Trophoblast retrieval and isolation from the cervix: origins of cervical trophoblasts and their potential value for risk assessment of ongoing pregnancies

Abstract

Background: Early during human development, the trophoblast lineage differentiates to commence placentation. Where the placenta contacts the uterine decidua, extravillous trophoblast (EVT) cells differentiate and invade maternal tissues. EVT cells, identified by expression of HLA-G, invade into uterine blood vessels (endovascular EVT), as well as glands (endoglandular EVT), and open such luminal structures towards the intervillous space of the placenta. Endoglandular invasion diverts the contents of uterine glands to the intervillous space, while glands near the margin of the placenta that also contain endoglandular EVT cells open into the reproductive tract. Cells of the trophoblast lineage have thus been recovered from the uterine cavity and endocervical canal. An emerging non-invasive technology [trophoblast retrieval and isolation from the cervix (TRIC)] isolates and examines EVT cells residing in the cervix to explore their origin, biology and relationship to pregnancy and fetal status.

Objective and rationale: This review explores the origins and possible uses of trophoblast cells obtained during ongoing pregnancies (weeks 5-20) by TRIC. We hypothesize that endoglandular EVT cells at the margins of the expanding placenta enter the uterine cavity and are carried together with uterine secretion products to the cervix where they can be retrieved from a Papanicolaou (Pap) smear. The advantages of TRIC for investigation of human placentation and prenatal testing will be considered. Evidence from the literature, and from archived in utero placental histological sections, is presented to support these hypotheses.

Search methods: We used 52 out of 80 publications that appeared between 1966 and 2017 and were found by searching the PubMed and Google Scholar databases. The studies described trophoblast invasion of uterine vessels and glands, as well as trophoblast cells residing in the reproductive tract. This was supplemented with literature on human placental health and disease.

Outcomes: The literature describes a variety of invasive routes taken by EVT cells at the fetal-maternal interface that could displace them into the reproductive tract. Since the 1970s, investigators have attempted to recover trophoblast cells from the uterus or cervix for prenatal diagnostics. Trophoblast cells from Pap smears obtained at 5-20 weeks of gestation have been purified (>95% β-hCG positive) by immunomagnetic isolation with nanoparticles linked to anti-HLA-G (TRIC). The isolated cells contain the fetal genome, and have an EVT-like expression profile. Similar EVT-like cells appear in the lumen of uterine glands and can be observed entering the uterine cavity along the margins of the placenta, suggesting that they are the primary source of cervical trophoblast cells. Cells isolated by TRIC can be used to accurately genotype the embryo/fetus by targeted next-generation sequencing. Biomarker protein expression quantified in cervical trophoblast cells after TRIC correlates with subsequent pregnancy loss, pre-eclampsia and fetal growth restriction. A key remaining question is the degree to which EVT cells in the cervix might differ from those in the basal plate and placental bed.

Wider implications: TRIC could one day provide a method of risk assessment for maternal and fetal disease, and reveal molecular pathways disrupted during the first trimester in EVT cells associated with placental maldevelopment. As perinatal interventions emerge for pregnancy disorders and inherited congenital disorders, TRIC could provide a key diagnostic tool for personalized precision medicine in obstetrics.

Figure 1

Extravillous trophoblast cells in the human uterine cavity. Immunocytochemical staining in utero with an antibody against HLA-G (and Hemalaun nuclear counterstain) in paraffin sections of an archived placenta (most likely early first trimester). The dark brown labeling of HLA-G serves as a marker for extravillous trophoblast (EVT) cells in the invasive zone between fetal and maternal regions. (a) An overview at the margin of the placenta showing villi and intervillous space, decidua basalis, decidua parietalis, decidua capsularis and the uterine cavity, as labeled. Details of the red insets in (a) follow: (b) demonstrates endoglandular EVTs (arrows) in the lumen of a gland near the edge of the placenta. (c) Shows an HLA-G positive EVT cell (arrow) located in the uterine cavity. (d) Shows an EVT cell (arrow) that has replaced the uterine epithelium, while others nearby approach the epithelium. (e) Shows another EVT cell located in the uterine cavity, possibly surrounded by glandular secretions.

Figure 2

EVT cells replace the uterine epithelium. Immunocytochemical double staining of invaded decidua (7 weeks gestational age) for cytokeratin 7 (Ck7, blue, serves as marker for glandular and uterine epithelium) and HLA-G (dark brown, serves as marker for EVT). No nuclear counterstain. (a) Overview shows the transitional zone between decidua capsularis (to the left) and decidua basalis (to the right) with uterine cavity above and intervillous space below. The decidua basalis includes prominent uterine glands (UG) with blue-labeled epithelia. (b) Inset shown in (a). Black arrows indicate the uterine epithelium. (c) Inset shown in (b). Higher magnification shows EVT cells (red arrows) breaking through the uterine epithelium at the opening of the UG, and an endoglandular EVT cell in the lumen of the gland.

Figure 3

Endoglandular and endovascular trophoblast cells express integrin ITGB1. Immunocytochemical single and double staining of serial sections of invaded decidua (11 weeks gestational age) for integrin subunit ITGB1 (INT B1, brown), CK7 (blue, serves as marker for glandular and uterine epithelium), HLA-G (dark brown, serves as marker for EVT), and von Willebrand Factor (vWF, blue, serves as marker for vascular endothelium). Colors of the labels are indicated in each panel. No nuclear counterstain in (a, b, d). Nuclei were counterstained with Hemalaun in (c, e, f). (a) Overview showing a UG partly surrounded by EVT, while in (b) a higher magnification of inset shown in (a) clearly demonstrates endoglandular EVT cells (red arrow) breaking through the glandular epithelium towards the glandular lumen. (c) Adjacent section to (b) labeled for ITGB1. Like the interstitial EVT, the endoglandular EVT (red arrows) express ITGB1 on their cell surface. (d) Shows endovascular EVT cells within a trophoblast plug (red arrows) of a uterine blood vessel lined with vWF-positive endothelia. (e) Shows the same trophoblast plug from an adjacent serial section stained for ITGB1. A higher magnification of the inset, shown in (f), reveals that endovascular EVT cells (red arrows) express ITGB1 on their cell surfaces.

Figure 4

Origin of cervical trophoblast cells during placental development. The developing conceptus is shown within the uterus at the implantation site (a) and later during the placentation period of weeks 5–12 of gestation (b). EVT cells originate from trophoblast cell columns at the base of the anchoring villi, and follow the interstitial, endovascular, and endoglandular invasion routes. The inset in (b) is expanded to the right, showing the transitional zone of the decidua basalis at the margin of the placenta. As demonstrated in Figs 1 and 2, interstitial EVT cells can invade and replace the uterine epithelium from the basal side, and then enter the uterine cavity. In the decidua basalis, endoglandular EVT cells invade and reach the lumen of UGs. We speculate that, at the lateral margin of the placenta, they are transported together with the glandular secretions into the uterine cavity. Once EVT cells have reached the uterine cavity, they could migrate towards the cervix, or be carried there by the uterine secretion products (arrows).