Blastocyst implantation that begins with attachment of the trophoblast to the uterus and terminates with formation of the placenta is a complex series of events in early pregnancy. To complete this series of events and to accomplish successful implantation and placentation, the embryo and the uterine endometrium must be synchronized during the limited period of uterine receptivity. Thus, the embryo and the endometrium induce changes in each other to promote receptivity.

Trophoblast cells that are indicated as extravillous trophoblast (EVT) are responsible for the anchoring of the placenta. The trophoblast, which arise from the proliferative cell columns, expresses the invasive phenotype that is necessary to penetrate deeply into the maternal decidualized endometrium. Thus, the trophoblast enables implantation of the human blastocyst and placentation by breaking through the maternal extracellular matrix.

Trophoblast cell invasion into the decidua is a complex and dynamic process that includes cell adhesion to matrix proteins, degradation of extracellular matrix (ECM), and cell motility. Trophoblast invasion is regulated in part by matrix metalloproteinases (MMPs), a family of zinc- and calcium-dependent proteolytic enzymes. MMPs and their activators and inhibitors play an important role during placental development, as well as during parturition, in which they are responsible for the degradation of ECM including type IV, V, VII, and X collagens, fibronectin, and gelatin. MMPs are synthesized intracellularly and secreted as inactive precursors that are activated in the extracellular space by various enzymes (including MMPs) in an autocrine or paracrine way and their activities are inhibited by tissue inhibitors of matrix metalloproteinases (TIMPs).

Regulation of MMP activity is controlled not only at the level of gene transcription and activation of the latent enzyme, but also locally in a tissue by TIMPs. These naturally occurring specific inhibitors of decidual or trophoblast cell origin have an important physiological role in regulating trophoblast invasion. The best-known TIMP is TIMP-1, a ubiquitous 28.5-kDa secreted glycoprotein that forms tight stoichiometric non-covalent complexes with the active forms of all known MMPs. In addition, it binds proMMP-2. TIMP-2 has an amino acid sequence identity with TIMP-1 and similar inhibitory activity against MMPs but preferentially binds to proMMP-9.

Many in vivo and in vitro studies have demonstrated that enzymes such as serine proteases and MMPs play pivotal roles in the invasion of the endometrium by trophoblasts. Special attention has been focused on the involvement of MMPs which degrade ECM components of decidua (endometrium) as well as on their inhibitors, TIMPs. Almost all studies focused on human pregnant endometrium are in vitro studies whereas almost all in vivo data are obtained in mouse, rat and monkey tissues. Until now, in vivo studies have not been focused on early human pregnancy. Therefore, our present study was aimed to supply in vivo data on the earliest stages of human pregnancy. We aimed to determine expression patterns of MMP-2, MMP-9, TIMP-1 and TIMP-2 in human decidual tissue samples of the very early stages of pregnancy in which expression of these molecules are of utmost importance. Therefore, we examined the relationship between trophoblast invasion and MMP-2, MMP-9, TIMP-1 and TIMP-2 expression as well as their distribution patterns using immunohistochemical methods to elucidate the mechanisms underlying trophoblast invasion during early pregnancy.

A total of 17 samples of endometrial decidual tissue (7 samples of 22–28 days (fourth week post coitus); 4 samples of 29–35 days (fifth week post coitus); 6 samples of 36–42 days (sixth week post coitus) in the first trimester of pregnancy were obtained by curettage of women with normal pregnancy and no apparent endometrial dysfunction or gynecologic diseases, who gave informed consent for the collection of tissue. Approval for this investigation was given by the Ethical Committee of Akdeniz University, Faculty of Medicine, Antalya, Turkey. All these tissue samples were fixed in 10% formalin (pH. 7.2) for 6 h and were dehydrated in ethanol, cleared in xylene, and embedded in paraffin. All samples were serially sectioned at 5 ?m thickness.

Paraffin tissue sections were attached to HistoGrip (00-8050, Zymed, San Francisco, CA, USA) coated glass slides and were deparaffinized and rehydrated through a graded series of ethanol. Sections were then treated in a microwave in 10 mM citrate buffer, pH 6.0, for 10 min. After three rinses in phosphate buffered saline (PBS), endogenous peroxidase activity was blocked by 3% hydrogen peroxide in methanol for 20 min and again washed three times in PBS. Afterwards, sections were incubated in a blocking serum (Ultra V Block, TP-060-HL; NeoMarker, Fremont, CA, USA) for 7 min in order to block non-specific binding. Subsequently, sections were incubated with the primary antibodies [rabbit polyclonal anti-MMP-2 (RB-1588-P1), dilution 1/800; rabbit polyclonal anti-MMP-9 (RB-1539-P1), dilution 1/800; rabbit polyclonal anti-TIMP-1 (RB-1531-P1), dilution 1/150; rabbit polyclonal anti-TIMP-2 (RB-1489-P1), dilution 1/200 and monoclonal anti-cytokeratin-7 (MS-283-P1), dilution 1/1500; all from NeoMarker] for 1 h. The anti-MMP-2 and anti-MMP-9 antibodies bind to both the inactive proform and the activated form of the enzymes. Sections were washed three times in PBS and treated with secondary antibody (TP-060-HL; NeoMarker) and a streptavidin-peroxidase complex (TP-060-HL; NeoMarker), respectively, for 15 min with each of the steps being followed by three rinses in PBS. Peroxidase activity was visialized with diaminobenzidine and hydrogen peroxide as substrates (K3466; Dako, Glostrup, Denmark), and sections were counterstained with Mayer’s hematoxylin (S3309; Dako), dehydrated and mounted. For controls, sections were treated with either normal rabbit IgG or normal mouse IgG depending on the primary antibody used in a dilution that gave the same final protein concentration as the primary antibody dilution. CK-7 antibody was used to detect extravillous trophoblast cells and epithelial structures in sections of decidual tissue.

Sections were evaluated with respect to MMP-2, MMP-9, TIMP-1, and TIMP-2 protein localization in a semiquantitative manner using a light microscope and selected areas were photographed using Kodak Gold ISO100 film (Kodak, Rochester, NY, USA). Staining intensity was scored as negative (?), weak (+), moderate (++), strong (+++) or very strong (++++). HSCORE values of MMP-2, MMP-9, TIMP-1 and TIMP-2 staining were obtained in a semiquantitative manner and included both intensity and distribution patterns of staining. Ten different fields of six sections per specimen at ×400 magnification were evaluated for the analysis of immunohistochemical MMP-2, MMP-9, TIMP-1 and TIMP-2 staining. Values were recorded as percentages of positively stained target cells in each of four intensity categories which were denoted as 0 (no staining), 1+ (weak), 2+ (moderate), 3+ (strong). For each tissue, an HSCORE value was derived by summation of the weighted intensity of staining [HSCORE=SP_i(i+1), where i is the intensity score and P_i is the corresponding percentage of the cells]. Comparison of HSCORE values was performed with Student-t tests and one-way-Anova tests. All statistical analyses were performed using Sigmastat for Windows version 3.0 (Jandel Scientific Corporation, San Rafael, CA, USA). Distribution patterns of MMP-2, MMP-9, TIMP-1 and TIMP-2 in cells of the endometrial decidua were also evaluated in the fourth, fifth and sixth weeks of pregnancy. Two blinded observers performed the HSCORE evaluation. The inter-individual variation was 15% for MMP-2, 12% for MMP-9, 13% for TIMP-1 and 17% for TIMP-2 staining.