1
METHOD FOR PREVENTING EMBRYO
IMPLANTATION
REFERENCE TO GOVERNMENT GRANTS 5
This work was supported in part by research grants from the Biomedical Research Support Grant Program, Division of Research Resources, the National Institutes of Health, grant number B.R.S.B. S07-RR-05415-29. The United States Government may have certain rights in this invention. 10
This is a continuation of application Ser. No. 08/126,063, filed Nov. 1, 1993, now abandoned, which is a division of Ser. No. 07/897,706 filed Jun. 12, 1992, now U.S. Pat. No. 5,279,941.
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BACKGROUND OF THE INVENTION
Over the past decade, investigators have come to recognize the importance of the extracellular matrix (ECM) in directing the growth, differentiation and function of the 20 overlying epithelium. Getzenberg et al., 'The Tissue Matrix: Cell Dynamics and Hormone Action", Endocrine Rev ,11: 399-417 (1990). The interaction between cell and extracellular matrix (or substratum) is mediated by several classes of cell adhesion molecules, one of the most important being the 25 integrins. Albelda et al., "Integrins and Other Cell Adhesion Molecules", FESEB J., 4: 2868-2880 (1990). Buck et al., "Integrin, a Transmembrane Glycoprotein Complex Mediating Cell-Substratum Adhesion", J. Cell Sci. Suppl, 8: 231-250 (1987). This diverse family of glycoprotein receptors is expressed on the cell membrane as heterodimeric a 30 and p subunits and is involved in both cell-cell and cellsubstratum adhesion. Specific recognition and binding of extracellular matrix (ECM) components such as fibronectin (FN), laminin (LM) and collagen (Col) transmit information to the cytoskeletal structure, an interaction which may have 35 major roles in promoting hormone responsiveness and genomic activation. Burridge et al., "Focal Adhesions: Transmembrane Junctions Between the Extracellular Matrix and the Cytoskeleton", Ann Rev. Cell. Biol. 4: 487-525 (1988) and Getzenberg et al. supra. 40
Although extensive information exists about specific integrin proteins, for example, Hemler, M. E. "VLA Proteins in the Integrin Family: Structures, Functions and Their Role on Leukocytes", Annu. Rev. Immunol: 365^100 (1990), little is known concerning the distribution of these receptors in the 45 female reproductive tract. In the uterus, the endometrium, composed of glandular epithelium and associated mesenchyme (stroma), maintains complex temporal and spatial functions in response to the cyclic hormonal milieu. The search for morphological or biochemical markers for uterine 5Q receptivity has been unsuccessful to date as reported by Rogers and Murphy, "Uterine Receptivity for Implantation: Human Studies", in Blastocyst Implantation, Yoshinaga, K. ed., Serono Symposia, pp. 231-238 (1989). Once such markers are identified, their role in endometrial phenomena including embryo implantation, fertility, contraception and 55 endometrial maturation and receptivity can likely also be identified. Thus, as some integrins appear to meet the criteria for markers of receptivity there is a great need for methods of detecting integrin cell adhesion molecules in endometrium. 60
SUMMARY OF THE INVENTION
The present invention is directed to methods of detecting receptivity of endometrium to embryo implantation by 65 detecting the |33 subunit of the ajfa integrin in endometrium with a monoclonal antibody.
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Methods of diagnosing fertility and methods of monitoring endometrial maturation in a mammal are also provided by monitoring the appearance of the (33 subunit of integrin in endometrium from a plurality of stages of the endometrial cycle. This is preferably done with a monoclonal antibody.
The present invention also provides methods of detecting the optimal window of embryo implantation in the endometrium by detecting the p3 subunit of integrin in an endometrial sample, preferably with a monoclonal antibody.
Further aspects of the invention include methods of preventing embryo implantation by contacting the p3 subunit of integrin in the endometrium with neutralizing Fab antibody fragments to p3. Methods of in vitro fertilization are also embodiments of the invention. These comprise detecting the fi3 subunit of integrin in an endometrial sample, fertilizing an egg in vitro, and introducing the zygote into the uterus having endometrial tissue expressing the P3 subunit.
Contraceptive and diagnostic kits are also contemplated hereby.
These and other aspects of the invention will become more apparent from the following detailed description when taken in conjunction with the following figures.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1F depict immunoperoxidase staining of normal endometrium. The photomicrographs depict the pattern of distribution for six different integrins that do not appear to change throughout the menstral cycle. Dark areas represent positive staining, light areas represent absence of stain (absence of specific integrin subunit). Immunohistochemical staining of the collagen/laminin receptor subunits: oc2(FIG. 1A), cc3(FIG. IB), ct6(FIG. 1C), and P4(FIG. ID) shows prominent staining of epithelium (<—) and microvessels (<—) without significant stromal staining (*) for ct2, cc3, and P4. Note basolateral staining cc6 and basal staining for P4. Staining for fibronectin receptor subunits cc4(FIG. IE), oc5(FIG. IF) show predominant mesenchyme staining (*) with decreased epithelial staining (<—). The immunoreactions (areas of dark staining) were developed by avidinbiotin-peroxidase complex using diaminobenzidine as a chromogen. For greater sensitivity, no counterstain was applied. Magnification: 125x.
FIGS. 2A-2C show photomicrographs of the immunohistochemical staining for the integrin subunit ay in proliferative vs. secretoryendometrium. The staining in the glandular epithelium (<—) was largely absent in the proliferative phase (FIG. 2A), and pronounced in all sections after menstrual cycle day 14 (FIG. 2B; day 20 endometrium). The microvasculature (<—) staining was also pronounced, and did not change throughout the menstrual cycle. The staining noted in secretory endometrial glands was significantly higher than that of background (FIG. 2C). Magnification: 125x.
FIGS. 3A-3D exhibit immunostaining of av and P3 (the two pairing subunits of the vitronectin receptor integrin) in proliferative phase vs. secretory phase endometrium. The staining intensity of a,, in the proliferative phase (FIG. 3A) was judged as "+" for the stromal cells (*) and "+" for glandular av (<—). Immunostaining for o^, in day 22 endometrium (FIG. 3B) demonstrates a significant increase in glandular staining (example of "++" staining intensity). Likewise, the staining for p3 was absent in proliferative epithelium (FIG. 3C; <-) and was notably increased in this
