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Recurrent Pregnancy Loss Is Associated with a Pro-Senescent Decidual Response During the Peri-Implantation Window Emma S Lucas1

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Recurrent Pregnancy Loss Is Associated with a Pro-Senescent Decidual Response During the Peri-Implantation Window Emma S Lucas1 bioRxiv preprint doi: https://doi.org/10.1101/368829; this version posted December 9, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 Recurrent pregnancy loss is associated with a pro-senescent decidual response during 2 the peri-implantation window 3 4 Emma S Lucas1,2†, Pavle Vrljicak1,2†, Joanne Muter1,2 , Maria M Diniz-da-Costa1,2, Paul J 5 Brighton2, Chow-Seng Kong2, Julia Lipecki3, Katherine Fishwick2, Joshua Odendaal2, Lauren 6 J. Ewington2, Siobhan Quenby1,2, Sascha Ott1,4, and Jan J Brosens1,2,* 7 8 1Tommy’s National Centre for Miscarriage Research, University Hospitals Coventry & 9 Warwickshire, Coventry, CV2 2DX, United Kingdom. 10 2Division of Biomedical Sciences, Clinical Sciences Research Laboratories, Warwick Medical 11 School, University of Warwick, Coventry CV2 2DX, United Kingdom. 12 3School of Life Sciences, Gibbet Hill Campus, University of Warwick, Coventry CV4 7AL, 13 United Kingdom. 14 4Department of Computer Science, University of Warwick, Coventry, CV4 7AL, United 15 Kingdom. 16 *Corresponding Author and Lead Contact: Jan Brosens M.D., Ph.D. Clinical Sciences 17 Research Laboratories, Warwick Medical School, University of Warwick, Coventry CV2 2DX, 18 United Kingdom. Tel: +44 2476968704; FAX: +44 2476968653; Email: 19 [email protected] 20 †These authors contributed equally 1 bioRxiv preprint doi: https://doi.org/10.1101/368829; this version posted December 9, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. 21 Abstract 22 Breakdown of the feto-maternal interface in early pregnancy causes miscarriage. The cycling 23 endometrium becomes poised to transition to a pregnant state during the midluteal 24 implantation window, coinciding with differentiation of stromal cells into decidual cells (DC) 25 and emergence of senescent decidual cells (snDC). Emerging evidence suggests that DC 26 engage uterine natural killer cells to eliminate their senescent counterparts, thus enabling 27 formation of a robust decidual matrix in pregnancy. To examine if failure to constrain snDC 28 during the peri-implantation window increases the risk of miscarriage, we reconstructed the 29 decidual pathway at single-cell level in vitro and demonstrated that, without immune 30 surveillance, secondary senescence rapidly transforms DC into progesterone-resistant cells 31 that abundantly express extracellular matrix remodelling factors. Additional single-cell analysis 32 of midluteal endometrium identified DIO2 and SCARA5 as marker genes of a diverging 33 decidual response in vivo. Finally, we report a conspicuous link between a pro-senescent 34 decidual response in luteal phase endometrium and recurrent pregnancy loss, suggesting that 35 pre-pregnancy screening and intervention may reduce the burden of miscarriage. 2 bioRxiv preprint doi: https://doi.org/10.1101/368829; this version posted December 9, 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. 36 Introduction 37 Approximately 15% of clinical pregnancies result in miscarriage 1, most often during the first 38 trimester. Fetal chromosomal abnormalities account for 50-60% of sporadic miscarriages 2, 39 although the incidence is lower in recurrent pregnancy loss (RPL) 3,4, defined as two or more 40 losses 5,6. Further, with each additional miscarriage, the frequency of euploid loss increases 41 whereas the likelihood of a successful pregnancy decreases 7, indicating that uterine factors 42 drive higher-order miscarriages. Unfortunately, few interventions improve live birth rates in 43 RPL 5, reflecting that in most cases the underlying mechanisms are incompletely understood. 44 Following the postovulatory rise in circulating progesterone levels, the endometrium 45 becomes transiently receptive to embryo implantation during the midluteal phase of the cycle. 46 This implantation window also heralds the start of intense tissue remodelling 8, driven in the 47 stroma by differentiation of endometrial stromal cells (EnSC) into specialized decidual cells 48 (DC) and accumulation of uterine natural killer (uNK) cells 9. Upon embryo implantation, DC 49 rapidly encapsulate the conceptus 10, engage in embryo biosensoring 11, and then form a 50 decidual matrix that controls trophoblast invasion 12. At a molecular level, decidual 51 transformation of EnSC encompasses genome-wide remodelling of the chromatin landscape 52 13, reprogramming of multiple signalling pathways 14-16, and activation of decidual gene 53 networks 17,18. This multistep differentiation process starts with an evolutionarily conserved 54 acute cellular stress response 19, marked by a burst of reactive oxygen species (ROS) and 55 release of proinflammatory cytokines 9,20,21. After a lag period of several days, EnSC lose their 56 fibroblastic appearance and emerge as secretory DC with abundant cytoplasm and prominent 57 endoplasmic reticulum 8. A hallmark of DC is resistance to multiple stress signals. Several 58 mechanisms underpin decidual stress resistance, including silencing of the c-Jun N-terminal 59 kinase (JNK) pathway and upregulation of various stress defence proteins and ROS 60 scavengers 8,15,22. In addition, DC highly express 11β-hydroxysteroid dehydrogenase type 1 23, 61 which converts inactive cortisone into cortisol, a potent anti-inflammatory glucocorticoid. Thus, 62 compared to EnSC, DC are exquisitely adapted to withstand the hyperinflammation and stress 63 associated with deep haemochorial placentation. 3 bioRxiv preprint doi: https://doi.org/10.1101/368829; this version posted December 9, 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. 64 Recently, we demonstrated that decidualization also results in the emergence of senescent 65 decidual cells (snDC), both in vitro and in vivo 9,24. In the stroma, the abundance of cells 66 expressing p16INK4, a tumour suppressor and canonical senescence marker, peaks transiently 67 during the midluteal phase before rising again prior to menstruation 9. Cellular senescence is 68 defined by a state of permanent cell-cycle arrest and prominent secretion of various bioactive 69 molecules, including ROS, extracellular matrix (ECM) remodelling proteins, proinflammatory 70 cytokines, chemokines and growth factors, referred to as senescence-associated secretory 71 phenotype (SASP) 25-27. Different types of senescent cells underpin pathological and 72 physiological processes. Chronic senescent cells accumulate progressively in response to 73 various stressors and cause gradual loss of organ function during ageing and in age-related 74 diseases mediated by the deleterious effects of the SASP on tissue homeostasis 25,27. By 75 contrast, acute senescent cells are linked to biological processes that involve programmed 76 tissue remodelling, including embryogenesis and wound healing 27-29. They are induced in 77 response to specific signals, produce a transient SASP with defined paracrine functions, and 78 are promptly cleared by immune cells 25. Recently we demonstrated that snDC exhibit 79 hallmarks of acute senescent cells 9. First, DC and snDC both emerge in response to FOXO1 80 activation, a pivotal decidual transcription factor downstream of the protein kinase A (PKA) 81 and progesterone signalling pathways. Second, their associated SASP critically amplifies the 82 initial decidual inflammatory response, which not only drives differentiation of EnSC but is also 83 linked to induction of key receptivity genes 21. Further, we have provided evidence that DC 84 recruit and activate uNK cells, which in turn may eliminate snDC through perforin- and 85 granzyme-containing granule exocytosis 9,24. 86 Although snDC constitute a relatively minor and variable stromal population in midluteal 87 endometrium, they have the potential to impact profoundly on the unfolding decidual response 88 in a manner that either promotes or precludes pregnancy progression. For example, a 89 transient SASP associated with acute senescent cells has been shown to promote tissue 90 plasticity by expanding resident progenitor populations 9,30. By contrast, senescent cells that 91 persist (i.e. chronic senescent cells) can induce senescence in neighbouring cells though 4 bioRxiv preprint doi: https://doi.org/10.1101/368829; this version posted December 9, 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. 92 juxtracrine signalling (termed ‘secondary’ or ‘bystander’ senescence), leading to 93 spatiotemporal propagation of the phenotype and loss of tissue function 25,31,32. 94 Recently we reported loss of clonal mesenchymal stem-like cells (MSC) in midluteal 95 endometrium of RPL patients but how this is linked to subsequent
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