Uterus and endometrium: Immunohistochemical localization of extracellular matrix proteins in luteal phase endometrium of fertile and infertile patients

The lack of expression of certain components involved in celladhesion and migration is believed to contribute to endometrialdysfunction and implantation failure. The purpose of this studywas to investigate whether luteal phase endometrium in womenwith unexplained infertility differs, with respect to specificextracellular matrix (ECM) proteins, from endometrium of normalfertile women. A panel of monoclonal antibodies to collagentype IV, fibronectin and laminin was used to characterize thelocalization of ECM components in the different endometrialcompartments. Precisely timed endometrial biopsies obtainedat 4, 7, 10 and 13 days following the luteinizing hormone surgewere obtained from 22 normal fertile women (group 1) and 24women suffering from unexplained infertility (group 2). Paraffin-embeddedsections were labelled using the streptavidin-biotin alkalinephosphatase technique. In group 1, collagen type IV, fibronectinand laminin were absent from the luminal epithelium but presentin stromal cells and the basement membrane of glands and bloodvessels. In group 2, these components were absent from all endometrialregions using equivalent titres of antibody to those used ingroup 1. This suggests that the endometrium of women with unexplainedinfertility demonstrates defects in the distribution of certainECM glycoproteins. A possible consequence of this defect maybe implantation failure.     

Mechanism of action of the intrauterine contraceptive device: evidence for a specific biochemical deficiency in the endometrium

The precise mechanism of action of the intrauterine contraceptivedevice (IUCD) is uncertain. In this study we compared the circulatingconcentrations of a specific endometrial protein, placentalprotein 14 (PP14), in 62 women with an IUCD and 16 controls.The concentrations of PP14 were substantially lower in IUCDusers. There was no difference in the concentrations of anotherand less specific endometrial protein, insulin-like growth factorbinding protein-1 (IGFBP-1). There was no difference in PP14concentrations between those women with and without intermenstrualbleeding. We conclude that the reduced concentrations of PP14in IUCD users reflect defective endometrial function in thesewomen, probably related to the contraceptive effect We proposethat the measurement of PP14 might be a means of comparing theefficiency of different devices.     

Two-dimensional gel analysis of human endometrial proteins: cyclic changes in the expression of specific proteins during the normal menstrual cycle

High resolution two-dimensional (2-D) gel electrophoresis wasused to compare the patterns of [³⁵S]methionine-labelled cellularproteins in endometrial tissue from healthy, normally menstruatingwomen. Samples of endometrial tissue were incubated with [³⁵S]methioninefor 20 h, and total cell lysates were processed for 2-D gelelectrophoresis. Using this technique it was possible to studyproteins with iso-electric points (pI) ranging from 3.5 to 11and relative molecular weights (M_r) ranging from 10 000 to 300000 Da. The fluorograms were compared by computer-aided analysiswhereby a total of 1095 [³⁵S]-labelled proteins were resolvedon the iso-electric focusing gels (IEF, pI 3.5–7) and488 on the non-equilibrium pH gradient electrophoresis (NEPHGE)gels (pI 6.5–11). Of the proteins on the IEF gels, 125showed differential expression during the menstrual cycle. Ofthese, 36 were maximally expressed in proliferative phase endometrium,26 in the interval phase and 63 in secretory and/or late secretoryphase endometrium. Correspondingly, on the NEPHGE gels a totalof 61 proteins exhibited cyclical variation, of which 30 weremore prominent in proliferative phase, 13 in interval phaseand 18 in secretory phase endometrium. This study shows that2-D gel electrophoresis is eminently suited to the identificationof proteins whose expression varies in a cyclical manner duringthe menstrual cycle. Further investigations should be carriedout to isolate and characterize these proteins with the aimof establishing useful markers for specific endometrial phasesof the menstrual cycle.     

Morphological events in the primate endometrium in the presence of a preimplantation embryo, detected by the serum preimplantation factor bioassay

BACKGROUND: Hormonal modulation of the endometrium towards receptivity is well established; however, the role of embryonic stimuli in modulation of the endometrium prior to implantation, especially in primates, is unknown. The aim of the present study was to evaluate the endometrial histology when the embryo was present in its vicinity prior to implantation. METHODS: Preimplantation factor (PIF) bioassay was used as a tool to detect the presence of an embryo in the uterine lumen of mated bonnet monkeys (Macaca radiata) (n=9). The control group comprised seven non-mated animals. The specificity of the PIF bioassay for the presence of an embryo was tested by studies in pregnant humans and monkeys. The effects of embryonic stimuli on the endometrial morphology were analysed by routine haematoxylin-eosin staining. The expressions of CD34, an endothelial cell marker, alpha-smooth muscle actin (alpha-SMA), a marker for blood vessel maturation, and prolactin, a marker of endometrial decidualization, were studied by immunohistochemistry. RESULTS: That PIF is embryo specific was established by its presence in sera of pregnant humans, monkeys and also in embryo culture media. Six mated bonnet monkeys were found to be PIF positive. Morphologically, the endometria from these PIF-positive animals showed the presence of the pre-epithelial plaque reaction, increased angiogenesis and stromal compaction. The significantly increased number of CD34- and alpha-SMA-positive blood vessels (P<0.05) in the endometria of PIF-positive animals indicated increased angiogenesis in response to embryonic stimuli. The endometrial expression of immunoreactive prolactin was also significantly increased (P<0.05) in the PIF-positive animals, indicating decidualization. CONCLUSIONS: Using PIF as a marker to detect early pregnancy in bonnet monkeys, we have shown that the embryo induces a pre-epithelial plaque type of reaction, increased angiogenesis and decidual reaction in the endometrium prior to implantation.     

Induction of a polarized micro-environment by human T cells and interferon-gamma in three-dimensional spheroid cultures of human endometrial epithelial cells

Expression of human leukocyte antigen (HLA)-DR molecules andproliferation of epithelium in human endometrium are polarized.We have suggested that the induction of such a polarized micro-environmentis T cell and interferon (IFN)-gamma dependent. The presentstudy was designed to demonstrate the induction of such a micro-environmentaround T cells and around the source of IFN-gamma. Spheroidsreminiscent of endometrial glands were formed by allowing three-dimensionalaggregation of endometrial epithelial cells of a cloned HLA-DRnegative endometrial carcinoma cell line (ECC1) over agarose.Both HLA-DR expression and inhibition of proliferation werefound to be directly dependent on the dose of IFN-gamma thatwas allowed to diffuse in the agarose beneath the spheroids.To show that the interaction of the epithelial cells with activatedT cells also induces HLA-DR molecules in a paracrine fashionin the epithelial cells, ECC1 spheroids were co-cultured withincreasing numbers of allogeneic peripheral blood T cells forvarious time-intervals. T cells bound to the ECC1 cells, andbecame activated as indicated by the expression of interleukin(IL)-2 receptor and HLA-DR molecules. A focal HLA-DR expressionbecame apparent in the ECC1 cells adjacent to the T cells. Asthe number of T cells added to spheroid cultures was increased,a concomitant increase in the number of HLA-DR positive ECC1cells occurred and HLA-DR immunoreactivity was enhanced in eachcell. There was a corresponding decrease in the proliferationof the ECC1 cells in T cell-ECC1 spheroid co-cultures. Basedon these data, we suggest that activation of T cells is associatedwith the induction of HLA-DR expression and inhibition of proliferationin a paracrine fashion in the epithelial cells and may be responsiblefor the creation of a polarized micro-environment in vivo.     

Prognostic value of serum proteins synthesized by breast carcinoma cells.

Several breast carcinoma cell lines or explants of such tumors, as well as examples of lactating or dysplastic breast tissue, synthesized three serum proteins (alpha 2-Zn-glycoprotein, alpha 1-antichymotrypsin, and alpha-lipoprotein) in vitro. These proteins were detected by immunoperoxidase techniques in 126 breast carcinomas that had been evaluated clinically for more than six years. Alpha-2-Zn-glycoprotein was present in 58% of the carcinomas, whereas alpha 1-antichymotrypsin was seen in 55% and alpha-lipoprotein in 52%. These markers showed a relationship with clinical outcome. Alpha-1-antichymotrypsin and alpha-lipoprotein were unfavorable determinants, whereas alpha 2-Zn-glycoprotein was detected in lesions with a favorable evolution. Taken individually, these markers have similar but rather weak associations with five-year survival rates; roughly 20% of patients in the "favorable" group died, compared with 33% in the "unfavorable" group. Alpha-2-Zn-glycoprotein in grade 1 tumors was a marker with marginally favorable significance, but alpha 1-antichymotrypsin significantly worsened the prognosis of grade 2 and 3 tumors. Furthermore, stratification of patients according to the number of positive unfavorable markers yielded striking results. Eight percent of patients with none of the unfavorable markers were dead at five years, compared with 55% of those whose lesions expressed three unfavorable markers.     

Identification of two de novo partial trisomies by comparative genomic hybridization

We report the use of comparative genomic hybridization (CGH) to define the extra chromosome region present in two de novo partial trisomies 15q25-qter and Xp21-pter, which could not be clarified by conventional G-banding. Investigation with fluorescence in situ hybridization (FISH) revealed that the partial trisomy corresponded to an unbalanced translocation between Y and 15 chromosomes in 1 patient and an unbalanced X/X reorganization in the other patient. The combination of classical karyotyping, CGH, and FISH is useful for the identification and characterization of partial trisomies in clinical diagnostic laboratories, in order to delineate the chromosome regions implicated in specific clinical disorders.     

Pregnancy: Differential increase in the maternal serum concentrations of the placental proteins human chorionic gonadotrophin, pregnancy-specific {beta}1-glycoprotein, human placental lactogen and pregnancy-associated plasma protein-A during the first half of normal pregnancy, elucidated by means of a mathematical model

The present study was performed to compare the increase in maternalserum concentrations of four placental proteins during the firsthalf of 240 normal pregnancies. The proteins were pregnancy-associatedplasma protein-A (PAPP-A), human chorionic gonadotrophin (HCG),human placental lactogen (HPL) hormone, and pregnancy-specific1-glyco-protein (PSG), all produced by trophoblast cells. Themedian increases were observed to be very close to exponentialgrowth curves. Based on simple assumptions, these growth curvescould be explained as being solely dependent on the growth ofthe placenta. The assumptions were that the proteins were producedin the placenta at a constant rate per gram of placental cellmass and secreted into the circulation shortly after synthesis.Our investigations showed that for two of the proteins, PSGand HPL, the rate constants were, in fact, close to the reportedgrowth rate of the placenta, whereas the PAPP-A production rateconstant was significantly higher than those of the others.The production curve for HCG was very different from that ofthe other proteins. PAPP-A and HCG must therefore have morecomplicated mechanisms for regulating the production. An equationwas constructed that permitted estimation of the molar productionof the placental proteins per gram of placental cell mass perday during the first half of normal pregnancy. The value washighest for HPL and lowest for PAPP-A.     

Flow cytometric analysis of specific binding of soluble Ia by I-region restricted alloactivated T cells

An antigen binding assay has been developed for quantitation by flow cytometry of vesicular and soluble Ia binding by alloactivated T cells. Binding of stimulator membrane vesicles was detected by anti-Ly-6.2 or anti-Ia monoclonal antibodies coupled to fluorescent latex beads. Vesicle binding by an I-A^k specific A.TH anti-A.TL T cell line occurred via I-A^k molecules, in that (a) vesicles expressing I-A^k molecules bound much more effectively than vesicles of H-2^b,q strains, and (b) inhibition of H-2^k vesicle binding occurred with anti-I-A^k, but not anti-K^k, anti-E^k, or anti-D^k antibodies. T cell receptor/Ia interactions were directly studied by inhibition of H-2^k vesicle binding by T cells with partially purified Ia glycoproteins. Inhibition of binding occurred via Ia molecules since (a) affinity column partially purified allogeneic I-A^k molecules inhibited binding much more effectively than syngeneic I-A^s molecules and (b) depletion of I-A^k but not E^k molecules in Ia^k containing glycoprotein fractions abrogated the inhibitory effect. The ability of this method to detect specific binding of soluble Ia with antigen activated T cells makes it a useful tool for studying interaction of membrane free major histocompatibility complex (MHC) products with native T cell receptor.     

The separation of human serum IgG into subclass fractions by immunoaffinity chromatography and assessment of specific antibody activity

Murine monoclonal antibodies ( McAbs ) with specificity for subclass-specific or subclass-restricted determinants on human IgG have been coupled to Sepharose to generate affinity columns. The judicial use of positive and negative chromatography and the exploitation of the special properties of individual McAb affinity columns has allowed the preparation of individual IgG subclasses from polyclonal IgG containing less than 1% contamination by any other IgG subclass. The specificity of the antibodies present in each polyclonal IgG subclass preparation has been assayed against a bacterial toxoid (tetanus), 2 bacterial cell wall antigens (E. coli and pneumococcal) and coat antigen(s) of a DNA virus (CMV). Antibodies were predominantly IgG1 to tetanus toxoid, IgG2 to pneumovax and E. coli cell walls, and IgG1, 2 and 3 to CMV coat antigens.