Ectopic pregnancy is the implantation of a fertilized ovum into tissues, outside the endometrial cavity. By far the most frequent sites of ectopic pregnancy are the fallopian tube (tubal pregnancy) and the ampullary region within the tube. Implantation rarely occurs in the peritoneum, cervix, ovary, or the broad ligament. One of the factors causing tubal pregnancy is the delay in progression of the ovum in the tuba uterina. Stimulation of receptors and closure of the isthmus are due to the release of prostaglandin F2 alpha (PGF2) which causes a reverse flow towards the fimbrial end, opposite to the flow of the tubal fluid. Furthermore, decreased amounts of ciliated cells nearby the isthmus as an effect of progesterone may also cause implantation of the fertilized ovum in the ampulla-isthmic area for 72 h. Moreover, delayed ovulation and fertilization may fail to suppress menstruation, which then flushes the embryo from the uterus back to the uterine tube.

Increased estrogen levels cause tubal implantation by destroying tubal motility. Tubal ectopic pregnancy usually occurs in the tube where anatomical and histological structures have been altered due to inflammation by interfering in the transport of the ovum. Failure of embryo development causing inappropriate secretion of molecules and incomplete definition of the implantation area may also increase ectopic pregnancy rates.

Defects in endometrial receptivity is the most important factor in the etiology of ectopic pregnancies. The endometrium undergoes cyclic changes in response to ovarian steroids during the reproductive cycle. How the endometrium becomes receptive in the midluteal phase is a complex phenomenon. Integrins, fibronectin and leukemia inhibitory factors are the most extensively characterized markers of endometrial receptivity. Implantation of the window phase is assured in tubal pregnancies during the delayed advance of the ovum in the tuba uterina due to the increased release of factors, tubal secretion in this area as well as the expression of integrins.

Extracellular matrix proteins play a significant role in adhesion and implantation. Integrins are a class of cell-adhesion molecules that interact with extracellular matrix, ligands and other cell-adhesion molecules, and participate in a wide variety of physiological processes. These molecules, which serve as receptors for the extracellular matrix, are present on virtually all cells and participate in cell-to-cell and cell-to-substratum attachment, signal transduction and interactions with the cytoskeleton. Specific combinations of integrins have emerged as reliable markers of implantation and fertility. Their absence in the endometrium has been associated with lack of the luteal phase, endometriosis, and unexplained infertility. Ligands for integrins expressed at the implantation site include fibronectin, laminin and collagen. Fibronectin is an extracellular adhesive glycoprotein that mediates cell–matrix adhesion. Thus, fibronectin may play an important role in implantation when adhesion between the embryo and maternal tissue is established.

We hypothesized that integrins and fibronectin which are involved in normal implantation may be expressed as well in tubal tissues during ectopic pregnancies, and their elevated expression may be causing tubal ectopic pregnancy. Thus, we investigated the expression of integrins (3, ?1, V, 2?1) and fibronectin in tubal samples with ectopic pregnancy and control tubal samples.

The study included formalin-fixed paraffin-embedded tissue blocks from 30 patients with tubal pregnancy and five patients who underwent tubal ligation (n=5). The latter group served as controls (group 1). In addition, normal areas in fallopian tubes with ectopic pregnancies were also used as a second control group (group 2). The ectopic site of tubal pregnancies was used as the study group (group 3). All samples were retrieved from the Ege Maternity Hospital, Department of Pathology, Izmir, Turkey. The Ethics Committee at the Ege Maternity Hospital approved our study protocol and all patients gave informed consents.

In each tubal specimen of patients with ectopic pregnancy, we investigated both the tubal implantation site and the tubal decidualization segment, away from the implantation site, that did not show any evidence of placental trophoblast. The tissue blocks were chosen carefully after histologic assessment of sections stained with hematoxylin–eosin (H&E). Serial sections (5 ?m thick) were cut from the selected blocks and prepared for indirect immunohistochemical staining.

For immunohistochemical staining, sections were first deparaffinized at 60°C overnight and then incubated in xylene for 30 min. After washing with a decreasing series of ethanol, sections were washed in distilled water and phosphate-buffered saline (PBS) for 10 min. Sections were then treated with 2% trypsin in Tris buffer (50 mM Tris base and 150 mM NaCl dissolved in deionized H₂O) at 37°C for 15 min and washed with PBS. Sections on the glass slides were delineated with a Dako pen (Dako, Glostrup, Denmark) and incubated in a solution of 3% H₂O₂ for 15 min to inhibit endogenous peroxidase activity. Then, sections were washed with PBS and incubated for 18 h at +4°C with primary antibodies: a polyclonal anti-integrin V antibody in a 1:100 dilution (Calbiochem, San Diego, CA, USA), a monoclonal anti-integrin 3 antibody in a 1:100 dilution (Oncogene Research, Cambridge, MA, USA), a monoclonal anti-integrin-?1 antibody in a 1:10 dilution (Oncogene Research, Boston, MA, USA), a monoclonal anti-2?1 antibody in a 1:100 dilution (Dako, Carpinteria. CA, USA) and a monoclonal anti-fibronectin antibody ready-to-use (Neomarkers, Fremont, CA, USA). Afterwards, sections were washed 3 times for 5 min each with PBS, followed by incubation with biotinylated IgG and then with streptavidin-peroxidase (Dako). All incubation steps were separated by three washing steps. After washing, 3 times for 5 min with PBS, sections were incubated with a substrate solution containing H₂O₂ and diaminobenzidine (Dako) for 5 min at room temp. and were counterstained with Mayer’s hematoxylin. Sections were covered with mounting medium and then analyzed with a BX 40 light microscope (Olympus, Tokyo, Japan). Control samples were processed in an identical manner, but the primary antibodies were omitted. Two observers, blinded to clinical information, evaluated the staining scores independently. Staining intensity was graded as absent (?), mild (+), moderate (++) and strong (+++), respectively. The Kruskal–Wallis non-parametric test was used to compare staining intensities, with p<0.05 as level of significance.

Histological analysis of the control group after staining with H&E showed that fallopian tubes consisted of an external serosal layer, an intermediate muscular layer and an internal mucosal layer. The serosa consisted of mesothelium and a thin layer of connective tissue. The muscular layer was organized into an inner circular layer and outer longitudinal layer. The mucosa exhibited longitudinal folds into the lumen of the ampulla; these folds showed a branching core of vascular components supporting the lamina propria (Lp) that was covered by a single layer of tall columnar epithelial cells, some of which were ciliated. The Lp of the mucosa was composed of connective tissue containing a network of reticular fibers, numerous fibroblasts and other connective tissue cells. It was separated from the epithelium by a thin basal lamina.

Figure 1. Photomicrographs of normal human fallopian tube (group 1) (A, B), control tissue from a fallopian tube with ectopic pregnancy away from the site of implantation (group 2) (C, D) and tissue at the site of implantation in the ectopic pregnancy group (group 3) (E, F). In sections of the ampullary region of fallopian tubes in group 1 and group 2, mucosa with epithelium (E) and lamina propria (Lp) is extensively folded projecting into the lumen of the tube, whereas the muscularis (M) is composed of a thick inner circular layer and an outer longitudinal layer. In cross sections of the ectopic site of pregnancy in fallopian tubes (group 3), stromal fibroblasts, known as decidual cells (arrows), are large and polygonally shaped and contain abundant amounts of glycogen and lipids. H&E staining. Magnifications, ×40 (A, C, E), ×200 (B, D, F).