Evidence from the very beginning: endoglandular trophoblasts penetrate and replace uterine glands in situ and in vitro

Abstract

Study question: How is histiotrophic nutrition of the embryo secured during the first trimester of pregnancy?

Summary answer: Rather than specifically focusing on invasion into spiral arteries, extravillous trophoblasts also invade into uterine glands (endoglandular trophoblast) from the very beginning and open them toward the intervillous space.

What is known already: Extravillous trophoblasts can be found in close contact and within the lumen of uterine glands, sometimes replacing glandular epithelial cells.

Study design, size, duration: As well as extensive screening of specimens from first trimester placentation sites in situ we used a previously established three-dimensional co-culture in vitro model system of first trimester villous explants with non-invaded decidua parietalis.

Participants/materials, setting, methods: First trimester placentas were obtained from elective terminations of pregnancies (n = 48) at 5-11 weeks of gestational age. A subset was processed for confrontation co-culture (n = 31). Invaded decidua basalis was obtained from 20 placentas. All tissues were sectioned, subsequently immunostained and immunodoublestained with antibodies against keratin 7 (KRT7), major histocompatibility complex, class I, G (HLA-G), matrix metallopeptidase 9 (MMP9), von Willebrand factor (VWF) and the appropriate Immunoglobulin G (IgG) negative controls. Replacement of endothelial/epithelial cells by extravillous trophoblasts was quantified semi-quantitatively. Additionally, hematoxylin and eosin-stained archival specimens from early implantation sites were assessed.

Main results and the role of chance: The earliest available specimen was from around Day 10 after conception; already at this stage trophoblasts had penetrated into uterine glands and had started to replace the epithelium of the glands. Endoglandular trophoblasts replaced uterine glands in vitro and in situ and could be found in the lumen of invaded glands. Quantitative analysis revealed significantly more replacement of epithelial cells in glands (63.8 ± 22.1%) compared with endothelial cells in vessels (26.4 ± 8.8%). Accumulated detached glandular epithelial cells could be repeatedly observed in the lumen of invaded glands. Additionally, in areas of trophoblast invasion the glandular epithelium seemed to be completely disintegrated compared with glandular epithelium in the non-invaded parts of the decidua. Whole tissue specimens were used in vitro and in situ instead of cell lines; these systems mostly maintain the context of the in vivo situation.

Limitations, reasons for caution: This is a descriptive study supported by in vitro experiments. However, a histological section will always only be a snapshot and quantification from histological sections has its limitations.

Wider implications of the findings: This study further strengthens the hypothesis of histiotrophic nutrition of the embryo prior to the establishment of the maternal blood flow toward the placenta. Invasion of uterine glands by endoglandular trophoblasts may have more impact on the outcome of early pregnancy than assumed up to now.

Keywords: co-culture; endoglandular trophoblast; implantation; invasion; model system; placenta; uterine gland.

Figure 1

Trophoblasts penetrate uterine glands during very early pregnancy. Hematoxylin and eosin stained section of a human implantation site at Day 10 (archival specimen). (a) Overview: the early conceptus (EC) has already implanted into the decidual stroma beneath the uterine epithelium. Lumina of the uterine glands beneath the early conceptus seem expanded and filled with fluid (probably a mixture of glandular secretion products and blood). Glandular epithelia nearby the invading early conceptus (asterisk) seem to be disintegrated compared with glandular epithelium in the non-invaded parts of the decidua (black dots). (b) Detail of the left hand inset in (a); arrows show the direction of trophoblasts invading the decidua and penetrating the uterine gland. (c) Higher magnification of the right hand inset in (a); arrows show single trophoblasts penetrating the uterine gland.

Figure 2

Trophoblasts penetrate uterine glands during very early pregnancy. Hematoxylin and eosin stained section of a human implantation site at gestational age 3 weeks (archival specimen). (a) Overview: The villous part has been disrupted from the decidua. In the broader overview under the microscope villi (V) and decidua (D) are clearly allocated (not shown). Arrows show the direction of trophoblasts invading the decidua and penetrating a uterine gland. (b) Higher magnification of the inset in (a); arrows show single trophoblasts penetrating a uterine gland.

Figure 3

Endoglandular trophoblasts invade and replace the epithelium of uterine glands and open the glands toward the intervillous space in situ. Immunohistochemical double staining of invaded decidua (gestational age 6–12 weeks) for (a–c) keratin 7 (KRT7) (blue, serves here as marker for glandular epithelial cells) and major histocompatibility complex, class I, G (HLA-G) (appears dark brown, serves here as marker for extravillous trophoblasts (EVT)) besides (d) for von Willebrand factor (VWF) (blue, serves here as marker for vascular endothelial cells) and HLA-G (brown). Note that there is no nuclear counterstain. (a–b) EVT (brown) invade into the decidua (arrow) and replace the epithelium (blue) of uterine glands (asterisk). (c–d) Serial sections show a villus attached to the decidua via a trophoblastic cell column, EVTs (brown) invade the decidual stroma and replace the glandular epithelium, the lumen of the gland is opened toward the intervillous space; arrows show the putative flow direction of glandular secretions toward the intervillous space (IVS). Asterisk in (c) marks residual glandular epithelial cells, triangle in (d) marks residual blood vessel (blue). (e) Hematoxylin and eosin staining of the invasive front during the first trimester (archival specimen) and (f) corresponding schematic drawing shows a similar picture like in (c and d). Trophoblasts originating from trophoblastic cell columns (green) invade into decidual tissues (light purple). The epithelium of the uterine gland seems to have been partly replaced by endoglandular trophoblasts, on the decidual side there is still residual glandular epithelium (light blue). Thus, the lumen of the gland is opened toward the intervillous space (red). This enables the release of glandular secretion products into the intervillous space (arrow).

Figure 4

Distinction between endoglandular and endovascular trophoblasts. Invaded decidua with immuno-double staining (gestational age 6–8 weeks). Rows are composed of serial sections (a–b, c–d), sections in the left column are immuno-double stained for keratin 7 (KRT7) (blue, serves here as marker for glandular epithelial cells) and major histocompatibility complex, class I, G (HLA-G) (appears dark brown, serves here as marker for extravillous trophoblasts (EVTs), in the right column for von Willebrand factor (VWF) (blue, serves here as marker for vascular endothelial cells) and HLA-G (brown). Only immunohistochemical double staining enables clear identification whether the invaded structure was of glandular (asterisk) or vascular (triangle) nature. No nuclear counterstain. (e) Quantitative analysis reveals significantly more replacement of epithelial cells in glands (63.8 ± 22.1%) compared with endothelial cells in vessels (26.4 ± 8.8%). Attachment of interstitial trophoblasts to glands or vessels was not significantly different.

Figure 5

Endoglandular trophoblasts invade and replace uterine glands in vitro (gestational age 6–12 weeks). Each row is composed of serial sections (a–c, d–f), sections in the left column are immuno-stained for major histocompatibility complex, class I, G (HLA-G), in the middle column immuno-double stained for keratin 7 (KRT7) (blue, serves here as marker for glandular epithelial cells) and HLA-G (appears dark brown, serves here as marker for extravillous trophoblasts (EVTs)), and in the right column immuno-double stained for von Willebrand factor (VWF) (blue, serves here as marker for vascular endothelial cells) and HLA-G (brown). (a and d) Overview of confrontations. V, Villi, D, Decidua. (b and e) Glandular epithelial cells appear blue. Extravillous trophoblasts (arrows) invade into decidual tissues, reach the uterine glands and replace uterine glandular epithelial cells as endoglandular trophoblasts. (c and f) Vascular cells appear blue. Immunohistochemical double staining clearly shows the glandular/vascular nature of the invaded structure. Nuclei were counterstained with Hemalaun (a and d) or no nuclear counterstain was used (b,c,e and f).

Figure 6

Endoglandular trophoblasts—peculiarities. Invaded decidua with immunohistochemical staining/double staining (gestational age week 5). Sections (a, c–e) are immuno-double stained for keratin 7 (KRT7) (blue, serves here as marker for glandular epithelial cells) and major histocompatibility complex, class I, G (HLA-G) (appears dark brown, serves here as marker for EVTs), section (b) is immuno-double stained for von Willebrand factor VWF (blue, serves here as marker for vascular endothelial cells) and HLA-G (brown), section (f) is immunostained for matrix metalloproteinase 9 (MMP9). (1) Trophoblastic equivocality: (a,b) Serial sections show a uterine gland, opened toward the lumen of an invaded vessel. Asterisk in (a) marks a uterine gland, where the epithelium is penetrated and partly replaced by endoglandular trophoblasts. The glandular epithelium is disintegrated. Triangle in (b) marks a uterine vessel, where the endothelium is partly replaced by endovascular trophoblasts. The arrow marks the presumable direction of glandular secretion/vascular plasma flow toward the intervillous space. (2) Disintegrated versus intact glandular epithelium in invaded versus non-invaded regions: (c) In the decidua basalis, virtually all glands are invaded by endoglandular trophoblasts. Glandular epithelia (asterisk) in the invaded parts appear disintegrated in comparison to (d) the non-invaded decidua parietalis from the same uterus. Here, the glandular epithelium (circle) appears compact and well-proportioned. (e–f) Serial sections, double staining shows an invaded gland (e), the epithelium is disintegrated, trophoblasts surrounding the uterine gland are positive for MMP9 (f). No nuclear counterstain (a–e) or nuclei were counterstained with Hemalaun (f).

Figure 7

Scheme of first trimester human placenta and its nutritional supply. Extravillous trophoblasts originate from cell columns of anchoring villi. During the first trimester of pregnancy extravillous trophoblasts invade into the decidual interstitium (1) to the first 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, thus being responsible for the establishment of the feto-maternal 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. Endoglandular trophoblasts (3) are defined as trophoblasts situated nearby uterine glands, replacing the glandular epithelium or trophoblasts present in the glandular lumen. Additionally, endoglandular trophoblasts may open uterine glands toward the lumen of an invaded spiral artery, secretion products of the uterine gland may thereby reach the intervillous space together with the blood plasma seeping through the trophoblast plug of the spiral arteries. Glandular secretion products in the intervillous space enable histiotrophic nutrition of the embryo prior to the establishment of the feto-maternal blood flow, i.e. hemotrophic nutrition.