bioRxiv preprint doi: https://doi.org/10.1101/595561; this version posted April 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC-ND 4.0 International license. 1 Complement in human pre-implantation embryos: attack 2 and defense 3 4 5 Martin P. Reichhardta,1, Karolina Lundinb, A. Inkeri Lokkic,d, Gaëlle Rechere, Sanna 6 Vuoristob, Shintaro Katayamaf, Juha Tapanainenb, Juha Keref,g,h , Seppo Meric,d,i,2, and 7 Timo Tuurib,2 8 9 aSir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom 10 11 bDepartment of Obstetrics and Gynecology, University of Helsinki and Helsinki University 12 Hospital, HUH, Helsinki, Finland 13 14 cDepartment of Bacteriology and Immunology, University of Helsinki, Helsinki Finland 15 16 dTranslational Immunology Research Program, Research Programs Unit, University of 17 Helsinki, Finland 18 19 eInstitut d’Optique Graduate School, CNRS - Université de Bordeaux, Talence, France 20 21 fDepartment of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden 22 23 gKing’s College London, School of Basic and Medical Biosciences, London, UK 24 25 hFolkhälsan Institute of Genetics, and University of Helsinki, Stem Cells and Metabolism 26 Research Program, Helsinki, Finland 27 28 iHumanitas University, Milan, Italy 29 30 31 32 33 1Corresponding author 34 35 2These authors share senior-authorship 36 37 38 1 bioRxiv preprint doi: https://doi.org/10.1101/595561; this version posted April 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC-ND 4.0 International license. 1 Abstract 2 3 It is essential for early human life that immunological responses to developing embryos are 4 tightly regulated. An imbalance in the activation and regulation of the human complement 5 system occurs in pregnancy complications, such as pre-eclampsia and recurrent miscarriage. 6 We hereby present the first full analysis of the expression and deposition of complement 7 molecules in human pre-implantation embryos. Thus far, immunological imbalance has been 8 considered in stages of pregnancy following implantation. We here show that complement 9 activation and deposition takes place on developing human embryos already at the pre- 10 implantation stage. Using confocal microscopy, we observed deposition of activation products 11 such as C1q, C3 and C5 on healthy developing embryos, which highlights the need for strict 12 complement regulation. The early embryos express the complement membrane inhibitors 13 CD46, CD55 and CD59 and bind the soluble regulators C4bp and factor H. These findings 14 show that complement targets human embryos, and indicate potential adverse pregnancy 15 outcomes, if regulation of activation fails. In addition, single-cell RNA sequencing of embryos 16 at oocyte, zygote, 4-cell and 8-cell stages showed expression of complement genes, e.g. C1s, 17 C2, C3, C5, factor B and factor D. This shows that the embryonic cells themselves have the 18 capacity to express C3 and C5, which may become activated and function as mediators of 19 cellular signaling. The specific local embryonic expression of complement components, 20 regulators, and deposition of activation products on the surface of embryos suggests that 21 complement has immunoregulatory functions and may impact cellular homeostasis and 22 differentiation at the earliest stage of human life. 23 24 Keywords 25 Complement, embryo, development, pre-implantation 26 27 Statement of significance 28 While canonical functions of the complement system relate to pathogen-defence, it is known 29 to drive certain immune pathologies. The work here described shows, for the first time, 30 expression and localization of a full range of complement molecules in human pre-implantation 31 embryos. We demonstrate complement attack against early embryos, and show presence of 32 embryonic defence mechanisms. Furthermore, we reveal early embryonic production of 33 complement activators, suggesting non-canonical roles in cell signalling and development. Our 34 findings thus reveal a fundamental role for complement at the earliest stages of human 35 embryogenesis. Our data opens up for future studies into the role of complement, both in 36 relation to infertility and pregnancy complications, as well as basic cellular processes during 37 early human development. 38 2 bioRxiv preprint doi: https://doi.org/10.1101/595561; this version posted April 8, 2019. 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-NC-ND 4.0 International license. 1 Introduction 2 3 The complement system is a part of the innate immune defense system, primarily involved in 4 anti-microbial defense, clearance of debris and immune regulation. This multi-lineage 5 enzymatic cascade functions as one of the earliest initiators of inflammation and a potent 6 inducer of adaptive immune responses. It may be initiated through the classical and lectin 7 pathways, driven by pattern-recognition (e.g. via C1q), or through the auto-activation of the 8 alternative pathway. All three pathways converge at the activation of C3 to C3a and C3b, with 9 the subsequent activation of C5 to C5a and C5b, and finally assembly of the pore-forming 10 membrane attack complex (MAC, C5b-C9). To avoid complement attack against self-tissue, 11 human surfaces express and/or recruit a number of membrane-bound and soluble regulators (1- 12 3). 13 14 Increased complement activation is observed in certain autoimmune diseases, but also during 15 pregnancy (4-6). Thus, the role of complement dysregulation has emerged as an important 16 contributing factor to pregnancy complications and infertility, e.g. in pre-eclampsia and 17 recurring miscarriage (7-10). Understanding how the clearance-function of complement is 18 regulated in the context of tolerating the “semiallograft” embryo is therefore of paramount 19 importance (11). 20 21 In addition to the manifest role of complement in immune targeting and clearance, novel 22 functions have been revealed in recent years. Studies across multiple species have revealed 23 unexpected roles of complement molecules in fertilization, embryonic growth and 24 organogenesis (12). The liver is the main site for synthesis of complement components, 25 however, these novel findings have been driven by the detection of local cell-derived 26 complement factors and functional links to basic cellular homeostasis and metabolism (3, 9, 27 13-19). Furthermore, a number of animal models have revealed an effect of complement on 28 mouse embryo hatching rate, Xenopus organogenesis as well as on rodent neuronal 29 development (20-24). 30 31 While our understanding of local cellular complement activities is increasing, knowledge of 32 complement expression and localization at the very early stages of human life, i.e. the pre- 33 implantation embryonic stage, is practically non-existing. To understand the impact of 34 complement on embryonic development, it is essential to investigate (i) potentially hazardous 35 embryonic targeting by maternal complement (complement clearance function), as well as (ii) 36 local embryonic production of complement components (cellular signaling and protection 37 against maternal complement attack). 38 39 In an effort to map the localization of complement molecules and understand the role of 40 complement activation in the early stages of human embryonic development, we here describe 41 the local cellular expression and surface deposition of complement components using single 42 cell RNA-transcriptomics and confocal microscopy of human non-fertilized oocytes, zygotes, 43 4-cell stage and 8-cell stage embryos. The current dogma in the field suggests that maternal 44 immune tolerance towards the conceptus is induced during and after implantation. However, 45 the findings presented here support a crucial role for innate immune mechanisms, such as 46 complement activation, already in the pre-implantation stage of human reproduction. 47 Furthermore, to the authors’ knowledge, these studies are the first to show the expression of 48 multiple activating and regulatory complement components during human embryogenesis. Our 49 findings thus suggest that complement signaling may be essential for early human 50 development, as observed in other mammals. 3 bioRxiv preprint doi: https://doi.org/10.1101/595561; this version posted April 8, 2019. 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-NC-ND 4.0 International license. 1 Results 2 3 Embryos express complement regulators, activators and receptors 4 To understand the role of complement during early embryogenesis, we analyzed single-cell 5 transcriptomes from embryos at different developmental stages. Included were non-fertilized 6 oocytes, zygotes, 4-cell stage embryos and 8-cell stage embryos. Using single-cell tagged 7 reverse-transcription sequencing (STRT), 5’- Transcript Far Ends (TFEs) were analyzed as 8 described previously (25). Specifically, transcripts from the 5’-untranslated region (UTR) and 9 upstream are reliable templates for proteins, and these were used for the analysis along with a 10 number of reliable reads from the coding sequences (CDs). Three separate libraries were 11 generated comparing oocyte to zygote, oocyte to 4-cell stage and 4-cell to 8-cell stage. To 12 avoid batch-bias, these libraries were not batch corrected. We analyzed the presence of specific 13 complement-related TFEs found in each of the four stages (Figure 1). 14 15 Single-cell RNA sequencing revealed expression of a varied set of complement related genes. 16 Transcripts correlating to a number of molecules known to protect cells from complement 17 attack were identified.