EP0513345A1 - Therapeutic and diagnostic applications of fetal fibronectin - Google Patents
Therapeutic and diagnostic applications of fetal fibronectinInfo
- Publication number
- EP0513345A1 EP0513345A1 EP92904308A EP92904308A EP0513345A1 EP 0513345 A1 EP0513345 A1 EP 0513345A1 EP 92904308 A EP92904308 A EP 92904308A EP 92904308 A EP92904308 A EP 92904308A EP 0513345 A1 EP0513345 A1 EP 0513345A1
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- European Patent Office
- Prior art keywords
- ffn
- fetal fibronectin
- mammal
- tissue
- fibronectin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to the field of mammalian reproduction and in particular, the invention relates to therapeutic and diagnostic applications through the manipulation of the protein, fetal fibronectin (FFN) . More particularly, therapeutic and diagnostic applications relating to fertility enhancement, contraception and contragestion are provided.
- FFN fetal fibronectin
- Infertility in humans is defined as one year of unprotected coitus without contraception. Although approximately 10-15% of couples are affected by infertility, the risk of infertility is doubled for women between the ages of 35 to 44 as compared to women between the ages of 30 and 34. Changes in fertility patterns such as result from increased maternal age, will have a significant impact on the make-up of populations. It has been calculated that, without immigration, the population in the United States will decline by the middle of the next century. Furthermore, the percent of people over the age of 65 will increase to over 23% in the next 100 years, resul ⁇ ting in an older and smaller work force.
- HSG hysterosalpolingogram
- hysteroscopy the direct examination of the uterus by a fiber optic device, can identity endometrial polyps, submucous leiomyomas M ⁇ llerian anomalies, and other abnormalities within the uterus itself.
- Another category of diagnostic procedures includes examination of ovarian function including ovulation and the secretion of progesterone during the luteal phase of the menstrual cycle.
- Ovarian function can be crudely assessed by measuring basal body temperatures during the menstrual cycle and cervical mucous testing around the time of ovulation. More accurate testing can be performed by measuring luteinizing hormone, a pituitary hormone which induces ovulation after a mid-cycle surge. Finally, serum progesterone levels can be measured to assess the adequacy of the luteal phase of the menstrual cycle.
- luteal phase defects In order to rule out inadequate ovarian progesterone effects in secretory endometrium, so-called luteal phase defects, the endometrium itself has been directly assessed by performing an endometrial biopsy three days before the suspected onset of menses. Endometrial biopsies are examined by hematoxylin and eosin staining of paraffin embedded specimens. For infertility patients, the reading of these biopsies may provide clinically useful information about the adequacy of the luteal phase, and other potential data, such as the presence of infection, inflammation, or neoplasia of the endometrium.
- the infertility patient could undergo endoscopic examination through an incision in the abdomen to directly visualize the external surfaces of the ovary, fallopian tubes and uterus, therefore identifying any gross pathology which was not detected by previous examinations.
- Endometriosis is associated with severe dysmenorrhea, and can result in infertility and debilitating pelvic pain.
- Mullerian anomalies such as unicornate and bicornate uteri can be associated with infertility and recurrent pregnancy loss.
- diagnosis of these abnormalities frequently requires operative procedures such as hysteroscopy and laparoscopy.
- a non-invasive method of delineating the anatomic distribution of functional Mullerian epithelium and thus anatomy of these structures would be clinically useful.
- In vitro fertilization (IVF) requires the removal of ova from a mammalian ovary, and exposure of these ova to sperm outside the body. Fertilization of each ovum requires that at least one living sperm penetrates the zona pellucida (outer covering) of the ovum and fuses with the pronucleus.
- the first is gamete intra-fallopian transfer (GIFT)
- the second is zygote intrafallopian transfer (ZIFT)
- GIFT gamete intra-fallopian transfer
- ZIFT zygote intrafallopian transfer
- the retrieved oocyte and sperm are mixed together and placed back into the fallopian tube where fertilization takes place.
- the fertilized zygote then travels down through the fallopian tube into the endometrial cavity, where implantation may or may not take place.
- the ZIFT procedure allows for fertiliz- ation to take place in vitro as in standard IVF, the fertilized zygote then is placed back in the fallopian tube where it travels down into the uterus to implant.
- hyper-stimulation protocols necessary to retrieve many oocytes from the donor women may have deleterious effects on the endometrium itself and decrease the rates of implantation.
- Two basic procedures have been utilized to help overcome this problem. The first is non-stimulated oocyte retrieval. A single egg is retrieved, allowed to be fertilized and placed back into the fallopian tube or uterus for implantation.
- the other technique involves the hyperstimulation portion of the IVF procedure to retrieve the eggs and allow for fertilization in vitro. The zygote is then frozen to be placed back into the patient after several normal cycles, with the hope that the endometrium will then be more receptive to implantation.
- Risk determination based on a combination of MSAFP value and maternal age allows for the detection of a third of DS fetuses while necessitating karyotype analysis in 5% of pregnant patients under 35. Wald et al., "Maternal Serum Screening for Down's Syndrome in Early Pregnancy", Brit. Med. J. (1988) 297:883-887.
- ectopic pregnancies and metastatic gestational trophoblastic disease can be challenging.
- the manifestations of tubal pregnancy include amenorrhea, vaginal spotting or bleeding, abdominal or pelvic pain and the presence of a pelvic mass.
- Laboratory testing for suspected ectopic pregnancy includes hemoglobin and hematocrit, white blood cell count, urine and serum human chorionic gonadotropin (HCG) pregnancy test, ultrasound including vaginal probe ultrasound, culdocentesis (a diagnostic procedure to detect blood in the peritoneum) , curettage of the endometrium to rule out the presence of products of conception within the uterus, laparoscopy and finally in emergency cases, laparotomy.
- HCG human chorionic gonadotropin
- the current modalities for detecting metastatic gestational trophoblastic disease include serum HCG level determination, chest X-ray, pelvic ultrasound, magnetic resonance imaging (MRI) computerized tomography (CT) scan of the abdomen, pelvis and head. Like in other solid tumors, small metastases can be missed by these procedures. The knowledge of the presence of metastases is critical for the successful treatment of these and other tumors. Therefore, a method which can localize and detect small quantities of trophoblast tissue would be very helpful for the management of this disease.
- contraceptive techniques include oral steroidal contraceptives, injected or implanted steroidal contraceptives, intrauterine devices, physical, chemical or physiocochemical barrier techniques, withdrawal, sexual abstinence around the time of ovulation, breast feeding and permanent sterilization.
- oral steroidal contraceptives In addition to the high failure rates of some of these methods, a number of these methods have serious pote ⁇ ntial complications for the users.
- metabolic changes induced by some oral contraceptives there is a potentially increased risk of untoward cardiovascular effects and thromboembolisms in susceptible patients.
- many of these techniques are expensive and require meticulous medical surveillance, and as such they are not applicable to poorer patients in the third world and underprivileged patients in the developed world.
- the present invention provides methods of regulating the reproductive potential of a mammal by regulating the amount of FFN to which the mammal's cells and/or tissues are exposed. Also provided are methods of monitoring the reproductive potential of a mammal by detecting the presence of, quantitating the amount FFN, or determining the ratio of total fibronectin to FFN to which the mammal's tissues are exposed. Further provided by the invention are methods of imaging and/or treating tissues expressing or exposed to FFN.
- the present invention provides methods of diagnosing fertility problems, methods of enhancing fertility and methods of decreasing fertility potential.
- FFN plays a significant role or roles in the mammalian reproductive process. While the inventor has developed certain theories regarding the role(s) thus played, the usefulness of the diagnostic and therapeutic methods disclosed herein do not necessarily depend upon the accuracy of any given theory. Indeed, certain theories may not be entirely consistent with one another.
- FFN has been determined to be localized in various reproductive tissue, including the implantation zone of the uterus. This would seem to indicate that FFN plays a role, and perhaps a substantial or critical one, of promoting or permitting the appropriate binding of cells, and in particular, the fertilization of the ova and the implantation of the blastocyst.
- FFN may enhance or control sperm-ova transport and/or contact, the adhesion of the blastocyst to the endometrial epithelia and the adherence of the placenta and membrane to the endometrial decidua, and may thus be a critical factor in determining whether fertilization and implantation occur and the manner and timing of placental and membrane separation, whether by way of premature abortion or full term delivery.
- the unique glycosylation properties of FFN might serve to enhance cell and ECM binding affinity with or without altering proteolytic susceptibility when compared to adult forms of fibronectin. Therefore enhancement of seminal, follicular, blastocyst or endometrial FFN synthesis could facilitate fertilization, implantation and placentation, while inhibition of FFN synthesis could serve as a contraceptive or contragestational agent.
- FFN might serve to inhibit inappropriate cell binding (e.g. tubal implantation) and thus promote fertilization or the pre-implant tion transport of the blastocys . -11-
- FFN FFN
- seminal and Mullerian tract epithelial FFN secretion could prevent sperm binding to the endocervix, endometrium or tubal epithelium prior to fertilization, thus enhancing the latter's probability of occurrence.
- tubal epithelial and follicular FFN could act to prevent oocyte adherence to the tubal epithelium with consequent failure of fertilization or, in the event of fertilization, ectopic implantation.
- endometrial glandular epithelial FFN synthesis and secretion in the very early luteal phase could prevent premature blastocyst adherence or implantation prior to the period of optimal endometrial morphological and biochemical receptivity (menstrual days 19-21) .
- Analo ⁇ gously, localization of FFN during pregnancy to the sites of future placental and membrane separation could facilitate separation of the placenta and membranes following delivery at term.
- reduced FFN cell and ECM binding affinity together with increased proteolytic susceptibility could facilitate preterm delivery secondary to either contractions or inflammation.
- FFN a FFN "lubricant”
- a FFN "lubricant” would serve to lessen the probability that the fertilized or unfertilized ova would implant prematurely, e.g. in the fallopian tube. It would further appear that an FFN "lubricant” would retard the premature implantation of the blastocyst in the uterus.
- FFN might also serve as a binding agent to promote trophoblast-decidual contact and the formation of placental and membrane connections to the uterus sufficiently strong to maintain pregnancy but sufficiently tenuous to facilitate expulsion of the placenta and membranes following expulsion of the fetus.
- FFN may serve one of two dissimilar functions i.e., either the enhancement or inhibition of cell-cell or cell-ECM bonding — or may serve both functions at different locations and/or times — e.g. retardation of cell binding in the Mullerian tract, and promotion of implantation in the uterus.
- the invention provides a novel method of monitoring the fertilization potential of a mammal by analyzing samples such as a semen sample or the post-coital cervico-vaginal mucus or follicle aspirates from a mammal to determine the relative likelihood that pre-coital or post-coital semen from that mammal is capable of fertilizing an ovum from a female mammal for the same species.
- samples such as a semen sample or the post-coital cervico-vaginal mucus or follicle aspirates from a mammal to determine the relative likelihood that pre-coital or post-coital semen from that mammal is capable of fertilizing an ovum from a female mammal for the same species.
- Other types of tissue which may be sampled include, but are not limited to, serum, sperm, ova, endometrium, placenta, uterine fluid and follicular fluid.
- the method of this invention provides information as to whether the mammal is producing FFN and in appropriate amounts for optimal fertilization potential.
- the method includes providing at least one sample of semen or post-coital or cervico-vaginal mucus and assaying the sample for FFN, whereby a relative change in the co ⁇ ncentration of FFN in said sample as compared to normal levels of FFN or the ratio of FFN to adult fibronectin in fertile mammals of the same species is indicative of the fertilization potential.
- Concentrations of FFN in follicular fluid are also substantial and may be correlated with fertility.
- the invention also provides a diagnostic procedure for ultrasound guided aspiration of follicles with measur ⁇ ement of FFN concentrations of follicle aspirates related to fertile mammals of the same species to determine the likelihood of fertilization. Similar comparison of FFN concentrations to adult fibronectin concentrations can also be utilized for assessing fertility potential.
- the invention further provides a novel infertility and recurrent abortion screening test comprising assaying at least one bodily fluid or cell type from a mammal suspected of being infertile or of having unexplained recurrent abortions for the presence of FFN autoantibodies.
- the association of the protein FFN with normal mammalian reproduction has now been reco- gnized.
- the method of this invention provides a tool for diagnosing mammals with an autoimmune infertility.
- This invention further provides a novel method for altering the fertility potential of a mammal by regulating the level of fibronectin in mammalian reproductive tract tissues or secretions.
- Increases in FFN can result from the infusion of exogenous FFN in semen, follicular fluids or onto tissues or by increasing the level of endogenous FFN by the use of pharmaceutically acceptable substances.
- the probability that a conceptus will become implanted in a mammalian uterus is altered by regulating the amount of FFN in the uterine cavity at about the time the uterus will be contacted with the conceptus.
- FFN has been localized in at least several locations including the implantation zone where trophoblastic cells make contact and attach to the uterus and within the chorionic membrane where trophoblastic cells make direct contact with the maternal decidua.
- decidualized endometrial stromal cells and endometrial glands synthesize and secrete FFN in vitro.
- the method of this invention changes the local environment of the surface of the endometrium at about the time the uterus will be contacted with the conceptus thereby altering the chance that a conceptus will become adherent and/or subsequently enhances FFN synthesis following implantation in the uterus.
- this invention is a novel method of regulating FFN synthesis so as to modify the probability of fertilization, blastocyst adhesion to the endometrium and/or implantation by regulating FFN synthesis in the mammalian uterus, ovarian follicle or seminal vesicle.
- the method comprises administering to said mammal a compound which regulates FFN synthesis by mammalian endometrial glands in culture. It has now been found that estrogen plus progesterone inhibit FFN synthesis in cultured endometrial glandular epithelia whereas the same hormonal treatment maintains FFN synthesis by cultured endometrial stromal cells. Either estrogen or progesterone alone inhibit stromal cell FFN synthesis.
- the invention includes a method of regulating FFN synthesis by administering systemically or topically a therapeutically effective amount of putative paracrine effectors which are known regulators of fibronectin synthesis such as transforming growth factor ⁇ (TFG-3) .
- putative paracrine effectors which are known regulators of fibronectin synthesis such as transforming growth factor ⁇ (TFG-3) .
- hormones including but not limited to estrogen, estrogen analogues, antagonists or agonists, progesterone analogues, antagonists or agonists can also be used, depending on the desired clinical effects.
- Analogous treatment can be administered to preovulatory women to regulate follicular FFN production.
- paracrine and/or hormonal manipulation can regulate seminal vesicle FFN production in the male. The importance of the protein FFN in reproduction has now been recognized; thus regulating the production of FFN by the method of this invention, provides inter alia methods of contraception and fertility enhancement.
- anti-FFN antibodies bind to FFN expressed by the mammal effectively decreasing the amount of available or free FFN regulating the ability of the sperm to fertilize the ova or the conception to implant.
- reproductive potential is regulated by administering to the mammal a FFN antigen in an amount sufficient to raise antibodies to FFN, whereby, for example, the probability that sperm will reach the fallopian tube, or fertilization will occur or that an adherent blastocyst will successfully migrate to the uterus so as to implant.
- the method comprises administering to a mammal a labeled antibody specific for FFN wherein the label is not radioactive so as to avoid irradiating reproductive tissues.
- the label has paramagnetic properties such that the label can be detected with MRI.
- the labeled antibody is used in an amount sufficient to bind to said trophoblastic tissue in an amount sufficient to detect the label bound to said FFN antibody, whereby the location of said trophoblastic tissue is dete ⁇ rmined.
- the amount of labeled antibody used and the port of entry vary according to the tissue to be imaged. For instance, in the case of suspected ectopic pregnancies or malignancies the port of entry may be by direct infusion into the bloodstream.
- the method comprises administering to a mammal a labeled antibody, wherein the label is not radioactive.
- the label has paramagnetic properties and is administered systemically or infused into the cervix and thence uterus, fallopian tubes and abdomen. For example, endometriosis could be identified in patients without resorting to operative diagnostic procedures. In the case of imaging of the uterus or other organs, direct topical application may be appropriate. As the concentration of the labeled antibody increases, the greater the nonspecific binding becomes allowing visualization of larger areas of tissue to determine for instance organ abnormalities along the Mullerian tract.
- Figure 1 is a set of graphs depicting immunoreactive FFN (ir-onfFN) , immunoreactive total FFN and immunoreactive prolactin in the media of cultured endometrial stromal cells treated as indicated in the figure.
- FFN Fetal Fibronectin
- FDC-6 refers to the monoclonal antibody defining the oncofetal structure of fibronectin as disclosed in U.S. Patent No. 4,894,326. U.S. 4,894,326 is incorporated by reference as if fully set forth herein.
- U.S. 4,894,326 discloses an IgGl monoclonal antibody (FDC-6) which defines a fibronectin structure or structures located between the "Hep-2" and the "Fib-2" domains in the COOH-terminal region of fibronectins isolated from hepatoma, sarcoma, and fetal fibroblasts. It is disclosed that this antibody indicates the presence of two classes of human fibronectin. First, fibronectin from fetal connective tissue, placenta, amniotic fluid, hepatoma and colon carcinoma as well as cell lines from fetal tissue hepatomas and sarcoma was characterized by the presence of the structure recognized by FDC-6 and by a slightly higher molecular weight.
- FDC-6 IgGl monoclonal antibody
- fibronectin from normal adult tissues and plasma was characterized by a slightly lower molecular weight and lack of reactivity with FDC-6, and is therefore devoid of the FDC-6 defined structure.
- the FDC-6 defined structure is referred to as the "oncofetal structure", or epitope and fibronectin containing this structure has been called “oncofetal fibronectin.”
- Fibronectin from most normal adult tissues and plasma lacking the oncofetal structure is characterized as "normal fibronectin.”
- Development of fibronectin from fetal to adult forms is disclosed to be associated with loss of the oncofetal structure defined by the FDC-6 antibody, and oncogenic transformation is disclosed to be associated with activation in synthesis of the oncofetal structure defined by the FDC-6 antibody.
- FDC-6 antibody expressed in oncofetal fibronectin is a useful marker of cancer, it is disclosed that the FDC-6 antibody and other antibodies raised against the oncofetal fibronectin structure will be useful for diagnosing human cancer and for monitoring and implementing various cancer treatments.
- the monoclonal antibody FDC-6 a product of hybridoma cell line ATCC No. HB9018, is suitable for use in this invention, as a specific probe for a variety of immunoassays such as, immunohistochemical localization, ELISA assays and immunoblot analyses.
- FDC-6 can be prepared substantially as described in U.S. 4,894,326. Briefly, the murine monoclonal antibody FDC-6 was established following immunization of mice with fibronectin isolated from human hepatoma cells. The hybridoma ATCC No.
- HB9018 was selected by positive reactivity of its antibody (FDC-6) with fibronectins from hepatoma, fibrosarcoma, and fetal fibroblasts and by negative reactivity with fibronectin from plasma.
- FDC-6 antibody
- Other antibodies recognizing trophoblastic and/or pregnancy-specific decidual ECM components are expected to be useful in this invention. It has now been found that FFN is present at sites of fertilization and implantation. Specifically, FFN is present in both seminal and follicular fluid and along the Mullerian tract in the apical cytoplasm and surface of epithelial cells indicating a role in fertilization. FFN has also been found in Mullerian, epithelial and trophoblastic tissue indicating a role in gamete, zygote and blastocyst transport as well as implantation and maintenance of pregnancy.
- FFN is produced by the method of this invention by recombinant DNA techniques and subsequent protein purification.
- a preferred method of protein purification is affinity column chromatography using FDC-6 as the affinity ligand.
- Recombinant DNA technology allows for production of large quantities of FFN as compared to endogenous production which in the case of cultured trophoblast tissue FFN synthesis decreases to minimal levels after 72 hours.
- the protein thus obtained can be subject to modifications such as altered glycosylation patterns or alterations in the amino acid sequence that can be affected by a variety of recombinant DNA techniques. Glycosylation can be achieved by selection of a particular host cell or by chemical modification after protein synthesis has taken place.
- oligonucleotide-directed site-specific mutagenesis can be used to alter various aspects of the protein. For instance specific activity can be changed by amino acid substitutions, deletions and insertions distant from the FFN epitope which result in either enhanced or reduced cell binding affinity respectively and thus either promote or inhibit respectively fertilization, implantation and placentation.
- the DNA sequence which is to be mutated must first be cloned into a vector which is capable of being replicated in both single-stranded and double-stranded forms.
- Appropriate vectors include the M13 cloning vectors which can be purified as either single-stranded bacteriophage or as double-stranded plasmids.
- a single-stranded oligodeoxyribonucleotide is then synthesized to contain both the desired mutation and a number of flanking nudeotide bases complementary to and sufficient to anneal to the single-stranded form of the DNA sequence to be mutated.
- the synthetic oligodeoxyribonucleotide is then annealed to the single-stranded vector and a new strand of DNA is synthesized by DNA polymerase using the synthetic oligodeoxyribonucleotide as the primer and the single-stranded vector as the template.
- the new double-stranded heteroduplex vector now contains one non-mutated strand and one mutated strand.
- the heteroduplex vector is then transfected into suitable host cells which replicate and amplify the double-stranded form of the vector. Due to semiconservative replication, some of the transformed progeny will contain vectors with only the mutant DNA, some will contain vectors with only the non-mutant DNA and others will contain both mutant and non-mutant vectors.
- the cells containing only the mutant vectors must then be selected from all others.
- the selection procedure most often used consists of radiolabeling the synthetic oligodeoxyribonucleotide used in the mutagenesis and hybridizing it to the transformed cells under stringent conditions. Cells which contain the mutant vector will retain the radiolabeled probe and become radioactive under such conditions. The cells carrying the mutant can therefore be distinguished from the non-mutant, non-radioactive cells. Cells containing both mutant and non-mutant vectors must be separated from those containing only mutant vectors necessitating another round of selection. The mutation must then be confirmed by sequencing. Further, since the DNA polymerase may have incorporated other mutations into the vector, the specifically mutated DNA is usually recloned into a new vector which has not been subject to the mutagenesis procedure.
- the unique glycosylation pattern of FFN may alter its susceptibility to proteases. Given the anatomic distribution of FFN in pregnancy (see Ex. 1) , such an enhanced susceptibility to proteolysis could contribute to preterm rupture of the membranes or premature placental separation (abruption) .
- the identification of specific proteases which selectively cleave FFN allows appropriate pharmacological interventions in order to prevent these untoward pregnancy events.
- Recombinant DNA techniques also allow for production of protein fragments and fusion proteins.
- recombinant DNA encoding FFN would be expressed in mammalian cell lines so as to conserve the native FFN glycosylation patterns. It is thought that the glycosylation pattern is responsible for FFN activity.
- FFN identified by the FDC-6 monoclonal antibody differs from adult fibronectin moieties only by the presence of a ⁇ -N-acetylgalactosamine at the threonine residue within the hexapeptide Val-Thr-His-Pro-Gly-Tyr sequence located within the IIICS region.
- Trophoblast derived ECM components can be extracted from the ECM deposited by trophoblasts in culture.
- endometrial glandular or decidualized stromal - specific ECM components can be deposited by glands and decidualized stromal cells in culture.
- This trophoblastic-specific or glandular- or decidual-specific ECM can be injected into mice or other animals for the generation of an antibody response and the preparation of many different polyclonal or monoclonal antibodies to these ECM components by known techniques. See e.g., "Antibodies, A Laboratory Manual," Cold Spring Harbor (1988), which manual is incorporated by reference as if fully set forth herein.
- anti-trophoblast- or decidualized stromal- or glandular-ECM-antibodies are generated, as described above, those antibodies which specifically bind to trophoblast- specific ECM or decidualized stromal- or glandular-specific ECM, but not other adult ECM components, can be identified.
- Measurement of secreted FFN in reproductive fluids is expected to be an important diagnostic test for a variety of conditions. Males and females with abnormal levels of FFN diagnosed by these methods can then be candidates for FFN therapy or pharmacologic manipulations to regulate FFN synthesis, as described in detail below.
- a method of diagnosing the fertilization potential of semen, post-coital or follicular fluid samples from a mammal to determine the relative likelihood that semen from that mammal is capable of fertilizing an ovum from a female mammal of the same species comprising the steps of providing at least one sample of semen or cervico-vaginal secretion or ovarian pre-ovulation follicular fluid; and assaying the sample for FFN, as described above, whereby a relative change in the concentration of FFN in said semen, post-coital or follicular fluid sample as compared to normal levels of FFN in fertile mammals of the same species is indicative of an altered fertilization potential.
- the amount of FFN present in the sample as compared with the quantity of FFN in a fertile control semen, post-coital or follicular sample can thus, e.g., be used to evaluate the fertilization potential of the sample in cases of unexplained male or female factor infertility. Additionally, males with abnormal levels of seminal FFN or couples with abnormal cervico-vaginal FFN levels as diagnosed by this method can then be considered as candidates for FFN therapy or pharmacologic manipulations to modulate FFN synthesis, as described in detail below. Further, the information provided by the method of this invention is useful in selecting semen and follicular samples more suitable for assisted reproduction technologies, e.g., selecting a semen sample with altered FFN levels for use in an in vitro fertilization procedure.
- the methods of this invention wherein FFN is assayed or imaged in reproductive fluids of non-pregnant females and semen and cervico-vaginal mucus, are then generally expected to be useful as diagnostic screens for mammals suspected of being infertile. Further, these assays or imaging techniques have important applicability for mammals undergoing assisted reproductive technologies in more precisely optimizing attempted fertilization.
- These methods of detecting the presence of FFN in endometrial, fallopian, cervical, vaginal, or other fluids of non-pregnant females permit optimizing transfer of sperm or fertilized or unfertilized ova to the uterine cavity or fallopian tube as judged by FFN levels in normal controls.
- Such assays or imaging techniques are expected to be easy, non-invasive ways to assess levels of FFN in mammals.
- a method to image potentially pathological trophoblastic tissue as well as physiological or pathological Miillerian tract epithelium in a mammal is administered an effective amount of an antibody specific for FFN, whereby the antibody is conjugated to a non-radioactive label.
- the labelled antibody is introduced into the mammal either by direct injection into the blood stream or by topical application such as douches or rinses.
- the labelled antibody couples to FFN and is imaged by an appropriate medical imaging device such as MRI.
- nonradiolabeled antibodies are the method of choice for imaging and treating tissue expressing FFN.
- MRI is used as the imaging method.
- FDC-6 is a suitable antibody for use in this invention.
- Other FFN antibodies prepared as described above are also expected to be suitable for use in the invention.
- trophoblastic tissue to be localized examples include but are not limited to ectopic pregnancies, placenta previa, other placental abnormalities and metastatic gestational trophoblastic disease and primary ovarian choriocarcinoma and other germ line tumors producing FFN as confirmed by evaluation of these malignant tissues.
- endometriosis or Mullerian tract anomalies can be similarly imaged.
- Suitable labels include but are not limited to paramagnetic labels such as microspheres with paramagnetic impurities or proteins with paramagnetic ligands.
- infertility and recurrent pregnancy loss screening tests are provided. At least one bodily fluid or cell type from a mammal suspected of being infertile or of having recurrent abortions is assayed for the presence of FFN autoantibodies.
- the presence of autoantibodies that bind to FFN can be assayed by basic enzyme-linked immunoassay (ELISA) (where purified FFN is immobilized on a plastic surface, and the subject's bodily fluid, e.g., serum, plasma, cervicouterine secretion, or seminal fluid, is applied to these wells in various dilutions) , or immunoblot techniques.
- ELISA basic enzyme-linked immunoassay
- a positive test would occur by using as a marker a secondary anti-human (IgM, IgA and IgG antigen) antibody, which would only bind in the assay if the patient's bodily fluids contained an anti-FFN autoantibody.
- Bodily fluids expected to be useful include but are not limited to plasma, serum, semen, follicular fluids, (peritoneal fluids) cervico-vaginal secretions and cervicouterine aspirates. Generally, any bodily fluid or cell type associated with FFN in a fertile control are believed to be useful.
- Certain causes of previously unexplained infertility are believed due to an autoimmune process leading to endogenous production of anti-FFN antibodies.
- this autoimmune process is believed to prevent the proper biological function of FFN, causing sperm aggregation, impeding fertilization and/or implantation or gamete transport and playing a role in repetitive miscarriage later in pregnancy.
- the first step in treating mammals with autoimmune infertility is a screening method to identify this group. Once identified, these patients might benefit from specific treatments used for patients with autoimmune diseases, such as immunoglobulin or immunosuppressive therapy.
- a method of altering the probability that gametes will fertilize or that a conceptus will become implanted in a mammalian uterus is provided.
- FFN is infused into the uterine cavity prior to coitus or about the time the uterus will be contacted with a conceptus, said infusing introducing a sufficient amount of FFN onto the surface of the uterine cavity to alter the probability that fertilization or implantation will occur.
- the infusion will be given at the time a uterus will be contacted with a conceptus e.g., at about the time of presumed implantation during a natural cycle of conception.
- agents found to increase endometrial glandular FFN synthesis can be employed (vide infra) .
- FFN could be infused in a variety of ways as are other agents for introduction into the uterus.
- FFN could be in a dissolved form, either in solution, within a gel, or in a slow release capsule to allow for an appropriate time-dependent concentration in the uterine cavity.
- An effective amount of FFN is that amount of FFN which when introduced onto the surface of the uterine cavity alters the probability that gamete fertilization or implantation of a conceptus will occur. Concentrations of FFN in the range from about 0.1 ⁇ g/ml to about 1 mg/ml are expected to be useful, since FFN concentrations in human reproductive fluids fall in this range.
- FFN is also present in sperm, semen and follicular fluid, it is believed to play a critical role in fertilization. Therefore therapeutic regimens utilizing FFN, e.g., the addition of FFN to a sample of semen intended for use in artificial insemination or into the vagina at the time of coitus, are believed applicable in helping to overcome certain male or female infertility problems due to insufficient FFN levels. Prior to/or following coitus FFN could be administered directly into the endometrial cavity to promote sperm migration or subsequent fertilization.
- Literature is known describing intrauterine infusions, gels or sponges for the treatment of a variety of conditions.
- putative stromal-epithelial paracrine effectors such as TGF-3, or ECM components are expected to be useful for enhancing FFN synthesis in mammalian Mullerian epithelium including endometrial glands. Endometrial glands in culture, within six days constitutively produce FFN de novo. while administration of estradiol and progesterone inhibits synthesis. Therefore, putative paracrine effectors and those compounds in plasma, ECM extracts, or maternal decidua which induce or inhibit FFN synthesis can be readily identified in the in vitro endometrium culture assay described herein. These may be employed in the method of this invention in an amount sufficient to augment or alter FFN synthesis in a mammal.
- Such compounds are expected to include steroids, peptides, and glycopeptide hormones, gonadotropins, growth factors, cytokines, antibodies, as well as portions of ECM proteins including other fibronectins, laminin, collagen types I and IV, vitronectin, or proteoglycans.
- FFN synthesis can be modified in a mammal about the time the uterus of the mammal will be contacted with a conceptus whereby altering the probability that the conceptus will implant in the uterus.
- a method of regulating FFN synthesis in a mammal comprising administering to said mammal a pharmaceutically acceptable compound which regulates FFN synthesis by mammalian Mullerian epithelial cells and other reproductive tract tissue in appropriate amounts.
- Compounds which regulate FFN synthesis by mammalian endometrial glands in culture can be selected from the group consisting of estrogen, estrogen analogues and progesterone inhibitors and antagonists.
- Modification of FFN synthesis in a mammal has a variety of utilities. For example, an infertile mammal determined to have a level of FFN different from that of a normal fertile control, may be a candidate for FFN synthesis modification. In contrast, FFN can be employed to achieve a method of contraception.
- Pharmacologic manipulation to yield a method of contragestion can also be achieved, as previously described, by introducing estrogen, estrogen analogues and progesterone inhibitors and antagonists to the local environment of the uterus through a suppository, gel or sponge or by direct systemic treatment. These manipulations are expected to be effective both for intrauterine and ectopic implantations, the majority of which are intratubal implantations.
- Pharmacologic manipulation of FFN is expected to be a useful non-surgical, non-invasive alternative to treatment of chromosomally abnormal pregnancies incompatible with life, missed abortions, incomplete abortions or for other indications. Termination of tubal ectopic pregnancies could occur by either systemic or direct installation of the pharmacologic agent to the tubal pregnancy via laparoscopic, ultrasonic, or retrograde cervicouterine irrigation.
- a method of decreasing the reproductive potential of a mammal is provided.
- the reproductive potential of the mammal is decreased by administering to the mammal a FFN antigen with or" without adjuvant in an amount sufficient to raise antibodies to FFN, whereby the probability that gametes will meet and fertilize or a conceptus will migrate to the uterus is decreased.
- FFN appears in the Mullerian tract epithelia, follicular fluid and trophoblasts during pregnancy.
- a novel method of permanent female sterilization based on FFN immunization is provided.
- FFN is present in the seminal fluid, it is believed that immunizing men or women against FFN will induce antibodies specific for unique regions of FFN, and that such antibodies should not have general systemic effects.
- These antibodies would bind to FFN secreted by the mammal e.g., Mullerian epithelia, follicular or trophoblastic cells, as well as the seminal fluid FFN.
- the presence of these antibodies may cause sperm agglutination or adherence to Mullerian epithelial cells, alter sperm motility, inhibit ovulation, impair ovum transport, prevent fertilization, and alter the potential for implantation by the blastocyst or otherwise induce sterilization.
- the presence of these antibodies is expected to prevent or impair attachment of the developing trophoblast to the endometrial decidua, thus ending the gestation.
- Male or female mammals could be immunized against FFN by using the whole molecule or just the IIICS domain of FFN with the proper glycosylation moieties.
- the protein can be dissolved at between about 1 to 50 ⁇ g/ml in sterile saline or saline with 0.4 mg aluminum hydroxide per ml as a vehicle.
- 0.5 to 1.0 ml of the protein solution is injected intramuscularly and then followed by booster injections at one and 6-12 months after the initial immunization.
- Such immunizations thus prevents antibodies from developing against portions of FFN in common with normal adult plasma or cellular fibronectin.
- a method of detecting the presence of chromosomally abnormal conceptuses during mammalian gestation comprising measuring maternal plasma concentrations of FFN from 6 to 20 weeks gestation. Approximately 5-10 ml of maternal blood is collected in a tube containing EDTA to produce a final concentration of EDTA of at least 5 mM. The plasma from this specimen is then collected and assayed for FFN concentrations by immunoassay or radioimmunoassay.
- the inventor has identified FFN in the cervical and vaginal secretions of patients with uncomplicated pregnancies prior to 21 weeks gestation. Cervico-vaginal FFN was rarely identified in patients with uncomplicated pregnancies between 21 and 37 weeks gestation. However, when present at this gestational age interval cervico-vaginal FFN was frequently associated with preterm delivery. This latter finding promoted the inventor to characterize the tissue source of FFN during pregnancy. Placentae, decidua and external membranes for immunohistochemical analysis were obtained from term pregnancies, a hysterectomy specimen and from elective first trimester abortions. Tubal ectopic pregnancy specimens with associate uterine decidual specimens were also obtained.
- Fetal tissue was obtained following midtrimester termin ⁇ ations.
- the presence and the localization of proteins recognized by FFN specific antibodies in non-gestational reproductive tract tissues was evaluated. Immunoperoxidase staining was performed according to the manufacturer's instructions, using an avidin-biotin peroxidase detection system (Vector Laboratories, Burlingame, CA) .
- Antisera were utilized at 4 ⁇ g/ml for FDC-6, a product of hybridoma cell line ATCC HB9018 (American Type Culture Collection, Rockville, MD provided by Adeza Biomedical, Sunnyvale, CA) , and undiluted for the negative rabbit and goat serum or control P3X63Ag8 mouse myeloma cell line supernatant. (American Type Culture Collection, Rockville, MD) .
- FFN FFN is secreted by the extravillous trophoblasts derived from the cytotrophoblastic shelf located in the decidua basalis and/or the decidual cells.
- the specific localization of FFN only to ECM in which both extravillous cytotrophoblasts and decidual cells are present suggests that paracrine interactions between these two cell types regulates placental FFN production.
- FFN FFN was identified in the cytoplasm of hepatocytes and renal collecting tubules and transitional epithelia.
- the presence of FFN in fetal kidneys may reflect excretion of a fibronectin fragment bearing the oncofetal epitope or synthesis of FFN in the genito-urinary tract.
- fetal urine is a source of the high levels of FFN present in the amniotic fluid since immunoassays of the urines of newborns failed to display evidence of FFN, despite high (12 ⁇ g/ml) cord plasma concentrations.
- Normal vagina no staining for FFN epitope, however, diffuse stromal staining for total fibronectin (tFN) .
- Normal cervix positive discrete juxta-luminal endocervical gland cytoplasmic staining for FFN in some patients' specimens, however diffuse stromal ECM staining for tFN.
- Tubo-ovarian complex no staining for FFN, but diffuse staining of stromal ECM for tFN.
- Normal fallopian tubes positive staining for FFN in the juxta-luminal cytoplasm of the tubal epithelium in most patients' specimens and diffuse staining of stromal ECM for tFN.
- Normal premenopausal endometrium a) Proliferative Phase: No FFN staining, but diffuse staining of stromal ECM for tFN.
- Secretory Phase - positive discrete juxta-luminal endometrial gland cytoplasmic staining, but only in tissues obtained from some patients in the early luteal phase.
- Menopausal endometrium no staining for FFN, diffuse staining of stromal ECM for tFN.
- Endometriosis of the colon intense specific staining for FFN in the juxta-luminal cytoplasm of the endometrial glands with diffuse staining of stromal ECM and histiocytes for tFN;
- Endometrial carcinomas and endometrial hyperplasia no staining for FFN, diffuse staining of stromal ECM for tFN.
- Endometriod tumor of the ovary no staining of the tumor for FFN, however, the fallopian tube in this specimen displayed an FFN staining pattern similar to #4 with luminal surface cytoplasmic staining of the tubal epithelium, throughout the specimen there was diffuse staining of stromal ECM for tFN.
- Squamous cervical carcinomas (moderate and poorly differentiated) and an endocervical adenocarcinoma: no staining for FFN, diffuse staining of stromal ECM for tFN.
- Mucinous cystadenoma of the ovary intense juxta-luminal staining of the epithelial cell cytoplasm for FFN with diffuse staining of both ovarian stromal ECM and a fibrin clot for tFN.
- Ovarian lymphoma no staining for FFN, whereas a normal lymph node demonstrated trace intracytoplasmic staining for FFN and tFN in macrophages the (the latter may represent a peroxidase artifact) .
- the cytotrophoblasts were cultured in Dulbecco's Modified Eagles' Medium (DMEM) containing 25 mM glucose and 25 mM HEPES (DMEMHG) supplemented with gentamicin (50 ⁇ g/ml) , glutamine (4 mM) , and 20% (v/v) heat-inactivated fetal calf serum.
- DMEM Dulbecco's Modified Eagles' Medium
- DMEMHG Dulbecco's Modified Eagles' Medium
- gentamicin 50 ⁇ g/ml
- glutamine 4 mM
- 20% v/v heat-inactivated fetal calf serum
- ELISA assay results showed that cell protein extract from freshly purified villous cytotrophoblasts contained barely detectable FFN on ELISA ( ⁇ 50 ng/mg cell protein) , in agreement with the negative villous staining for FFN in the placenta. A small quantity of FFN was present intracellularly after 24 h, (125 ng/mg cell protein) , suggesting that FFN synthesis had been initiated by the cultured cells. After 96 h, trophoblasts contained 18 fold more FFN (2200 ng/mg cell protein) than the 24 hour cells, representing 0.2% of total trophoblast cell protein. Thus cytotrophoblasts, while not synthesizing FFN in vivo r are induced in culture to produce significant FFN.
- follicular fluid obtained from mature follicles aspirated under ultrasound guidance during (IVF) oocyte retrieval was assayed for FFN and total fibronectin. Concentrations of 2.9 ⁇ g/ml and 24.6 ⁇ g/ml for FFN and total fibronectin, respectively, were observed. Thus follicular fluid from mature follicles appear to contain both adult and oncofetal forms.
- the original tissue specimen was formalin fixed, paraffin embedded, sectioned and stained with hematoxylin-eosin and assigned a menstrual date according to the criteria of Noyes et al., "Dating the Endometrial Biopsy", Fertil. Steril. (1950) 1:3-2.
- BM Basal Media
- SCS Basal Media
- E2 lOnM estradiol
- MPA 1 ⁇ M Medroxyprogesterone acetate
- E2 + MPA BM + 10% SCS with 0.1% ethanol (the vehicle control).
- the cultures were maintained in a 37°C 95% air 5% C0_ incubator. The medium was replenished every 4 - 5 days with the corresponding experimental or control medium.
- the conditioned medium was concentrated and assayed for levels of Prolactin (PRL) , (Amersham RIA) total fibronectin (Total Fibronectin Assay®, Adeza Bio edical) and (Romcheck®, Adeza Biomedical) FFN.
- PRL Prolactin
- Aminase inhibitors PI: soybean trypsin inhibitor, pepstatin, leupeptin, aprotinin, PMSF, all at a final concentration of 10 ⁇ g/ml
- the cell pellets were resuspended in HBSS + PI, and divided into aliquots to measure levels of protein by Modified Bradford Assay (Bio-Rad) , total fibronectin and FFN.
- Figure 1 shows the effects of E2, MPA and E2 + MPA on levels of PRL, fibronectin and FFN in the conditioned medium of primary stromal cell cultures, and normalized to protein content in the harvested cells.
- Virtually no PRL (Fig. 1A) was detected in control or E2-supplemented medium, whereas MPA elicited only a small increase in PRL levels.
- the combination of E2 + MPA produced a marked synergism in PRL output by the 5-9 days collection period with further increases evident by 13-17 days. This elevation in PRL production appears to be progesterone-regulated, while the action of E2 likely reflects maintenance or enhancement of progesterone receptors.
- Isolated endometrial glands were distributed among 16 Millicell (Millipore) chambers containing collagen I gels in BM + lTS + +2% SCS and grown under polarizing conditions. These were divided into control (+ 0.1% ethanol) and experimental (lOnM E2 + 1 ⁇ M MPA) groups and, placed in a 37°C incubator. After a 7 day incubation, the media were collected from all 16 chambers and pooled into control subgroups A and B, and experimental groups A and B, then concentrated and frozen. The cells in the A subgroups were harvested by collagenase I and the cell pellets stored frozen, whereas in the B subgroups, the medium was replenished with corresponding fresh medium and the cultures returned to the incubator.
- the medium from the B subgroup was collected and the cells harvested as above.
- the collected media were analyzed for FFN and total fibronectin content and the cell pellets were assayed for protein content (after subtracting collagen blank values ( ⁇ 8% of total cell protein) ) , FFN and total fibronectin content.
Abstract
Cette invention concerne des utilisations de la fibronectine foetale dans les domaines de la thérapie et du diagnostic pour surveiller et réguler le potentiel de reproduction d'un mammifère.The present invention provides uses of fetal fibronectin in therapy and diagnostics to monitor and regulate the reproductive potential of a mammal.
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US621780 | 1984-06-18 | ||
US62178090A | 1990-12-04 | 1990-12-04 |
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EP0513345A1 true EP0513345A1 (en) | 1992-11-19 |
EP0513345A4 EP0513345A4 (en) | 1994-09-14 |
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EP9292904308A Withdrawn EP0513345A4 (en) | 1990-12-04 | 1991-11-27 | Therapeutic and diagnostic applications of fetal fibronectin |
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EP (1) | EP0513345A4 (en) |
AU (1) | AU1150892A (en) |
CA (1) | CA2075300A1 (en) |
WO (1) | WO1992010199A1 (en) |
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GB0201273D0 (en) * | 2002-01-21 | 2002-03-06 | Isis Innovation | Fibronectin variants and screening methods |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0261798A2 (en) * | 1986-08-22 | 1988-03-30 | O'Neill, Christopher | Fertility control |
EP0316919A2 (en) * | 1987-11-17 | 1989-05-24 | Adeza Biomedical Corporation | Vaginal sample test and reagents |
US4894326A (en) * | 1986-04-09 | 1990-01-16 | Fred Hutchinson Cancer Research Center | Monoclonal antibody defining oncofetal structure of fibronectin |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
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US4442119A (en) * | 1980-07-07 | 1984-04-10 | The Board Of Regents For The University Of Oklahoma | Cyclopropyl analogs as estrogenic and anti-fertility agents |
AT385658B (en) * | 1985-03-28 | 1988-05-10 | Serotherapeutisches Inst Wien | METHOD FOR PRODUCING A FIBRONEKTIN SOLUTION SUITABLE FOR HUMAN USE |
US5084277A (en) * | 1988-08-30 | 1992-01-28 | Greco John C | Vaginal progesterone tablet |
-
1991
- 1991-11-27 CA CA 2075300 patent/CA2075300A1/en not_active Abandoned
- 1991-11-27 WO PCT/US1991/008986 patent/WO1992010199A1/en not_active Application Discontinuation
- 1991-11-27 AU AU11508/92A patent/AU1150892A/en not_active Abandoned
- 1991-11-27 EP EP9292904308A patent/EP0513345A4/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4894326A (en) * | 1986-04-09 | 1990-01-16 | Fred Hutchinson Cancer Research Center | Monoclonal antibody defining oncofetal structure of fibronectin |
EP0261798A2 (en) * | 1986-08-22 | 1988-03-30 | O'Neill, Christopher | Fertility control |
EP0316919A2 (en) * | 1987-11-17 | 1989-05-24 | Adeza Biomedical Corporation | Vaginal sample test and reagents |
Non-Patent Citations (4)
Title |
---|
FILE SERVER STN KARLSRUHE,FILE MEDLINE ABSTRACT NO.87126765 & ARCH BIOCHEM BIOPHYS, (1987 JAN) 252 (1) 1-6 * |
FILE SERVER STN KARLSRUHE,FILE MEDLINE ABSTRACT NO.91157960 & AM J PATHOL, (1991 MAR) 138 (3) 537-43 * |
FILE SERVER STN KARLSRUHE,FILE MEDLINE ABSTRACT NO.91333673 & N ENGL J MED, (1991 SEP 5) 325 (10) 669-74 * |
See also references of WO9210199A1 * |
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EP0513345A4 (en) | 1994-09-14 |
WO1992010199A1 (en) | 1992-06-25 |
AU1150892A (en) | 1992-07-08 |
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