Extravillous trophoblasts invade more than uterine arteries: evidence for the invasion of uterine veins

Abstract

During the first trimester of pregnancy, extravillous trophoblasts (EVTs) invade into the decidual interstitium to the first third of the myometrium, thereby anchoring the placenta to the uterus. They also follow the endovascular and endoglandular route of invasion; plug, line and remodel spiral arteries, thus being responsible for the establishment of hemotrophic nutrition with the beginning of the second trimester and invade and open uterine glands toward the intervillous space for a histiotrophic nutrition during the first trimester. The aim of this study was to provide proof that uterine veins are invaded by EVTs similar to uterine arteries and glands in first trimester of pregnancy. Therefore, serial sections from in situ first trimester placenta were immuno-single- and immuno-double-stained to distinguish in a first step between arteries and veins and secondly between invaded and non-invaded vessels. Subsequently, invasion of EVTs into uterine vessels was quantified. Our data show that uterine veins are significantly more invaded by EVTs than uterine arteries (29.2 ± 15.7 %) during early pregnancy. Counted vessel cross sections revealed significantly higher EVT invasion into veins (59.5 ± 7.9 %) compared to arteries (29.2 ± 15.7 %). In the lumen of veins, single EVTs were repeatedly found, beside detached glandular epithelial cells or syncytial fragments. This study allows the expansion of our hitherto postulated concept of EVT invasion during first trimester of pregnancy. We suggest that invasion of EVTs into uterine veins is responsible the draining of waste and blood plasma from the intervillous space during the first trimester of pregnancy.

Keywords: Endoglandular trophoblasts; Endovascular trophoblasts; Extravillous trophoblasts; Invasion; Placenta; Uterine veins.

Fig. 1

Uterine artery along with uterine vein invaded by extravillous trophoblasts (EVTs). Serial sections of invaded myometrium with immunohistochemical staining/double staining (gestational age 7 weeks). Images in a, b, e, f are immuno-double-stained for von Willebrand factor (VWF) (blue serves as marker for vascular endothelial cells) and major histocompatibility complex, class I, G (HLA-G) (brown serves as marker for EVT), c immuno-staining for smooth muscle actin (brown serves as marker for a muscle layer and as additional control for desmin staining; besides sm actin-positive myofibroblasts and smooth muscle cells of the myometrium stain positive), d immuno-staining for desmin (brown serves as marker for muscle layer). a, e Overview: Two sections through an invaded spiral artery (triangle) along with an invaded vein (circle). b, f Detail of the inset in a allows a closer look to the contour and shape of the invaded vessels. c, d Serial sections show the residual smooth muscle layer of the converted artery (triangle), but the complete absence of a muscle layer in the neighboring vein (circle). Arrows in b, f show single EVTs already situated in the lumen of the artery and vein. No nuclear counterstain (a, b, e, f) or nuclei were counterstained with hemalaun (c, d)

Fig. 2

Uterine veins invaded by extravillous trophoblasts (EVTs). Columns are composed of serial sections of invaded decidua (gestational age 7 weeks). Sections are stained as follows: a, b immuno-double staining for von Willebrand factor (VWF) (blue serves as marker for vascular endothelial cells) and major histocompatibility complex, class I, G (HLA-G) (brown serves as marker for EVTs); c, d immuno-staining for desmin (brown serves as marker for a muscle layer); e, f immuno-staining for smooth muscle actin (brown serves as marker for a muscle layer and as additional control for desmin staining; besides some sm actin-positive myofibroblasts are seen). a, b Uterine vessels (blue), the decidual stroma is invaded by EVTs (brown), and the endothelium of the vessel is partly replaced by EVTs (a), whereas the arrow in b points to a single EVT situated in the lumen of the vein. c–f The absence of a muscular layer confirms that the vessels in a, b are uterine veins (circle). No nuclear counterstain (a, b) or nuclei were counterstained with hemalaun (c–f). Circles uterine veins

Fig. 3

Extravillous trophoblasts (EVTs), uterine veins and EphB4. Sections of invaded decidua with immunohistochemical staining/double staining (gestational age 7–8 weeks). Images in a–d are immuno-double-stained for EphB4 (blue serves as marker for venous endothelial cells) and major histocompatibility complex, class I, G (HLA-G) (brown serves as marker for EVT), sections in e–h are immuno-stained for EphB4. a Overview: Eph4-positive endothelium (blue) of a vein (circle); the surrounding decidual stroma is invaded by EVTs (brown). b Detail of the inset in a allows a closer look to the invaded vein (circle), arrow points to EVT within the endothelium. Arrows in c, d indicate additional EVTs within the venous lumen and/or the venous endothelium. e–h Morphologically unambiguous arteries (triangles) usually stain negative for EphB4 (e, f), but occasionally the arterial endothelium displays positive staining for EphB4 (g, h). No nuclear counterstain (a–d) or nuclei were counterstained with hemalaun (e–h). Circles uterine veins. Triangles uterine arteries

Fig. 4

Uterine artery not invaded by extravillous trophoblasts (EVTs). Serial sections of invaded decidua (gestational age 7 weeks). Images are stained as follows: a immuno-double staining for von Willebrand factor (VWF) (blue serves as marker for vascular endothelial cells) and major histocompatibility complex, class I, G (HLA-G) (brown serves as marker for EVTs), b immuno-staining for desmin (brown serves as marker for a muscle layer), c immuno-staining for smooth muscle actin (brown serves as marker for a muscle layer and as additional control for desmin staining, besides some sm actin-positive myofibroblasts are seen), d hematoxylin and eosin staining. a Uterine vessels (blue triangles); the decidual stroma is strongly invaded by EVTs (brown). b–d The presence of a muscular layer confirms that the vessel is a uterine artery (triangle). No nuclear counterstain (a) or nuclei were counterstained with hemalaun (b–d). Triangles uterine artery

Fig. 5

Examples for unclassified vessels (asterisk). Invaded decidua with immuno-staining and double staining (gestational age 7–8 weeks). Rows are composed of serial sections a–b, c–d, e–f, and sections in the left column are immuno-double-stained for von Willebrand factor (vWF) (blue serves as marker for vascular endothelial cells) and major histocompatibility complex, class I, G (HLA-G) [brown serves as marker for extravillous trophoblasts (EVTs)], in the right column immuno-stained for desmin (brown serves as marker for muscle layer). Asterisks mark unclassified vessels, triangle marks an artery, and circles mark veins. a, b Examples for unclassified vessels with a diameter below 10 µm. c–f Examples for unclassified vessels completely surrounded by EVTs; a possible layer of smooth muscle cells may have already been replaced by EVTs. No nuclear counterstain (a, c, e) or nuclei were counterstained with hemalaun (b, d, f)

Fig. 6

Quantification of trophoblast invasion: Figure shows that the number of invaded venous cross sections is higher than the number of invaded arterial cross sections. a Counted vessel cross sections of 16 placentas revealed significantly higher EVT invasion into veins compared to arteries (two observers). EVTs attached to the respective vessel showed similar results with no significant differences in numbers. In all assessed specimens, significantly more uninvaded arteries were found compared to uninvaded veins. b The comparison between manual and automated image selection (one observer) did not reveal any significant differences within the subset of three placentas, neither in invaded arteries, nor in invaded veins. Manual counting was set to 100 %. *p < 0.05; **p < 0.01

Fig. 7

Glandular epithelial cells and syncytial fragments in uterine veins. Serial sections of invaded decidua (gestational age 8 weeks). Sections in the left column (a, d, g) are immuno-double-stained for keratin 7 (KRT7) (blue serves as marker for glandular epithelial cells and syncytial fragments) and major histocompatibility complex, class I, G (HLA-G) (appears dark brown serves as marker for extravillous trophoblast), in the middle column (b, e, h) von Willebrand factor (vWF) (blue, serves as marker for vascular endothelial cells) and major histocompatibility complex, class I, G (HLA-G) (brown), in the right column (c, f, i) immuno-staining for desmin (brown serves as marker for muscle layer). (a, d, g) In the lumen of the vein (circle) detached glandular epithelial cells (d, g) and putative syncytial fragments can be found (d) (arrows, KRT7-positive (blue), but HLA-G-negative). b, e, h The serial sections in the middle and right column show that this luminal structure is a vessel (circle in b, c), arrows in e, h show the endothelium (vWF-positive), and the endothelium is partly replaced by EVTs. c, f, i The right column confirms the venous origin of the vessel (circle) by negative staining for desmin. The endothelium in this vein is incomplete. No nuclear counterstain (left and middle column) or nuclei were counterstained with hemalaun (right column)

Fig. 8

Concept of extravillous trophoblast (EVTs) invasion in first trimester human placenta. EVTs originate from cell columns of anchoring villi. During the first trimester of pregnancy, EVTs invade into the decidual interstitium (1) reaching the inner third of the myometrium, thereby anchoring the placenta to the uterus. They also follow the (2) endovascular route of invasion, plug, line and remodel spiral arteries (2, red), thus being responsible for the establishment of the maternal–fetal blood flow starting with the beginning of the second trimester. Prior to the opening of spiral arteries toward the intervillous space, maternal blood plasma is seeping through the trophoblastic plugs. Endovascular trophoblasts also reach and invade uterine veins and replace the venous endothelium (2, blue). Via the opened and dilated veins, maternal blood plasma and glandular secretion products are drained from the intervillous space into the maternal circulation. Endovascular trophoblasts invade and replace the tunica media of vessels, which in some cases leads to the fact that classification into artery or vein is no longer possible (2, red and blue). Endoglandular trophoblasts (3) are situated nearby uterine glands, replace the glandular epithelium and open the lumen of uterine glands toward the intervillous space. Scheme adapted from (Moser et al. 2015)