bioRxiv preprint doi: https://doi.org/10.1101/2020.08.14.248807; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 PRG2 and AQPEP are misexpressed in fetal membranes in placenta previa and percreta 2 3 Elisa T. Zhang1, Roberta L. Hannibal1,6, Keyla M. Badillo Rivera1,7, Janet H.T. Song1,8, Kelly 4 McGowan1, Xiaowei Zhu1,2, Gudrun Meinhardt3, Martin Knöfler3, Jürgen Pollheimer3, Alexander 5 E. Urban1,2, Ann K. Folkins4, Deirdre J. Lyell5, Julie C. Baker1,5* 6 7 1DepartMent of Genetics, Stanford University School of Medicine, Stanford, California, United 8 States of AMerica. 9 2DepartMent of Psychiatry and Behavioral Sciences, Stanford University School of 10 Medicine, Stanford, California, United States of AMerica. 11 3DepartMent of Obstetrics and Gynaecology, Reproductive Biology Unit, Medical University of 12 Vienna, Vienna, Austria. 13 4DepartMent of Pathology, Stanford University School of Medicine, Stanford, California, United 14 States of AMerica. 15 5DepartMent of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, 16 California, United States of AMerica. 17 6Present address: Second GenoMe, Inc., Brisbane, California, United States of AMerica. 18 7Present address: Eversana Consulting, South San Francisco, California, United States of 19 AMerica. 20 8Present address: Division of Genetics and GenoMics, Boston Children’s Hospital, Harvard 21 Medical School, Boston, Massachusetts, United States of AMerica. 22 23 *Correspondence: [email protected] 24 300 Pasteur Dr. 25 Alway M337 26 Palo Alto, CA 94305-5120 27 28 Running title: Molecular signatures of placenta previa and PAS 29 Keywords: Placenta, fetal MeMbranes, placenta accreta spectruM, placenta previa, PRG2, 30 AQPEP 31 Summary sentence: 3SEQ and iMMunofluorescence reveal that extravillous trophoblast 32 factors, Most notably PRG2 and AQPEP, define the diseases placenta previa and placenta 33 accreta spectruM (PAS) in both the chorioamniotic MeMbranes and the placental disk. 34 Grant support: This work was funded by an NIH grant (NICHD R01 HD094513), a Stanford 35 Discovery and Innovation Foundation Grant, and a Stanford SPARK grant to J.C.B. E.T.Z was 36 supported by an A.P. Giannini Foundation postdoctoral fellowship, a Stanford Child Health bioRxiv preprint doi: https://doi.org/10.1101/2020.08.14.248807; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 37 Research Institute postdoctoral award, and a Stanford Dean’s Postdoctoral Fellowship. J.H.T.S. 38 was supported by a National Science Foundation Graduate Research Fellowship and Stanford 39 Graduate Fellowship. M.K. and J.P. are supported by grants froM the Austrian Science Fund 40 (P31470-B30 and P-33485-B30). D.J.L. was supported by The Arline and Pete HarMan Faculty 41 Scholar Fund froM the Stanford Maternal Child Health Research Institute and the forMer 42 Stanford Child Health Research Institute. 43 44 45 Abstract 46 The obstetrical conditions placenta accreta spectruM (PAS) and placenta previa are a 47 significant source of pregnancy-associated Morbidity and Mortality, yet the specific Molecular 48 and cellular underpinnings of these conditions are not known. In this study, we identified 49 Misregulated gene expression patterns in tissues froM placenta previa and percreta (the Most 50 extreMe forM of PAS) coMpared with control cases. By coMparing this gene set with existing 51 placental single-cell and bulk RNA-Seq datasets, we show that the upregulated genes 52 predoMinantly Mark extravillous trophoblasts. We perforMed iMMunofluorescence on several 53 candidate Molecules and found that PRG2 and AQPEP protein levels are upregulated in both 54 the fetal MeMbranes and the placental disk in both conditions. While this increased AQPEP 55 expression reMains restricted to trophoblasts, PRG2 is Mislocalized and is found throughout the 56 fetal meMbranes. Using a larger patient cohort with a diverse set of gestationally aged-matched 57 controls, we validated PRG2 as a Marker for both previa and PAS and AQPEP as a Marker for 58 only previa in the fetal MeMbranes MeMbranes. Our findings suggest that the extraeMbryonic 59 tissues surrounding the conceptus, including both the fetal meMbranes MeMbranes and the 60 placental disk, harbor a signature of previa and PAS that reflects increased trophoblast 61 invasiveness. 62 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.14.248807; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 63 Introduction 64 Placenta accreta spectruM (PAS) is a condition that is increasing at an alarMing rate1, 65 yet despite its significant harM to Mothers and infants, the Molecular and cellular origins are not 66 well-understood. PAS is characterized by excessive invasion of the placental cells into the 67 MyoMetriuM or Muscle wall of the uterus. Deliveries associated with PAS are extreMely 68 challenging because the placenta becoMes pathologically attached to the uterus, leading to 69 heMorrhage and high rates of maternal and neonatal Morbidity and Mortality1,2. PAS is 70 characterized by the subtypes accreta, increta, and and percreta delineating the extent of this 71 invasion1. Percreta is the Most extreMe case, in which invasion extends through the uterine wall 72 and soMetiMes into neighboring organs such as the bladder1,3. Nearly all accreta patients are 73 also afflicted with previa, a condition in which the placenta is located in the lower segMent of the 74 uterus, directly covering the cervix4,5. This placeMent thus precludes vaginal delivery and puts 75 the Mother at risk of intra- and post-partum hemorrhage6–9. Both PAS and previa are associated 76 with uterine daMage or dysfunction as a result of previous cesarean deliveries, endoMetrial 77 ablation, in vitro fertilization (IVF), or advanced Maternal age1,10–13. A coMbination of Multiple 78 previous cesarean deliveries and previa elevates the risk of accreta to 67%, strongly supporting 79 the correlation between uterine daMage, previa, and PAS5. 80 81 While placental trophoblast cells appear to be at the heart of PAS, the role they play is 82 unknown. The prevailing Model is that uterine scar tissue, coMbined with placental placement in 83 the lower uterus (previa), leads to deficient decidualization and the absence of appropriate 84 regulatory signals to the trophoblasts, resulting in abnorMal and aggressive placental invasion. 85 Consistent with this Model, PAS is characterized by expanded nuMbers of invasive extravillous 86 trophoblasts (EVT)s14–16. EVTs are also expanded in previa, even though placental overinvasion 87 is not characteristic of this disease17. The Misexpression of several Molecules within 88 trophoblasts have been shown to correlate with disease, including lower levels of β-catenin in bioRxiv preprint doi: https://doi.org/10.1101/2020.08.14.248807; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 89 previa and accreta18, higher levels of HMG1 and VEGF in previa19, higher levels of DOCK4 in 90 accreta trophoblasts20, and absence of osteopontin staining in percreta21. An in-depth 91 exaMination of the involveMent of trophoblasts, even years after uterine daMage, is thus 92 intriguing and critical for understanding disease etiology. 93 94 Although the placental disk plays a vital role in Many diseases of pregnancy, the fetal 95 membranes have also been iMplicated in soMe conditions. These extraplacental meMbranes, 96 which line the fluid-filled aMniotic sac to protect the fetus, are susceptible to infection and/or 97 rupture that can jeopardize fetal health and lead to preterM birth. While not previously thought to 98 be affected in placental diseases like preeclaMpsia, the fetal MeMbranes have been found to 99 contain cellular and Molecular pathologies22. For exaMple, in severe preeclaMpsia, the 100 trophoblast layer is expanded in thickness in the fetal MeMbranes. This is further accoMpanied 101 by Misexpression of Many oxidative stress and iMMune genes, including key pregnancy 102 proteins such as PAPPA, HTRA4, and CSH122. In addition, recent studies report cases of 103 physically adherent MeMbranes during delivery, as well as the presence of MyoMetrial 104 Myofibers attached to these meMbranes15,23. These cases are often associated with either 105 concurrent, subsequent, or prior PAS, thus raising the idea of fetal meMbrane involveMent in 106 this disease24. Therefore, while trophoblast overinvasion and expansion of EVTs in PAS is seen 107 priMarily in the placental disk16, the MeMbranes May also be coMproMised. 108 109 Here we exaMined the fetal MeMbranes of PAS and previa coMpared to gestationally 110 age-Matched controls to identify Molecules that could discriMinate between these conditions. 111 We found that 74 transcripts were significantly upregulated in cases of percreta and previa. 112 These genes are highly enriched for extracellular Molecules that are EVT-specific Markers. We 113 validated two of these Molecules, Proteoglycan 2 (PRG2) and AMinopeptidase Q (AQPEP; 114 otherwise known as Laeverin), using iMMunofluorescence in the MeMbranes of PAS, previa, bioRxiv preprint doi: https://doi.org/10.1101/2020.08.14.248807; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.