161 5 REPRODUCTIONREVIEW Investigation of infertility using endometrial organoids Konstantina Nikolakopoulou 1,2 and Margherita Y Turco 1,2 1Department of Pathology, University of Cambridge, Cambridge, Cambridgeshire, UK and 2Centre for Trophoblast Research, University of Cambridge, Cambridge, Cambridgeshire, UK Correspondence should be addressed to M Y Turco; Email: [email protected] Abstract Infertility is a common problem in modern societies with significant socio-psychological implications for women. Therapeutic interventions are often needed which, depending on the cause, can either be medical treatment, surgical procedures or assisted reproductive technology (ART). However, the treatment of infertility is not always successful due to our limited understanding of the preparation of the lining of the uterus, the endometrium, for pregnancy. The endometrium is of central importance for successful reproduction as it is the site of placental implantation providing the interface between the mother and her baby. Due to the dynamic, structural and functional changes the endometrium undergoes throughout the menstrual cycle, it is challenging to study. A major advancement is the establishment of 3D organoid models of the human endometrium to study this dynamic tissue in health and disease. In this review, we describe the changes that the human endometrium undergoes through the different phases of the menstrual cycle in preparation for pregnancy. We discuss defects in the processes of endometrial repair, decidualization and acquisition of receptivity that are associated with infertility. Organoids could be utilized to investigate the underlying cellular and molecular mechanisms occurring in non-pregnant endometrium and early pregnancy. These studies may lead to therapeutic applications that could transform the treatment of reproductive failure. Reproduction (2021) 161 R113–R127 Introduction blockage, due to damage from infections by Chlamydia trachomatis or Neisseria gonorrhoea. Suboptimal The World Health Organization describes infertility as function of the endometrium, the mucosal lining of the a ‘disease of the reproductive system defined by the uterus, failing to achieve optimal decidualization and failure to achieve a clinical pregnancy after 12 months become receptive are a major cause of primary infertility or more of regular unprotected sexual intercourse’ (Cha et al. 2012). (Zegers-Hochschild et al. 2017). It affects around 10% The efficiency of reproduction is lower in humans of couples of reproductive age and is a consequence compared to other mammals with only 30% of of female or male reproductive dysfunction and/or low conceptions resulting in a live birth and the majority embryo quality (Vander Borght & Wyns 2018). In women, of these losses occurring preclinically (Macklon et al. infertility is classified as primary or secondary, based 2002). In ART, methods for the selection of ‘good’ on whether a clinical pregnancy has been previously embryos prior to transfer include the assessment of achieved or not (Zegers-Hochschild et al. 2017). Despite embryo quality using a grading system that assesses the the advances in assisted reproductive technology (ART), number of cells, fragmentation, speed and symmetry of infertility is an increasing global public health issue. cell division etc. The grading system changes in regard Primary infertility of female origin is attributed to to the blastocyst stage. Yet, live birth rates after ART still hormonal, functional or anatomical dysfunction of remain low (Gleicher et al. 2019). Ovarian stimulation the organs of the reproductive tract. It is a complex for fresh embryo transfer might impair endometrial disorder with a range of contributing factors including receptivity and lead to asynchrony between the embryo systemic diseases (diabetes and obesity), environmental and the endometrium in comparison to the transfer (endocrine disruptors) and lifestyle (chronic stress of frozen-thawed embryos in a natural cycle which and delayed childbearing) influences that can affect results in higher pregnancy rates (Shapiro et al. 2013). reproductive function (Vander Borght & Wyns 2018). Although later evidence shows that choosing to transfer Up to 30% of cases of infertility remain unexplained fresh over frozen embryos and vice versa does not seem (Gelbaya et al. 2014). Known causes include ovarian to significantly change the live birth rates (Wong et al. dysfunction due to premature ovarian insufficiency, poor 2017), the matter is still under continuous investigation. egg quality, polycystic ovary syndrome or fallopian tubal Understanding how the normal endometrium is regulated © 2021 The Authors This work is licensed under a Creative Commons ISSN 1470–1626 (paper) 1741–7899 (online) Attribution 4.0 International License. https://doi.org/10.1530/REP-20-0428 Online version via https://rep.bioscientifica.com -20-0428 R114 K Nikolakopoulou and M Y Turco to interact with the implanting blastocyst is essential to are embedded into an extracellular matrix (typically the define the cellular and molecular mechanisms that are commercially available Matrigel) and are grown in a altered in a suboptimal endometrium. chemically-defined medium designed to sustain WNT The endometrium is a unique mucosa that consists and MAPK signalling whilst inhibiting TGFB and BMP of surface epithelium overlying glands with intervening pathways (Boretto et al. 2017, Turco et al. 2017). The stromal and immune cells. All these undergo profound organoids are spheroidal structures, around several phenotypic and transcriptional changes during the hundred microns in size, with a central lumen that is menstrual cycle in response to ovarian hormones (Garry lined by a single columnar epithelium with microvilli et al. 2009, Munro et al. 2010, Ruiz-Alonso et al. 2012). on their apical surface (Fig. 1). They can be expanded Current knowledge of human endometrial function is long-term (>6 months) without genetic alterations. The based on decades of studies using endometrial tissues organoids respond functionally to ovarian hormones and a range of in vivo and in vitro models. Animal models, and placental factors, demonstrated by their expression including non-human primates (monkeys, baboons) of oestrogen and progesterone receptors and production (Stouffer & Woodruff 2017) and rodents, have provided of uterine glandular products (Turco et al. 2017). The important insights (Fullerton et al. 2017). However, ability of the organoids to recapitulate the changes of the animal studies are labour and cost-intensive and not endometrial epithelium across the menstrual cycle has all aspects of human endometrium are recapitulated. also been demonstrated by comparing the hormonally In vitro models offer a more reproducible alternative. stimulated organoids to endometrial tissue by single-cell Immortalized human endometrial stromal cells have RNA sequencing (Garcia-Alonso et al. 2021). Organoids been used as reporter lines to study genes involved in can also be derived from pathological endometrial tissues decidualization (Haller et al. 2019). Commonly used including endometriosis and endometrial carcinomas cell lines derived from endometrial adenocarcinomas (Boretto et al. 2019). Collectively, these in vitro models (Ishikawa cells and ECC1) are useful to test the effects allow the study of endometrial physiology and disease of oestrogen and progesterone as well as to model the in a reproducible and standardized manner and are a luminal epithelium to study implantation in vitro (Mo promising tool for the investigation of infertility. et al. 2006, Zhang et al. 2012). These systems suffer from a failure to fully reflect the normal physiological Organoids for the study of the normal endometrium characteristics of the endometrial epithelium. In addition, Menstruation, tissue repair and regeneration their 2D nature prevents the cell–cell interactions which are characteristic of the complex microenvironment in During the menstrual cycle, which in humans last 26–32 vivo. The establishment of tissue-derived 3D culture days, the endometrium undergoes shedding, repair, systems or organoids of the female reproductive tract regeneration and remodelling to provide a ‘fertile soil’ for and the placenta offer powerful models for the study of implantation of the embryo (Evans et al. 2016). Because these tissues and pregnancy in vitro (Alzamil et al. 2020, of this extraordinary plasticity, it has been challenging to Heidari-Khoei et al. 2020, Ojosnegros et al. 2021). study as access to samples throughout the cycle is needed, The physiology of the endometrium is a determining together with models that can recapitulate this process. factor in the establishment and maintenance of Menstruation, the shedding of the functional layer of pregnancy as it provides the site of implantation and the endometrium (days 1–5), occurs in the absence of early nutrition to the conceptus during the first few conception and embryonic implantation. The trigger weeks of pregnancy (Burton et al. 2002). In this review, for menstruation is the regression of the corpus luteum we describe the functional and morphological changes leading to a reduction in levels of ovarian hormones, of the endometrium during the menstrual cycle and oestrogen and progesterone (Fig. 2). Vasoconstriction in pregnancy. We discuss how both the endometrial of uterine blood vessels and breakdown of the stratum physiology and the defects that might arise in distinct functionalis then follow (Maybin & Critchley 2015).