 169 interferon beta-1 a biologic drug 0.0236 DPB1 ,LILRA2,LTF,NMI,SERPINA1 170 ZFP36 transcription regulator 0.0237 ABCG4,AHRR,E2F8,FOSL1,LTF,MMP1,TFRC,TNF BCL2,CALB 1,CLTC,DHX36,EPS8,INPPL 1,L YZ,MAF 171 HOXA9 transcription regulator 0.0237 B,OSBPL8,PIK3R2,PTPRC,RAP1 B,SATB 1 172 MDK !growth factor 0.0238 BCL2,GZMB,PTPRC 173 DDX17 enzyme 0.0238 FOSL1,HTRA1,mir-515 174 IL23 complex 0.0247 BCL2,ICOS,IL4Il,TBX21,1NF BCL2,CXCL 1O,CXCL 11 ,CXCL9 ,HLA-A,HLA- B,HSH2D,KCNK17 ,LILRB2,NLGN2,NPTX2,RBL2,SL 175 IFNA2 cytokine 0.025 C18A2,STAT4,TBX21,1NF 176 EFNA2 kinase 0.0251 DSC1 ,FOSL1,KRT13,NKX3-1 ,PXN,SOX9 miR-146a-5p (and other miRNAs w/seed BRCA1,DMBT1,IL1RL2,IRAK2,LTF,TBX21 ,TNF,TR 177 GAGAACU) mature microma 0.0251 AF6 BCL2,CXCL10,CXCL9,FOSL1,FXYD1,GZMB,MAG, 178 S100A8 other 0.0257 MMP1,MYH8,PTGES,SIX1,SLC26A1 ,1NF 179 NPC2 transporter 0.0267 MAP1LC3B,PTGES,STAT4,1NF 180 SCD enzyme 0.0271 ACACB,ACADL,BCL2,CXCL10,SLC27A1,1NF 181 IL12B cytokine 0.0271 CCR5,CXCL1 O,EOMES,GZMB,STAT4,1NF AXIN2,BCL2,CDX2,CLDN1 ,HOXA3,KCNJ15,MUC4, 182 CDX2 transcription regulator 0.0274 SOX9,TCF7L2,USP12 AXIN2,CLEC4E,CXCL 1O ,DMBT1 ,PTGES,RALGDS,T 183 NOD2 other 0.0274 NF 184 Fcgr3 group 0.028 IT GAL, TBX21 ,TNF 185 Iga complex 0.028 CXCL10,FCGR1A,TNF 186 dactolisib chemical drug 0.028 BCL2,CXCL5,RICTOR chemical - endogenous 187 phosphatidylinositol mammalian 0.029 MMP1,1NF 188 SLC5A8 transporter 0.029 BCL2, TNFRSF 1OA 189 ARID5A transcription regulator 0.029 TBX21,1NF 190 ZC3HAV1 other 0.029 CXCL10,1NF 191 IgG2a complex 0.029 BCL2,1NF 192 CYP11A1 enzyme 0.029 CRH,PNMT 193 ABCD1 transporter 0.029 ABCD3,1NF 194 ST8SIA4 enzyme 0.029 P AX6,PIK3CA 195 WISP1 other 0.029 AXIN2,MMP1 196 RAPGEF1 other 0.029 PXN,RAP1A miR-491-5p (and other 197 miRNAs w/seed mature microma 0.029 GIT1 ,PXN GUGGGGA) 198 RASGRP4 other 0.029 IL13RA2,TNF 199 TAPBP transporter 0.029 HLA-A,HLA-B 200 NEU3 enzyme 0.029 BCL2,MMP1 201 TSPO transmembrane receptor 0.029 BCL2,TNF 202 dacarbazine chemical drug 0.029 CXCL10,CXCL9 203 lemairamin chemical reagent 0.029 TNF,TRAF6 204 chemical toxicant 0.029 BCL2,TNF 205 diacerein chemical drug 0.029 BCL2,TNF chemical - endogenous 206 geranylgeraniol mammalian 0.029 MMP1,RAP1A 207 isotretinoin biologic drug 0.0293 ATP12A,BCL2,CD19,CD38,MMP1,PRKCA,TFRC ABCC8,BCL2,C2orf40,CD38,GRAP,KLF7,MAP4K2,M UM1L1,PCDH7,PHF1 ,RAPGEF1,RICTOR,SLC38A2,S 208 LMNA other 0.0297 MARCC2,YTHDF2,ZNRF2 209 GNAI2 enzyme 0.0298 CCR5,ITGAL,PRKCA, TNF BTK,CD19,CHI3L1 ,CSF2RA,CXCL10,CXCR1 ,FES,H OTAIRM1 ,L TF ,L YZ,mir- 25,MMP1 ,0SCAR,PTPRC,TFEC,TNF,TUBA3C/TUBA 210 SPil transcription regulator 0.0321 3D ATRNL1 ,C4A/C4B,CNTNAP2,CSMD 1,CSNK1 G2,EF NB 1, GRM5,GTF2H2,HLA- 211 topotecan chemical drug 0.0324 DQA1,0SBPL8,PRKN,UBE2B,UBE3A 212 mir-143 m1crorna 0.0327 BCL2,MAPK7 ,OSBPL8 213 CXCL3 cytokine 0.0327 CXCL10,GRK2,TNF chemical- endogenous 214 antimycin A non-mammalian 0.0327 BCL2,MMP1 ,TNF 215 cuprizone chemical toxicant 0.0327 CXCL10,MAG,TNF 216 ALOX15 enzyme 0.0332 AXIN2,BCL2,FOSL 1, TNF 217 CITED2 transcription regulator 0.0332 CDX2,FGF5,MMP1 ,PCGF2 218 linsidomine chemical drug 0.0332 CRH,MMP1 ,PDGFA ,TNF 219 PDPK1 kinase 0.0332 BCL2,CXCL 1O,PDGF A, TNF 220 TNFSF14 cytokine 0.0332 CXCL10,CXCL11,CXCL5,TNF 221 VTN other 0.0332 BCL2,MMP 1, PXN, TNF 222 fluvoxamine chemical drug 0.0332 CCR5,CXCR1 ,HTR7,TNFRSF10C N-acetylmuramyl-L-alanyl- chemical - endogenous 223 D-isoglutamine non-mammalian 0.0337 AOX1 ,AXIN2,CLDN1 ,CXCL11 ,DMBT1,TNF 224 HIC1 transcription regulator 0.0337 ADORA2B,AHNAK2,AN02,EPHA2,SIRT1 ,SOX9 ATG7,BCL2,CDX2,CXCL10,CXCL11 ,CXCL9,PXN,SI 225 PBK (family) group 0.0344 RTl,TNF BCL2,BLK,CD74,EOMES,EVI2A,EVL,LYZ,NAV1 ,PI 226 SOX4 transcription regulator 0.035 TRMl ,SLC17 A8,SORT1 ,TMEM2,TMEM255A BCL2,CCR5,CXCL10,CXCL11 ,CXCL9,FCGR3A/FCG 227 IL18 cytokine 0.0362 R3B,GZMB,ITGAL,MMP1 ,TBX21 ,TNF,XCL1 228 CORT other 0.0368 CCL19,CCR5,CXCL10,TNF 229 bafilomycin A 1 chemical drug 0.0368 CXCL10,TNF,TNFRSF10A,ULBP2 ADAMDEC1 ,ADORA2B,CCR5,CHBL1,CLEC4E,CX CL5,EVL,FCGR1A,HTRA1 ,IL13RA2,JAKMIP2,KCNJ 15 ,LILRB2,MCURl,MMPl,PDGFA,PITRMl,PTGES,S 230 IL13 cytokine 0.0374 ERPINA1 ,SORT1 ,TFEC,TNF,TRPV1 231 PPTl enzyme 0.0376 CXCLlO,TNF 232 SlOOAlO other 0.0376 MMPl,TNF 233 Ul snRNP complex 0.0376 SLAMF7,TNF 234 HLA-DQBl other 0.0376 HLA-DQAl,TNF 235 MAOA enzyme 0.0376 BCL2,SLC 18A2 236 CLTC other 0.0376 CD74,HLA-A 237 MAD2L1 other 0.0376 BCL2,SOX9 238 CXCL16 cytokine 0.0376 CD38,TNF 239 ULBPl transmembrane receptor 0.0376 HLA-A,TNF 240 vadimezan chemical drug 0.0376 CXCL10,TNF 241 alpha-tocopherol succinate chemical drug 0.0376 BCL2,TNF chemical - endogenous 242 thymidine mammalian 0.0376 MMP1 ,MUS81 243 Pde4 group 0.0377 CRH,CXCL5,TNF 244 MAPK8IP1 other 0.0377 EOMES, TBX21 ,TNF 245 NODI other 0.0377 CDX2,CYLD,TNF 246 triflusal chemical drug 0.0377 CD74,TNF,XCL1 247 idarubicin chemical drug 0.0377 BCL2,TP53BP2,TP53INP1 chemical- endogenous 248 sodium chloride mammalian 0.0386 CALB 1,CRH,PTGES,SLC 12A3, TNF ,TRPV6 Salmonella enterica serotype abortus equi ADAMDEC 1,ADORA2B,CFB,CXCL5,FOSL1 ,LRRC8 249 chemical toxicant 0.0391 C,MAD 1L1 ,MMP 1, NME7 ,SLAMF7, TEX14,TNF BRCA1,CD74,CRHBP,CTXN1,EPS8,GBP5,HLA- DQA1,KIF1B,LRRK2,MMP1 ,SLC18A2,TENM1 ,TME 250 SNCA enzyme 0.0392 M117,TMEM255A,TNF ATG7,AXIN2,BTK,CD19,CD74,CHI3L1,CLDN1,FGG, FOSLl,HLA- 251 PRDMl transcription regulator 0.0392 DQA1 ,MAFB,MMP25,SLC16A5,TBX21,TNF chemical - endogenous 252 taurolithocholic acid mammalian 0.0397 TNF 8-cyclopentyl-1 ,3 - 253 dipropylxanthine chemical reagent 0.0397 TNF 254 Pdel Igroup 0.0397 CRH 255 CEACAM16 other 0.0397 TECTB 256 CGP 57380 chemical reagent 0.0397 TNF 257 carteolol chemical drug 0.0397 TNF 258 thymidine phosphorylase Igroup 0.0397 MARCKS 259 TFIIF complex 0.0397 POLB coactivator-Dtx-Notch- 260 Rbpsuh complex 0.0397 MAG chemical - endogenous 261 soraphen-Alalpha non-mammalian 0.0397 RICTOR chemical - endogenous 262 MALP-2R non-mammalian 0.0397 TNF chemical- endogenous 263 Silybum marianum extract non-mammalian 0.0397 TNF 264 sodium thiosulfate chemical drug 0.0397 BCL2 265 TOX2 transcription regulator 0.0397 TBX21 266 DACT1 other 0.0397 DVL2 267 RGN enzyme 0.0397 BCL2 268 TOX3 other 0.0397 BRCA1 269 TET3 enzyme 0.0397 MGAT5B 270 KREMEN1 other 0.0397 AXIN2 271 GKN1 ~owth factor 0.0397 SOX9 272 CTSO peptidase 0.0397 BRCA1 273 MARCH4 enzyme 0.0397 TNFRSF10A 274 SNORD21 other 0.0397 HLA-A 275 NYX other 0.0397 TRPM1 276 TADA2B peptidase 0.0397 CD19 277 CXCL17 cytokine 0.0397 GPR35 278 WASHC5 other 0.0397 ATP7A 279 NLGN2 enzyme 0.0397 GAD2 280 TLR2/3/4 group 0.0397 STAT4 281 NFKBIL1 transcription regulator 0.0397 TNF 282 KRT71 other 0.0397 ATP7A g-protein coupled 283 GPR18 receptor 0.0397 TNF 284 CLEC16A other 0.0397 PRKN 285 HCG18 other 0.0397 TRAF6 286 PX-866 chemical drug 0.0397 SORT1 287 MIIP other 0.0397 HDAC6 288 !phosphodiesterase 4 inhibitor chemical drug 0.0397 TNF 289 POU5F2 transcription regulator 0.0397 CRH 290 PHF2 enzyme 0.0397 HTRA1 291 MARCH9 other 0.0397 TNFRSF10A 292 METAP2 !peptidase 0.0397 BCL2 293 Tcp4 other 0.0397 HTRA1 294 LILRA3 other 0.0397 TNF

295 Caveolin I group 0.0397 TNF 296 NSC 270012 chemical reagent 0.0397 TNF 297 dalcetrapib chemical drug 0.0397 TNF 298 MT-NDl enzyme 0.0397 HLA-G 299 ivabradine chemical drug 0.0397 CXCLll 300 abexinostat chemical drug 0.0397 MUC4 301 HCV 796 chemical drug 0.0397 MAP1LC3B 302 voxtalisib chemical drug 0.0397 BCL2 303 MEDI-547 biologic drug 0.0397 EPHA2 304 CYP21A2 enzyme 0.0397 PNMT 305 RASGRF2 other 0.0397 TNF 306 SYTL4 transporter 0.0397 STXlA 307 MED25 other 0.0397 MMPl 308 TPR other 0.0397 MADILl 309 NAPA transporter 0.0397 BCL2 310 RGS9 enzyme 0.0397 GNB5 311 OTP transcription regulator 0.0397 CRH 312 GR 82334 biologic drug 0.0397 CXCLlO 313 PRL-3 inhibitor I chemical reagent 0.0397 ULBP2 314 recombinant interferon beta biologic drug 0.0397 HLA-G 315 Slpi (includes others) other 0.0397 TNF 316 ADAMTS7 'peptidase 0.0397 SOX9 317 mir-302 m1croma 0.0397 HDAC4 miR-545-3p (miRNAs 318 w/seed CAGCAAA) mature microma 0.0397 BRCAl 319 mir-329 m1croma 0.0397 GAD2 320 mir-873 m1croma 0.0397 CDK3 321 mir-219 m1croma 0.0397 ICOS 322 KREMEN2 other 0.0397 AXIN2 323 MAOB enzyme 0.0397 IL411 324 DYNLT3 other 0.0397 BCL2 325 GBPl enzyme 0.0397 MMPl 326 Raet 1d/Raet 1e other 0.0397 HLA-A 327 thiazide diuretic chemical drug 0.0397 SLC12A3 328 MMD kinase 0.0397 TNF 329 RGSll enzyme 0.0397 GNB5 330 IFITl other 0.0397 CXCL10 331 AKAP8 other 0.0397 TNF 332 RGS7 enzyme 0.0397 GNB5 333 MT-C01 enzyme 0.0397 HLA-G 334 R-(+)-p-aminoglutethimide chemical reagent 0.0397 TNF 335 INVS transcription regulator 0.0397 NKX3-2 336 DDXl enzyme 0.0397 HTRA1 337 ZFP36L1 transcription regulator 0.0397 TNF 338 HNRNPAO other 0.0397 TNF 339 SKPl transcription regulator 0.0397 BRCAl 340 UBE2Jl enzyme 0.0397 TNF 341 RITl enzyme 0.0397 BCL2 342 DLGAP3 other 0.0397 GRM5 343 BCL2L14 other 0.0397 TNF 344 APOC2 transporter 0.0397 TNF 345 SMARCD1 transcription regulator 0.0397 MMPl 346 CD300E other 0.0397 TNF 347 anagliptin chemical drug 0.0397 TNF 348 THRIL other 0.0397 TNF - 349 CORT-108297 chemical reagent 0.0397 CRH 350 TPD52 other 0.0397 BCL2 351 SLC3A1 transporter 0.0397 SLC7A13 352 Hmgn2 (includes others) other 0.0397 SOX9 353 KLHL3 other 0.0397 WNK4 354 Cd209d other 0.0397 TNF 355 LM03 other 0.0397 ASCL1 g-protein coupled 356 Ccr2 receptor 0.0397 TNF 357 RBM39 transcription regulator 0.0397 HTRAl 358 MARCKS other 0.0397 TNF 359 PTPRCAP other 0.0397 PTPRC 360 sapanisertib chemical drug 0.0397 CCR5 361 GABPB2 transcription regulator 0.0397 BRCA1 362 MED4 transcription regulator 0.0397 HTRA1 363 FAU other 0.0397 TNF 364 LMT-28 chemical reagent 0.0397 TNF chemical - kinase 365 SRI-29132 inhibitor 0.0397 TNF 366 typhoid vaccine Ty21 a biologic drug 0.0397 PTPRC chemical - kinase 367 merck C inhibitor 0.0397 TNF 4,4'- 368 diaminodiphenylmethane chemical toxicant 0.0397 CXCL10 369 8-carboxamidocyclazocine chemical reagent 0.0397 CCR5 chemical- endogenous 370 ~usticidin A non-mammalian 0.0397 TNF 371 mitoguazone chemical drug 0.0397 IT GAL 372 deoxyspergualin chemical drug 0.0397 TNF 373 BG60 chemical reagent 0.0397 TNF chemical - endogenous 374 methaneselenol non-mammalian 0.0397 ULBP2 375 RP 48740 chemical drug 0.0397 TNF 376 melarsoprol chemical drug 0.0397 BCL2 377 propagermanium chemical drug 0.0397 TNF 378 aminooxypentane-rantes chemical reagent 0.0397 CCR5 379 PKA-I complex 0.0397 TNF 380 POP-1 ',3'-propanediol chemical reagent 0.0397 TNF 381 POP-1 ',2'-propanediol chemical reagent 0.0397 TNF 382 POP-1 ',4'-butanediol chemical reagent 0.0397 TNF chemical - endogenous 383 POP-1 ',3'-butanediol mammalian 0.0397 TNF POP-2',2'- 384 dimethylpropanediol chemical reagent 0.0397 TNF 385 phosphatidylpropanol chemical reagent 0.0397 TNF chemical - endogenous 386 phytosphingosine non-mammalian 0.0397 CLDNl 387 SH-130 chemical reagent 0.0397 TNF 388 NSC 95397 chemical reagent 0.0397 TNF chemical - kinase 389 SU9597 inhibitor 0.0397 PAX6 390 allotrap 1258 chemical drug 0.0397 TNF chemical- endogenous 391 polysaccharide mammalian 0.0397 TNF chemical- endogenous 392 methyl palmitate mammalian 0.0397 TNF 393 tirofiban chemical drug 0.0397 TNF chemical- endogenous 394 L-cystine mammalian 0.0397 TNF chemical - endogenous 395 !glucuronic acid mammalian 0.0397 SOX9 396 cyanoketone chemical reagent 0.0397 TNF 397 6alpha-fluorotestosterone chemical toxicant 0.0397 BCL2 398 dexanabinol chemical drug 0.0397 TNF chemical - endogenous 399 L-tartaric acid mammalian 0.0397 TNF S-nitroso-N- 400 acetylpenicillamine chemical reagent 0.0406 BCL2,BRCA1 ,MSH3,TNF 401 GAB2 other 0.0406 BCL2,IL 13RA2,P AEP ,TNF 402 SETDBl enzyme 0.0406 AN02,CSMD1 ,EPS8,TP53BP2 403 aldesleukin biologic drug 0.041 BCL2,FCGR3A/FCGR3B,HLA-B,HLA-G,TNFRSF10C 404 RNA polymerase iii complex 0.043 mir-154,mir-25,XP01 miR-296-5p (miRNAs 405 w/seed GGGCCCC) mature microrna 0.043 BCL2,HGS,MMP 1 406 IDOl enzyme 0.043 FCGRlA,HLA-G,TNF 407 TLR1 transmembrane receptor 0.043 CSF2RA,FCGR1A,TNF 408 LGALS9 other 0.043 CCR5,FCGR3A/FCGR3B,TNF BCL2,CXCL5,FCGR1A,GZMB,HLA-DPA1 ,HLA- 409 CSF3 cytokine 0.0437 DQA1 ,HOXB7,LTF,LYZ,PRKCA,TFRC,TNF 410 entinostat chemical drug 0.044 BCL2,CD33,CSF2RA,MMP1 ,0LFM4,TNFRSF10A 411 EFNA1 other 0.044 DSC1 ,EPHA2,FOSL1 ,KRT13,PXN,SOX9 412 NDRG1 kinase 0.0446 BCL2,CXCL5,MAP1LC3B,MMP1 413 MEIS1 transcription regulator 0.0446 ASCL1 ,DCUN1D1 ,HOXA3,TSC22D2 chemical - endogenous 414 bile acid mammalian 0.0446 APOC2,CDX2,MUC4,TNF AHRR,BCL2,CRH,CXCL 1O ,ITGAL,MMP 1, SIRT1 ,SO 415 quercetin chemical drug 0.0467 X9,TNF,TRPV6 416 2',3'-dialdehyde ATP chemical reagent 0.0471 CXCL10,TNF 417 tannic acid chemical toxicant 0.0471 BCL2,CFB 418 cytidylyl-3'-5'-guanosine chemical reagent 0.0471 BCL2,TNF 419 FCGR3A/FCGR3B transmembrane receptor 0.0471 FCGR3A/FCGR3B, TNF 420 RSP03 kinase 0.0471 AXIN2,TNF 421 NEK7 kinase 0.0471 CXCL10,RGCC 422 AZD8055 chemical drug 0.0471 CXCL10,TNF miR-142-3p (and other miRNAs w/seed 423 GUAGUGU) mature microrna 0.0471 PRKCA,TNF 424 CTSZ [peptidase 0.0471 LILRB2,TNF 425 RFXAP transcription regulator 0.0471 HLA-B,HLA-DQA1 426 RAMP1 transporter 0.0471 FLT4,TNF 427 RPS6KB2 kinase 0.0471 BCL2,CXCL10 428 TIA1 other 0.0471 SIRT1 ,TNF 429 IFNL4 cytokine 0.0471 CXCL10,CXCL11 430 PDYN transporter 0.0471 GRM5,TNF 431 LILRB1 transmembrane receptor 0.0471 BCL2,TNF cyclic guanosine chemical - endogenous 432 monophosphate-adenosine non-mammalian 0.0471 CXCL10,TNF monophosphate 433 brimonidine chemical drug 0.0471 BCL2,TNF chemical- kinase 434 PD 168393 inhibitor 0.0471 BCL2,CXCL10 chemical - kinase 435 tyrphostin AG 127 inhibitor 0.0471 BCL2,TNF chemical - kinase 436 N -chlorotaurine inhibitor 0.0471 MMPl,TNF 437 PPRCl transcription regulator 0.0476 CLDNl,EPHBl,IL13RA2,0SCAR,TNF

438 ETS I group 0.0488 CHI3L 1, CRH,TNF 439 PERl transcription regulator 0.0488 NKX3-1 ,SLC12A3,WNK4 440 SMARCEl transcription regulator 0.0488 BCL2,BRCA1,CYLD chemical- endogenous 441 mangiferin non-mammalian 0.0488 BCL2,MMP1 ,TNF 442 alendronic acid chemical drug 0.0488 BCL2,EFNB 1,EPHB 1 443 IL17F cytokine 0.0489 CXCLl O,CXCL5,MMP1 ,TRAF6 444 HMGB1 transcription regulator 0.0495 BRCAl,CXCL5,HLA-G,MMPl,PTGES,SIRT1 ,TNF 445 SYK kinase 0.0495 BCL2,CXCL10,FOSL1 ,PIK3CA,RGCC,TNF,TP53INP1 BCL2,CCR5,FCGR3A/FCGR3B,GZMB,ICOS,mir- 446 IL12 (family) group 0.0498 25,STAT4,TBX21 ,TNF Supplemental Table 3: Upstream regulators of differentially expressed genes, spontaneous versus NIFT pregnancies. Input genes: D"ffi1 eren f 1a11 y expresse d genes WI"th P -va 1ue <0 05 Ill. RNA-seq . Rank Upstream Regulator Molecule Type P-value Target molecules in dataset 1 KRT10 other 0.000315 IKBKB,IKBKG,TNF 2 NLRP12 other 0.000921 CXCL13,HLA-A,HLA-F,NR3C1 ,TLR4,TNF 3 AP1M1 transporter 0.00103 ATP7A,HLA-A 4 ACTC1 enzyme 0.00103 ACTA1 ,ACTA2 5 CIDEA other 0.00103 TNF,XDH 6 ERRFI1 other 0.00105 MIXL 1, NAIP,TNF 7 CASZ1 enzyme 0.00124 ACTA1 ,ENPP1 ,FGF18,MYH3,RAMP1 ,SLC24A2 ADGRV1 ,BCL11A,CALB1 ,CCL21 ,FCGR1A,NECAB2 8 FFAR3 lg-protein coupled receptor 0.00126 ,SPOCK1 , TLR7 ,TRPC4

9 USP7 I peptidase 0.00151 FOXP3,TNF,TNNT2,TRPC4,USP7 10 TEAD3 transcription regulator 0.00164 ACTA1 ,DES,TNNT2 CRH,CRYAA/CRYAA2 ,GAST,HOXA5,IGFBP1 ,LIF,P 11 :J>horbol esters chemical - other 0.0026 RKCA,TNF,VIP 12 IL1RL1 transmembrane receptor 0.00279 CD28,TLR1 ,TLR4,TNF 13 CNPY3 other 0.00303 TLR1 ,TLR4 14 Ap1 gamma group 0.00303 CD28,HLA-A 15 Raet1b other 0.00303 HLA-A,TNF 16 H60a other 0.00303 HLA-A,TNF 17 C20-D3-vitamin A chemical drug 0.00303 C3 ,CFB 18 mtansenn chemical drug 0.00303 CRH,NR3C1 19 ADCY7 enzyme 0.00335 TLR1 ,TLR4,TNF 20 CD200 other 0.00409 FOXP3,MRC1 ,TLR4,TNF 21 GNA15 enzyme 0.00429 ACTA2,BMP3,C3 ,CP,ENPP1 ,FHL1 ,MAFK,PFN2,XDH ACTA2,CAV3,EHF,FOXP3,HDAC6,MUC5AC,PHKG 1 22 NR4A1 ligand-dependent 0.00507 ,PPP1R3C,RAMP3,SLC39A11 ,TNF,TRIM41 ,UCP2 chemical - endogenous 23 mosme mammalian 0.00568 AIF1 ,C3,C4A/C4B,CFB,CXCL13,TNF 24 mir-506 mtcroma 0.00593 GLB,TNF 25 TRPV5 ion channel 0.00593 CALB 1,TRPV 6 26 chlorothiazide chemical drug 0.00593 CALB 1, TRPV 6 chemical- endogenous non- 27 medicarpin mammalian 0.00593 CD28,TNF 28 N-methyl-D-aspartate chemical drug 0.00686 C3,CALB1,CNTF,GRM1,LIF,TNF CALB1 ,CPLX1 ,GABBR2,IGFBP7,PPP2R2C,RBP7,TN 29 SBDS other 0.00735 C,ZNRF1 30 HLA-A other 0.00743 CD244,HLA-A,KLRG 1 31 B4GALNT1 enzyme 0.00743 C3,C4A/C4B,TNF 32 GRIN1 ion channel 0.00743 NTF3,SMN 1/SMN2,TNF 33 TEAD1 transcription regulator 0.00781 ACTA1 ,ACTA2,DES,TNNT2 ACTA2,AQP3,C4A/C4B,CD53,CP,CPT1A,DGKA,DO CK1 ,FGB,GATAD2B,HAVCR1,IGFBP1,MARCKS,M LFI ,MRCI,NCAMI,NOX1 ,PCDH20,PCDHB9,PCGF2, PIK3CA,PLEKHMI,PRKCA,RPS9,SLC22A8,SMN1 /S MN2,TLR4,TNF,TNK2,UCP2,WNT3,WT1 ,ZMAT3,ZN 34 cisplatin chemical drug 0.00794 F33A 35 coomassie brilliant blue chemical drug 0.00923 CNTF ,LIF ,TNF N-carbobenzyloxy-leucine- 36 leucine-norvalinal chemical- protease inhibitor 0.00923 CAV3 ,SERPINB 1O ,USF2 37 loxoribine chemical drug 0.00923 TLR4,TLR7,TNF 38 CD6 transmembrane receptor 0.00967 FOXP3,TNF 39 ACAN other 0.00967 FOXP3,TNF 40 Mucl transmembrane receptor 0.00967 MUC5AC,TNF 41 MARCHI enzyme 0.00967 MARCHI,TNF 42 idazoxan chemical drug 0.00967 CRH,NR3Cl chemical - endogenous 43 starch mammalian 0.00967 CALBI,TRPV6 44 Pln other 0.00974 ACTA1 ,BLK,DES,MYH3,PRKCA,SPTB 1-methyl-4-phenyl-1 ,2,3,6- 45 tetrahydropyridine chemical toxicant 0.0103 AIF1 ,CALB 1,GRMI ,LIF ,PTPRC,TLR4,TNF 46 Z-551 chemical reagent 0.0113 TLR4,TNF,UCP2 47 latrunculin B chemical reagent 0.0113 ACTA1 ,ACTA2,DES 48 B biologic drug 0.0118 CSF2RA,CXCL13,LIF,mir-95,PFN2,TNF,WT1 ABCD3,ABLIM1 ,ACTA 1,CALB 1,DPYSL3 ,GZMA,NR 49 tacrolimus chemical drug 0.0119 3Cl,PASK,PPP3CB,PRICKLE1 ,SLIT2,TNC,TNF ACTA1 ,ACTA2 ,CD 177 ,HLA- 50 MKL1 transcription regulator 0.0131 A,MAFK,PFN2,RAMP 1,SERPINB 1O,SPOCK3, TNC ACTAl,ACTA2,ACTG2,AZI2,CALBl,CD177,CYLD, DES,FHLl,GATA4,GP5,NLRP1 ,PCDH15,RAMPl,SER 51 SRF transcription regulator 0.0132 PINB10,TCAP,TNC,TNNT2,WT1 52 CA4 enzyme 0.0133 ACTA1 ,KRT13,ROS1 ,SPTB 53 budesonide chemical drug 0.0135 BLK,MAP2K7 ,MUC5AC,NR3C 1, PRKCA,PRKD 1,TNF 54 cobalt chloride chemical reagent 0.0135 ACTA2,ATP7A,CP,DEC1 ,GAST,IREB2,WT1 55 RAB7B [peptidase 0.0142 TLR4,TNF 56 PHLPP2 enzyme 0.0142 PRKCA,TNF 57 Msx3 transcription regulator 0.0142 MRC1 ,TNF 58 DDIT4 other 0.0142 MIXLl,TNF 59 C4A/C4B [peptidase 0.0142 C4A/C4B,TNF 60 GDF5 growth factor 0.0142 FRZB,WNT3 61 NEDD4L enzyme 0.0142 PIK3CA,TNK2 62 mito-TEMPO chemical reagent 0.0142 ACTA2,TNF 2,3-dioxo-6-nitro-7- sulfamoylbenzo(f)quinoxalin 63 e chemical reagent 0.0142 GRIK4,NTF3 chemical- endogenous non- 64 xestospongin C mammalian 0.0142 CRH,TNF 65 RWJ 67657 chemical - kinase inhibitor 0.0142 ACTA2,TNF 66 propyl gallate chemical toxicant 0.0142 MUC5AC,TNF 67 ingenol mebutate chemical drug 0.0142 PRKCA,PRKD1 12-hydroxyeicosatetraenoic chemical - endogenous 68 acid mammalian 0.0142 NOX1,PTPRC 69 CCNK kinase 0.0149 DGKA,PLTP , TAF8,UCP2 70 glycyrrhizic acid chemical drug 0.0149 ADRB2,AIF1 ,MUC5AC,TNF 71 NCOA4 transcription regulator 0.0149 EHF ,GATA4 ,NR3C 1, PL TP 72 D chemical toxicant 0.0156 ACTA1 ,ACTA2,CAV3,CXCR1 ,TNF 73 STAP2 other 0.0161 CXCR1,FGB,TNF 74 FKBP4 enzyme 0.0161 C3,LIF,TNF 75 HOTAIRM1 other 0.0161 CFH,GRB 1O,HOTAIRM1 76 SMARCA2 transcription regulator 0.0167 ACTA1,CP,CXCL13,CYLD,DCT,DES 77 OTX2 transcription regulator 0.0179 DCT,NCAM1,0CA2,SLC16A8,TNC,TRPM1 ACTA1 ,CAV3 ,CD53,CP,FOXP3,K.RT13 ,MUC5AC,TC 78 NOS2 enzyme 0.018 AP, TLR4,TNF, TNK2,TNNT2 79 deferasirox chemical drug 0.0186 F13A1,FAM20C,GAS7,TNF 80 TFEC transcription regulator 0.0188 BCL11A,DCT,Fl3A1 81 ADRB3 lg-protein coupled receptor 0.0188 ADRB2,TNF,UCP2 82 EREG !growth factor 0.0188 CALB1 ,TNC,TNF 83 SGK1 kinase 0.0192 KCNJl ,KCNQ4,KCNQ5,LIF, TNF ,TRPV 6 84 Ptk Igroup 0.0194 NR3C1 ,TNF 85 MED28 other 0.0194 ACTA2 ,ACTG2 86 ABCD1 transporter 0.0194 ABCD3,TNF 87 TSHR lg-protein coupled receptor 0.0194 SLC16A10,SLC01C1 88 SLC22A1 transporter 0.0194 CPT1A,SSTR1 89 CLIC4 ion channel 0.0194 SLC16A8,TNF 90 lemairamin chemical reagent 0.0194 TLR4,TNF ACTA1,ACTA2,CD177,HLA- 91 MKL2 transcription regulator 0.0195 A,MAFK,RAMP 1,SERPINB 10 92 LTBR transmembrane receptor 0.0201 ACTA2,CCL21 ,CXCL13,GPM6B,TNF 93 Pde4 group 0.0218 CD163,CRH,TNF 94 MST1R kinase 0.0218 ACTA2,ROS1,TNF 95 vancomycm biologic drug 0.0219 AASS,C3,C4A/C4B,CP ,FGB,IGFBP 1,MRC 1 ABCD3,BCL11A,C3,CALB1,CAV3,CFB,CNTF,CYLD ,DMBT1,EHF,ENPP1,FCGR1A,FOXP3,GAST,GLI2,G PR34,HDAC6,HLA-A,HLA- F,LIF,MUC5AC,NCAM1,PASK,PKP1,RBPJ,SLIT2,TC 96 NFkB (complex) complex 0.0222 F 12,TLR4, TNF, TRIM38 ACTA2,FOXP3,FRZB,GZMA,MIXL1,MMP17,RAB17, 97 EOMES transcription regulator 0.0243 WNT3 ACTA1,EPM2AIP1,ESRP2,FOXP3,GATA4,HOXB13, 98 DNMT3B enzyme 0.0243 LRRC34,MUC4,PPM1J,PPP1R3C,TNNT2 99 TLR1 transmembrane receptor 0.0251 CSF2RA,FCGR1A,TNF 100 leptomycin B chemical drug 0.0251 ACTA1,GATA4,TNF 101 MSTN growth factor 0.0253 ACTA2,CAV3,DES,TNC,TRIM63 chemical- endogenous 102 L-phenylalanine mammalian 0.0254 GAST,IGFBP1 6-amino-4-( 4- phenoxyphenylethylamino )q 103 uinazoline chemical reagent 0.0254 C3 ,TCF12 104 atrazine chemical toxicant 0.0254 GATA4,NR3C 1 105 S100A10 other 0.0254 HTR4,TNF 106 CD5L transmembrane receptor 0.0254 TLR4,TNF 107 TTN kinase 0.0254 FHL1 ,TRIM63 108 RAB1A enzyme 0.0254 ADRB2,PRKCA 109 ACTA2 other 0.0254 ACTA1,ACTA2 110 NDP growth factor 0.0254 CNTF,LIF 111 ULBP1 transmembrane receptor 0.0254 HLA-A,TNF chemical- endogenous 112 benzoic acid mammalian 0.0254 FOXP3,TNF 113 magnesium sulfate chemical drug 0.0254 AQP3,TNF AKT3,CD244,GFI1B,IKBKB,INPPL1 ,PHKG1,PIK3CA 114 EBF1 transcription regulator 0.0257 ,PPP1R3C,TLR4 115 PTK2 kinase 0.0273 ACTA1,ACTA2,GATA4,TLL1,TNC

116 JAG1 I growth factor 0.0276 ACTA2,GLI2,NOTCH4, WNT3 AKT3,ARHGAP33,CDA,CPT1A,CRH,DGKA,EHD4,F OXP3,GPR63,HLA- A,IGSF9B,KCNIP2,KIAA 1549L,MIND Y3 ,mir- 329,MRC1,MUC5AC,NKAIN2,NKX6- 2,PAX8,PCDH20,RP1 ,SLC18A2,SMN1/SMN2,SPOCK 117 CREB1 transcription regulator 0.028 3,SSTR1 ,TNF 118 NLRC5 transcription regulator 0.0286 HLA-A,HLA-F, TNF 119 ST8SIA1 enzyme 0.0286 C3 ,C4A/C4B,TNF 120 IRAK1 kinase 0.0302 FOXP3,MUC5AC,NOX1,TNF chemical - endogenous CD163,CRH,GABBR2,IGFBP1 ,IGFBP7,NARFL,NOT 121 hydrocortisone mammalian 0.0304 CH4,NR3C1,ZNF143 CRH,EPHA10,GRIK4,GRM1,KCNQ4,KCNQ5,KIAA1 122 REST transcription regulator 0.0304 549L,NKAIN2,VIP 123 cyclophosphamide chemical drug 0.0316 C4A/C4B,CP,FGB,FOXP3,IGFBP1 ,TNF voltage-gated calcium 124 channel complex 0.032 ACTA1 ,PTPRC 125 TGFBR [group 0.032 ACTA2,NOX1 126 PI3Ky [group 0.032 TLR4,TNF 127 TOLLIP other 0.032 TLR4,TNF 128 PB other 0.032 ACTA2,TNF miR-142-3p (and other miRNAs w/seed 129 GUAGUGU) mature microrna 0.032 PRKCA,TNF 130 PDYN transporter 0.032 GRM1 ,TNF 131 [poloxarner chemical reagent 0.032 ABCD3,CPT1A 12(R)- chemical - endogenous 132 hydroxyeicosatetraenoic acid mammalian 0.0321 MUC5AC chemical - endogenous 133 leukotriene A4 mammalian 0.0321 TNC 8-cyclopentyl-1 ,3- 134 dipropylxanthine chemical reagent 0.0321 TNF chemical-endogenous 135 3 -arninoiso butanoate mammalian 0.0321 CPT1A 136 tobrarnycin chemical drug 0.0321 SMN1/SMN2 chemical - endogenous 137 taurolithocholic acid mammalian 0.0321 TNF 138 Pde1 group 0.0321 CRH 139 cortivazol chemical reagent 0.0321 NR3C1 140 CGP 57380 chemical reagent 0.0321 TNF 141 ARRDC3 other 0.0321 ADRB2 142 carteolol chemical drug 0.0321 TNF 143 TRIM72 enzyme 0.0321 ACTA2 144 thymidine phosphorylase !group 0.0321 MARCKS 145 indoprofen chemical drug 0.0321 SMN1/SMN2 146 KCNIP4-IT1 other 0.0321 KCNIP4 chemical - endogenous non- 147 MALP-2R mammalian 0.0321 TNF chemical- endogenous non- 148 Silybum marianum extract mammalian 0.0321 TNF 149 OPHN1 other 0.0321 GRM1 150 MCOLN1 ion channel 0.0321 GAST 151 REPIN1 other 0.0321 MIXL1 152 SNORD21 other 0.0321 HLA-A 153 GGN other 0.0321 ADRB2 154 NYX other 0.0321 TRPM1 155 CXCL17 cytokine 0.0321 GPR35 156 WASHC5 other 0.0321 ATP7A 157 RDH11 enzyme 0.0321 NR3C1 158 NFKBIL1 transcription regulator 0.0321 TNF 159 KRT71 other 0.0321 ATP7A 160 GPR18 lg-protein coupled receptor 0.0321 TNF 161 CEP290 transcription regulator 0.0321 GLI3 162 GGT7 enzyme 0.0321 ADRB2 163 MIIP other 0.0321 HDAC6 164 !phosphodiesterase 4 inhibitor chemical drug 0.0321 TNF 165 POU5F2 transcription regulator 0.0321 CRH 166 Nkx6-3 other 0.0321 GAST 167 BTN2A2 other 0.0321 FOXP3 168 LILRA3 other 0.0321 TNF 169 MBNL2 other 0.0321 ABLIM1 170 Caveolin group 0.0321 TNF 171 NSC 270012 chemical reagent 0.0321 TNF 172 dalcetrapib chemical drug 0.0321 TNF 173 abexinostat chemical drug 0.0321 MUC4 174 UPF3B transporter 0.0321 UPF1 175 RASGRF2 other 0.0321 TNF 176 UQCRC2 enzyme 0.0321 MUC5AC 177 MYLPF other 0.0321 ACTA2 178 SRSF9 enzyme 0.0321 NR3C1 179 OTP transcription regulator 0.0321 CRH 180 CLN3 other 0.0321 HOOK1 181 ATP4A transporter 0.0321 GAST 182 Slpi (includes others) other 0.0321 TNF 183 JAKMIP1 other 0.0321 GABBR2 184 CFI peptidase 0.0321 C3 185 TSTA3 enzyme 0.0321 ACTA2 miR-502-5p (and other miRNAs w/seed 186 UCCUUGC) mature microma 0.0321 KMT5A 187 mir-490 m1croma 0.0321 FOXP3 188 PROTOR !group 0.0321 FOXP3 189 Raet 1d/Raet 1e other 0.0321 HLA-A 190 MMD kinase 0.0321 TNF 191 DPF2 other 0.0321 CXCL13 192 PISD enzyme 0.0321 PCYT2 193 AKAP8 other 0.0321 TNF 194 ITIH5 other 0.0321 ACTA2 195 FEM1B transcription regulator 0.0321 RAD9A 196 VGLL4 other 0.0321 ACTA1 197 R -( +)-p-aminoglutethimide chemical reagent 0.0321 TNF 198 ZFP36L1 transcription regulator 0.0321 TNF 199 NPIPA7 (includes others) other 0.0321 ADRB2 200 CHL1 other 0.0321 NCAM1 201 HNRNPAO other 0.0321 TNF 202 SH3GL1 other 0.0321 GRM1 203 PAGRl other 0.0321 NR3Cl 204 UBE2Jl enzyme 0.0321 TNF 205 NOXAl other 0.0321 NOXl 206 TPO enzyme 0.0321 FOXP3 207 CFD Ipeptidase 0.0321 C3 208 MPG enzyme 0.0321 HPRTl 209 BCL2L14 other 0.0321 TNF 210 ALX4 transcription regulator 0.0321 NCAMl 211 RXFPl g-protein coupled receptor 0.0321 ACTA2 212 APOC2 transporter 0.0321 TNF 213 CD300E other 0.0321 TNF 214 anagliptin chemical drug 0.0321 TNF 215 THRIL other 0.0321 TNF 216 PDCD5 other 0.0321 FOXP3 217 GABRA2 ion channel 0.0321 TLR4 218 CORT-108297 chemical reagent 0.0321 CRH 219 SLC3Al transporter 0.0321 SLC7A13 220 TIP IN other 0.0321 HAVCRl 221 KLHL3 other 0.0321 WNK4 222 Cd209d other 0.0321 TNF 223 Ccr2 g-protein coupled receptor 0.0321 TNF 224 MARCKS other 0.0321 TNF 225 PTPRCAP other 0.0321 PTPRC 226 GRASP other 0.0321 GRMl 227 FAU other 0.0321 TNF 228 LMT-28 chemical reagent 0.0321 TNF 229 NSC117079 chemical reagent 0.0321 FGF18 230 SRI-29132 chemical - kinase inhibitor 0.0321 TNF 231 typhoid vaccine Ty21 a biologic drug 0.0321 PTPRC 232 3-mercaptopicolinic acid chemical - kinase inhibitor 0.0321 NR3Cl 233 merck C chemical - kinase inhibitor 0.0321 TNF 234 i usticidin A chemical - endogenous non- 0.0321 TNF mammalian 235 salinazid chemical reagent 0.0321 IREB2 236 deoxyspergualin chemical drug 0.0321 1NF 237 nafoxidine chemical drug 0.0321 C3 238 BG60 chemical reagent 0.0321 1NF 239 bathophenanthroline chemical reagent 0.0321 CP 240 RP 48740 chemical drug 0.0321 1NF 241 lpropagermanium chemical drug 0.0321 1NF 242 PKA-I complex 0.0321 1NF 243 POP-1',3'-propanediol chemical reagent 0.0321 1NF 244 POP-1',2'-propanediol chemical reagent 0.0321 1NF 245 POP-1',4'-butanediol chemical reagent 0.0321 1NF chemical - endogenous 246 POP-1',3'-butanediol mammalian 0.0321 1NF POP-2',2'- 247 dimethylpropanediol chemical reagent 0.0321 1NF 248 phosphatidylpropanol chemical reagent 0.0321 1NF 249 SH-130 chemical reagent 0.0321 1NF 250 NSC 95397 chemical reagent 0.0321 1NF 251 allotrap 1258 chemical drug 0.0321 1NF chemical - endogenous 252 polysaccharide mammalian 0.0321 1NF chemical - endogenous non- 253 limonoate D-ring-lactone mammalian 0.0321 TLR4 254 ipratropium chemical drug 0.0321 VIP chemical - endogenous 255 methyl palmitate mammalian 0.0321 1NF 256 tirofiban chemical drug 0.0321 1NF chemical - endogenous 257 L-cystine mammalian 0.0321 1NF chemical - endogenous 258 fucose mammalian 0.0321 ACTA2 chenlical-endogenous 259 dihydroxyacetone mammalian 0.0321 NR3Cl 260 cyanoketone chemical reagent 0.0321 TNF 261 dexanabinol chemical drug 0.0321 TNF chemical - endogenous 262 L-tartaric acid mammalian 0.0321 TNF 263 OXT other 0.0323 CRH,GATA4,NR3C1 264 PBX3 transcription regulator 0.0323 ERBIN,HOXA5,HOXA6 265 IL15RA transmembrane receptor 0.0323 CD244,PTPRC, TNF 266 SERPINE2 other 0.0329 CCL21 ,CXCL13,GLI3,TNF 267 PLN transporter 0.0336 BLK,DES,MYH3,PRKCA,SPTB 268 ethionine chemical toxicant 0.0358 C4A/C4B,CP ,FGB,IGFBP 1 269 hexachlorobenzene chemical toxicant 0.0359 C4A/C4B,CP,FGB,IGFBP1 ,MRC1 270 bosentan chemical drug 0.0363 ACTA2,GATA4,TNF 271 TRPS1 transcription regulator 0.0363 ENPP1 ,RAMP 1, RANBP3L 272 oxymatrine chemical drug 0.0363 PTPRC,TLR4,TNF 273 MBP other 0.0363 FOXP3,PTPRC,TNF ACTA1 ,CALBI,CAMTA2,CHRDL1 ,GABRG3,IGFBP1 274 HDAC4 transcription regulator 0.0366 ,PRKCA,TNF 275 monocrotaline chemical toxicant 0.0388 FHL 1, NCAM1 ,NOX1, TNF 276 PDE4B enzyme 0.0391 MUC5AC,TNF 277 MEOX1 transcription regulator 0.0391 GATA4,GLI2 278 NFKBIE transcription regulator 0.0391 LIF,TNF 279 CAMK2A kinase 0.0391 SMN1/SMN2,WNT3 280 IL17D cytokine 0.0391 CXCL13,FOXP3 281 TNFSF18 cytokine 0.0391 FOXP3,MAP2K7 chemical - endogenous 282 allopregnanolone mammalian 0.0391 CRH,TNF 283 CHD7 enzyme 0.0405 GLI2,GLI3,RBPJ 284 BGN other 0.0405 CXCL13,PLTP,TNF 285 GW7647 chemical drug 0.0405 ABCD3,CPT1A,IGFBP1 286 heme chemical - endogenous 0.0405 CD163,MRC1 ,TNF mammalian miR-21-5p (and other miRNAs w/seed 287 AGCUUAU) mature microma 0.0408 ACTA2,CFL2,MARCKS,SLC16A10,TNF ABCD3,ACMSD,CCL21 ,COQ3,CPT1A,FGB,GNB4,N 288 fenofibrate chemical drug 0.041 R3C1 ,PDZK1 ,PLTP,PTPRC,TNF,TRIM63,UCP2 289 !phenylbutazone chemical drug 0.0433 C4A/C4B,CP,FGB,IGFBP1 ,MRC1 290 MKNK1 kinase 0.0442 CELF4,CP,DPYSL3,IFRD1 ,KIF5A,PLTP,TNF ACSM3,AKT3,C3 ,CFB,CLCN7,EHF,F13A1 ,FHL1 ,FO XP3 ,HLA- 291 IL10RA transmembrane receptor 0.0445 A,HOXB 13 ,HTR4,NLRC5,RIPK3,SULT1 C2,TNF 292 CLEC7A transmembrane receptor 0.045 MUC5AC,TLR4,TNF 293 CRX transcription regulator 0.045 ARR3,RP1 ,RS 1

294 I phenacetin chemical drug 0.0452 CP ,FGB,IGFBP 1, MRC 1 295 fasudil chemical drug 0.0452 ACTA2,MRC 1, TLR4,TNF 296 S-nitrosoglutathione chemical toxicant 0.0452 CNTF,FOXP3,LIF,TNF 297 ADAM17 peptidase 0.0452 ACTA1 ,CXCR1 ,MUC5AC,TNF 298 SST other 0.0452 CRYAA/CRY AA2,GAST ,NR3C1 ,SSTR1 299 NRIP1 transcription regulator 0.046 CYP4B1 ,GYS2,0PLAH,SLC16A10,TNF 300 CXCL8 cytokine 0.046 COL 12A1 ,CXCR1 ,IFRD 1, NOX1 ,NR3C 1, TNF (E)-4-hydroxy-3-methyl-but- chemical - endogenous non- 301 2-enyl pyrophosphate mammalian 0.0468 CXCL13,TNF 302 T 0070907 chemical reagent 0.0468 DGKA,TNF 303 ABCA4 transporter 0.0468 C3 ,CFB 304 HSPB1 other 0.0468 ACTA2,TNF 305 CNOT3 other 0.0468 IGFBP1 ,UCP2 306 ferric ammonium citrate chemical drug 0.0468 C3 ,IREB2 307 manumycin A chemical reagent 0.0468 MUC5AC,TNF chemical - endogenous non- 308 rosmarinic acid mammalian 0.0468 ACTA2,TNF 309 · albuterol chemical drug 0.0468 GRK2,TNF 310 FOXA2 transcription regulator 0.0479 ACTA2,ACTG2,C3 ,CPT1A,DCT,FGB,GATA4,IGFBP1 ,MUC4,MUC5AC,UCP2 chemical - endogenous non- GRK2,IKBKB,IKBKG,NR3C1 ,PCDHAC1 ,RIPK3,SLC 311 Igeldanam ycin mammalian 0.048 16AlO,TNF,USP51 312 NCF1 enzyme 0.0496 ACTA2,HYAL3,TNF chemical- endogenous 313 linolenic acid mammalian 0.0496 ABCD3,TNF,UCP2