Trophoblast organoid systems to study human placentation.

Human trophoblast organoids provide a valuable in vitro system to investigate human placental development and function. In this issue of Developmental Cell, Shannon et al. benchmark two organoid models against primary trophoblast at single-cell resolution, identifying their strengths and limitations.

Human uterine natural killer cells regulate differentiation of extravillous trophoblast early in pregnancy.

In humans, balanced invasion of trophoblast cells into the uterine mucosa, the decidua, is critical for successful pregnancy. Evidence suggests that this process is regulated by uterine natural killer (uNK) cells, but how they influence reproductive outcomes is unclear. Here, we used our trophoblast organoids and primary tissue samples to determine how uNK cells affect placentation. By locating potential interaction axes between trophoblast and uNK cells using single-cell transcriptomics and in vitro modeling of these interactions in organoids, we identify a uNK cell-derived cytokine signal that promotes trophoblast differentiation at the late stage of the invasive pathway. Moreover, it affects transcriptional programs involved in regulating blood flow, nutrients, and inflammatory and adaptive immune responses, as well as gene signatures associated with disorders of pregnancy such as pre-eclampsia. Our findings suggest mechanisms on how optimal immunological interactions between uNK cells and trophoblast enhance reproductive success.

Primitive haematopoiesis in the human placenta gives rise to macrophages with epigenetically silenced HLA-DR.

The earliest macrophages are generated during embryonic development from erythro-myeloid progenitors (EMPs) via primitive haematopoiesis. Although this process is thought to be spatially restricted to the yolk sac in the mouse, in humans, it remains poorly understood. Human foetal placental macrophages, or Hofbauer cells (HBC), arise during the primitive haematopoietic wave ~18 days post conception and lack expression of human leukocyte antigen (HLA) class II. Here, we identify a population of placental erythro-myeloid progenitors (PEMPs) in the early human placenta that have conserved features of primitive yolk sac EMPs, including the lack of HLF expression. Using in vitro culture experiments we demonstrate that PEMP generate HBC-like cells lacking HLA-DR expression. We find the absence of HLA-DR in primitive macrophages is mediated via epigenetic silencing of class II transactivator, CIITA, the master regulator of HLA class II gene expression. These findings establish the human placenta as an additional site of primitive haematopoiesis.

Spatial multiomics map of trophoblast development in early pregnancy.

The relationship between the human placenta-the extraembryonic organ made by the fetus, and the decidua-the mucosal layer of the uterus, is essential to nurture and protect the fetus during pregnancy. Extravillous trophoblast cells (EVTs) derived from placental villi infiltrate the decidua, transforming the maternal arteries into high-conductance vessels1. Defects in trophoblast invasion and arterial transformation established during early pregnancy underlie common pregnancy disorders such as pre-eclampsia2. Here we have generated a spatially resolved multiomics single-cell atlas of the entire human maternal-fetal interface including the myometrium, which enables us to resolve the full trajectory of trophoblast differentiation. We have used this cellular map to infer the possible transcription factors mediating EVT invasion and show that they are preserved in in vitro models of EVT differentiation from primary trophoblast organoids3,4 and trophoblast stem cells5. We define the transcriptomes of the final cell states of trophoblast invasion: placental bed giant cells (fused multinucleated EVTs) and endovascular EVTs (which form plugs inside the maternal arteries). We predict the cell-cell communication events contributing to trophoblast invasion and placental bed giant cell formation, and model the dual role of interstitial EVTs and endovascular EVTs in mediating arterial transformation during early pregnancy. Together, our data provide a comprehensive analysis of postimplantation trophoblast differentiation that can be used to inform the design of experimental models of the human placenta in early pregnancy.

Natural Killer Cells in the Human Uterine Mucosa.

The presence of granulated lymphocytes in the human uterine mucosa, known as decidua during pregnancy, or endometrium otherwise, was first noted in the nineteenth century, but it was not until 1990 that these cells were identified as a type of natural killer (NK) cell. From the outset, uterine NK (uNK) cells were found to be less cytotoxic than their circulating counterparts, peripheral NK (pNK) cells. Recently, unbiased approaches have defined three subpopulations of uNK cells, all of which cluster separately from pNK cells. Here, we review the history of research into uNK cells, including their ability to interact with placental extravillous trophoblast cells and their potential role in regulating placental implantation. We go on to review more recent advances that focus on uNK cell development and heterogeneity and their potential to defend against infection and to mediate memory effects. Finally, we consider how a better understanding of these cells could be leveraged in the future to improve outcomes of pregnancy for mothers and babies.

Local immune recognition of trophoblast in early human pregnancy: controversies and questions.

The role of the maternal immune system in reproductive success in humans remains controversial. Here we focus on the events that occur in the maternal decidua during the first few weeks of human pregnancy, because this is the site at which maternal leukocytes initially interact with and can recognize fetal trophoblast cells, potentially involving allorecognition by both T cells and natural killer (NK) cells. NK cells are the dominant leukocyte population in first-trimester decidua, and genetic studies point to a role of allorecognition by uterine NK cells in establishing a boundary between the mother and the fetus. By contrast, definitive evidence that allorecognition by decidual T cells occurs during the first trimester is lacking. Thus, our view is that during the crucial period when the placenta is established, damaging T cell-mediated adaptive immune responses towards placental trophoblast are minimized, whereas NK cell allorecognition contributes to successful implantation and healthy pregnancy.

Why mothers die at a busy tertiary urban hospital in Kampala, Uganda: a comprehensive review of maternal deaths 2016-2018 and implications for quality improvement to reduce deaths.

Background: Reviewing maternal deaths and drawing out lessons for clinical practice is part of an effective cohesive intervention strategy to reduce future deaths. Objective: To review maternal deaths at the National Referral hospital in Kampala over a 3-year period (2016-2018) to determine causes of death, extent of preventability, proportion of deaths notified and audited as per national guidelines. Methods: Trained-multidisciplinary panels (obstetricians and senior midwives) conducted retrospective reviews of maternal deaths that occurred. Results: Major causes of deaths: obstetric haemorrhage (158/350; 45%), hypertensive disorders of pregnancy (87/350; 25%) and infection (95/350; 27%). Overall, 294/350 (84%) of maternal deaths were considered preventable. In 95% (332/350) of cases, delays within healthcare facilities were identified (64%; 226/350). We note that only 115/350 (33%) cases had been audited. This proportion did not change during the studied period. In 48% (167/350) of cases, notification to the Ministry of Health occurred, but only 11% of deaths (39/350) were notified within the recommended 24-hours. Conclusions: A high proportion (84%) of deaths were preventable. Significant delays to care occurred within health-care facilities. Results suggest that a well-supported, and timely maternal death review process with targeted and pragmatic interventions might be effective in reducing maternal deaths in this setting.

Learning from maternal deaths due to uterine rupture: review of cases from peri-urban Uganda.

BACKGROUND: Maternal deaths from uterine rupture continue to occur globally, with particularly high rates in sub-Saharan Africa. Maternal death reviews have been shown to be an effective part of cohesive strategies to prevent future deaths. OBJECTIVE: This study aimed to conduct maternal death reviews for all deaths following uterine rupture in the study center, to assess preventability, and to synthesize key learning points that may help to prevent future maternal deaths following uterine rupture. STUDY DESIGN: Thorough case reviews of all maternal deaths from 2016 to 2018 at the study center (a national referral hospital in urban Uganda) were conducted by trained multidisciplinary panels of obstetricians and midwives. Medical records of women who died following uterine rupture (n=37, 10.6% of all maternal deaths) were extracted for further analysis. RESULTS: Most maternal deaths due to uterine rupture (36/37, 97%) were preventable, with most having been still potentially preventable after the women reached the study center (24/36, 67%). Obstructed labor was the leading cause of uterine rupture, accounting for 73% (27/37) of cases. Previous cesarean delivery was confirmed in 38% (14/37) of cases. The incidence of grand multiparity was 11% (4/37), and 11% (4/37) were primiparous. Most women (28/37, 76%) died within 24 hours of admission. On arrival at the study center, 19 (51%) were critically ill. Exploratory laparotomy was performed in 54% (20/37) of cases, and a further 35% (13/37) died while awaiting laparotomy. Four women died shortly after arrival at the study center (within 1 hour) and received basic resuscitative treatment; 27% (10/37) of women who died had received antenatal planning or preparation. CONCLUSION: Most deaths due to uterine rupture were preventable. The key lessons that emerged from the reviews were: (1) careful birth preparation and complication awareness for women with known risk factors, (2) early recognition of obstructed labor, (3) close monitoring of obstetrical interventions known to be associated with uterine rupture, and (4) treating incipient or suspected uterine rupture as a time-critical obstetrical emergency. The recommendations emerging from our narrative reviews are suitable for implementation in low-resource obstetrical settings, where high numbers of deaths involving uterine rupture occur.

Single-cell roadmap of human gonadal development.

Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.

Preventing death following unsafe abortion: a case series from urban Uganda.

BACKGROUND: Maternal deaths from unsafe abortion continue to occur globally, with particularly high rates in Sub-Saharan Africa where most abortions are classified as unsafe. Maternal death reviews are an effective part of cohesive strategies to prevent future deaths while abortion remains illegal. OBJECTIVE: This study aimed to conduct maternal death reviews for all deaths occurring following unsafe abortion during the study period, to assess preventability, and to synthesize key learning points that may help to prevent future maternal deaths following unsafe abortions. STUDY DESIGN: Full case reviews of all maternal deaths (350 cases from Jan 2016 to Dec 2018) at the study center (a national referral hospital in urban Uganda) were conducted by specially trained multidisciplinary panels of obstetricians and midwives. We extracted the reviews of women who died following unsafe abortions (13 [2.6%]) for further analysis. RESULTS: Most maternal deaths owing to unsafe abortion were found to be preventable. The key recommendations that emerged from the reviews were (1) that clinicians should maintain a high index of suspicion for delayed presentation and rapid decompensation in cases where unsafe abortion has occurred, (2) that a low threshold for early intravenous antibiotic therapy should be applied, and (3) that any admission with complications following an unsafe abortion merits review by an experienced clinician as soon as possible. CONCLUSION: Postabortion care is part of essential emergency medical care and should be provided with high standards, especially in areas where there is limited or no legal access to abortion care. Implementing the recommended learning points is likely to be feasible even in low-resource obstetrical settings and, given the high rates of preventability found in maternal deaths owing to unsafe abortion, is likely to be effective.

Barriers and facilitators to maternal death surveillance and response at a busy urban National Referral Hospital in Uganda.

Background: Preventable maternal and newborn deaths remain a global concern, particularly in low- and- middle-income countries (LMICs) Timely maternal death surveillance and response (MDSR) is a recommended strategy to account for such deaths through identifying contextual factors that contributed to the deaths to inform recommendations to implement in order to reduce future deaths. Implementation of MDSR is still suboptimal due to barriers such as inadequate skills and leadership to support MDSR. With the leadership of WHO and UNFPA, there is momentum to roll out MDSR, however, the barriers and enablers for implementation have received limited attention. These have  implications for successful implementation. The aim of this study was: To assess barriers and facilitators to implementation of MDSR at a busy urban National Referral Hospital as perceived by health workers, administrators, and other partners in Reproductive Health. Methods: Qualitative study using in-depth interviews (24), 4 focus-group discussions with health workers, 15 key-informant interviews with health sector managers and implementing partners in Reproductive-Health. We conducted thematic analysis drawing on the Theory of Planned Behaviour (TPB).   Results: The major barriers to implementation of MDSR were: inadequate knowledge and skills; fear of blame / litigation; failure to implement recommendations; burn out because of workload   and inadequate leadership- to support health workers. Major facilitators were involving all health workers in the MDSR process, eliminate blame, strengthen leadership, implement recommendations from MDSR and functionalize lower health facilities (especially Health Centre -IVs). Conclusions: The barriers of MDSR include knowledge and skills gaps, fear of blame and litigation, and other health system factors such as erratic emergency supplies, and leadership/governance challenges. Recommendation: Efforts to strengthen MDSR for impact should use health system responsiveness approach to address the barriers identified, constructive participation of health workers to harness the facilitators and addressing the required legal framework.

Mapping the temporal and spatial dynamics of the human endometrium in vivo and in vitro.

The endometrium, the mucosal lining of the uterus, undergoes dynamic changes throughout the menstrual cycle in response to ovarian hormones. We have generated dense single-cell and spatial reference maps of the human uterus and three-dimensional endometrial organoid cultures. We dissect the signaling pathways that determine cell fate of the epithelial lineages in the lumenal and glandular microenvironments. Our benchmark of the endometrial organoids reveals the pathways and cell states regulating differentiation of the secretory and ciliated lineages both in vivo and in vitro. In vitro downregulation of WNT or NOTCH pathways increases the differentiation efficiency along the secretory and ciliated lineages, respectively. We utilize our cellular maps to deconvolute bulk data from endometrial cancers and endometriotic lesions, illuminating the cell types dominating in each of these disorders. These mechanistic insights provide a platform for future development of treatments for common conditions including endometriosis and endometrial carcinoma.

Characterization of primary models of human trophoblast.

Two recently developed models, trophoblast organoids and trophoblast stem cells (TSCs), are useful tools to further the understanding of human placental development. Both differentiate from villous cytotrophoblast (VCT) to either extravillous trophoblast (EVT) or syncytiotrophoblast (SCT). Here, we compare the transcriptomes and miRNA profiles of these models to identify which trophoblast they resemble in vivo. Our findings indicate that TSCs do not readily undergo SCT differentiation and closely resemble cells at the base of the cell columns from where EVT derives. In contrast, organoids are similar to VCT and undergo spontaneous SCT differentiation. A defining feature of human trophoblast is that VCT and SCT are human leukocyte antigen (HLA) null, whereas EVT expresses HLA-C, -G and -E molecules. We find that trophoblast organoids retain these in vivo characteristics. In contrast, TSCs express classical HLA-A and HLA-B molecules, and maintain their expression after EVT differentiation, with upregulation of HLA-G. Furthermore, HLA expression in TSCs differs when grown in 3D rather than in 2D, suggesting that mechanical cues are important. Our results can be used to select the most suitable model for the study of trophoblast development, function and pathology.

How Do Uterine Natural Killer and Innate Lymphoid Cells Contribute to Successful Pregnancy?

Innate lymphoid cells (ILCs) are the most abundant immune cells in the uterine mucosa both before and during pregnancy. Circumstantial evidence suggests they play important roles in regulating placental development but exactly how they contribute to the successful outcome of pregnancy is still unclear. Uterine ILCs (uILCs) include subsets of tissue-resident natural killer (NK) cells and ILCs, and until recently the phenotype and functions of uILCs were poorly defined. Determining the specific roles of each subset is intrinsically challenging because of the rapidly changing nature of the tissue both during the menstrual cycle and pregnancy. Single-cell RNA sequencing (scRNAseq) and high dimensional flow and mass cytometry approaches have recently been used to analyse uILC populations in the uterus in both humans and mice. This detailed characterisation has significantly changed our understanding of the heterogeneity within the uILC compartment. It will also enable key clinical questions to be addressed including whether specific uILC subsets are altered in infertility, miscarriage and pregnancy disorders such as foetal growth restriction and pre-eclampsia. Here, we summarise recent advances in our understanding of the phenotypic and functional diversity of uILCs in non-pregnant endometrium and first trimester decidua, and review how these cells may contribute to successful placental development.

Menstrual flow as a non-invasive source of endometrial organoids.

Assessment of the endometrium often necessitates a biopsy, which currently involves an invasive, transcervical procedure. Here, we present an alternative technique based on deriving organoids from menstrual flow. We demonstrate that organoids can be derived from gland fragments recovered from menstrual flow. To confirm they faithfully reflect the in vivo state we compared organoids derived from paired scratch biopsies and ensuing menstrual flow from patients undergoing in vitro fertilisation (IVF). We demonstrate that the two sets of organoids share the same transcriptome signature, derivation efficiency and proliferation rate. Furthermore, they respond similarly to sex steroids and early-pregnancy hormones, with changes in morphology, receptor expression, and production of 'uterine milk' proteins that mimic those during the late-secretory phase and early pregnancy. This technique has wide-ranging impact for non-invasive investigation and personalised approaches to treatment of common gynaecological conditions, such as endometriosis, and reproductive disorders, including failed implantation after IVF and recurrent miscarriage.

BAP1/ASXL complex modulation regulates epithelial-mesenchymal transition during trophoblast differentiation and invasion.

Normal function of the placenta depends on the earliest developmental stages when trophoblast cells differentiate and invade into the endometrium to establish the definitive maternal-fetal interface. Previously, we identified the ubiquitously expressed tumour suppressor BRCA1-associated protein 1 (BAP1) as a central factor of a novel molecular node controlling early mouse placentation. However, functional insights into how BAP1 regulates trophoblast biology are still missing. Using CRISPR/Cas9 knockout and overexpression technology in mouse trophoblast stem cells, here we demonstrate that the downregulation of BAP1 protein is essential to trigger epithelial-mesenchymal transition (EMT) during trophoblast differentiation associated with a gain of invasiveness. Moreover, we show that the function of BAP1 in suppressing EMT progression is dependent on the binding of BAP1 to additional sex comb-like (ASXL1/2) proteins to form the polycomb repressive deubiquitinase (PR-DUB) complex. Finally, both endogenous expression patterns and BAP1 overexpression experiments in human trophoblast stem cells suggest that the molecular function of BAP1 in regulating trophoblast differentiation and EMT progression is conserved in mice and humans. Our results reveal that the physiological modulation of BAP1 determines the invasive properties of the trophoblast, delineating a new role of the BAP1 PR-DUB complex in regulating early placentation.

The CD94/NKG2A inhibitory receptor educates uterine NK cells to optimize pregnancy outcomes in humans and mice.

The conserved CD94/NKG2A inhibitory receptor is expressed by nearly all human and ∼50% of mouse uterine natural killer (uNK) cells. Binding human HLA-E and mouse Qa-1, NKG2A drives NK cell education, a process of unknown physiological importance influenced by HLA-B alleles. Here, we show that NKG2A genetic ablation in dams mated with wild-type males caused suboptimal maternal vascular responses in pregnancy, accompanied by perturbed placental gene expression, reduced fetal weight, greater rates of smaller fetuses with asymmetric growth, and abnormal brain development. These are features of the human syndrome pre-eclampsia. In a genome-wide association study of 7,219 pre-eclampsia cases, we found a 7% greater relative risk associated with the maternal HLA-B allele that does not favor NKG2A education. These results show that the maternal HLA-B→HLA-E→NKG2A pathway contributes to healthy pregnancy and may have repercussions on offspring health, thus establishing the physiological relevance for NK cell education. VIDEO ABSTRACT.

Diversity of KIR genes and their HLA-C ligands in Ugandan populations with historically varied malaria transmission intensity.

BACKGROUND: Malaria is one of the most serious infectious diseases in the world. The malaria burden is greatly affected by human immunity, and immune responses vary between populations. Genetic diversity in KIR and HLA-C genes, which are important in immunity to infectious diseases, is likely to play a role in this heterogeneity. Several studies have shown that KIR and HLA-C genes influence the immune response to viral infections, but few studies have examined the role of KIR and HLA-C in malaria infection, and these have used low-resolution genotyping. The aim of this study was to determine whether genetic variation in KIR and their HLA-C ligands differ in Ugandan populations with historically varied malaria transmission intensity using more comprehensive genotyping approaches. METHODS: High throughput multiplex quantitative real-time PCR method was used to genotype KIR genetic variants and copy number variation and a high-throughput real-time PCR method was developed to genotype HLA-C1 and C2 allotypes for 1344 participants, aged 6 months to 10 years, enrolled from Ugandan populations with historically high (Tororo District), medium (Jinja District) and low (Kanungu District) malaria transmission intensity. RESULTS: The prevalence of KIR3DS1, KIR2DL5, KIR2DS5, and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6 vs 13.2%: p = 0.006, 57.2 vs 66.4%: p = 0.005, 33.2 vs 46.6%: p < 0.001, and 19.7 vs 26.7%: p = 0.014, respectively) or Jinja (7.6 vs 18.1%: p < 0.001, 57.2 vs 63.8%: p = 0.048, 33.2 vs 43.5%: p = 0.002, and 19.7 vs 30.4%: p < 0.001, respectively). The prevalence of homozygous HLA-C2 was significantly higher in populations from Kanungu (31.6%) compared to Jinja (21.4%), p = 0.043, with no significant difference between Kanungu and Tororo (26.7%), p = 0.296. CONCLUSIONS: The KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes may partly explain differences in transmission intensity of malaria since these genes have been positively selected for in places with historically high malaria transmission intensity. The high-throughput, multiplex, real-time HLA-C genotyping PCR method developed will be useful in disease-association studies involving large cohorts.