CA2075300A1 - Therapeutic and diagnostic applications of fetal fibronectin - Google Patents

Abstract

Therapeutic and diagnostic applications are provided to monitor and regulate the reproductive potential of a mammal.

Description

W092/10l99 -207530~
PCT/VS9l/08986 Des~ription Therapeutic And Diagnostic Applications Of Fetal Fibronection :~.
Field of the Invention 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 5 fibronectin (FFN). More particularly, therapeutic and `~
diagnostic applications relating to fertility enhancement, contraception and contragestion are provided.
, Back~rou~d of the Invention 10In the field of mammalian reproduction, many diagnostic procedures exist to aid the obstetrician-gynecologist in making a diagnosis and choosing an appropriate course of action.
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 re~ult from increased maternal age, will have a significant impact on the make-up of populations. It has been calculated that, without immigxation, the population in the United States will decline by t:he middle of the next century.
Furthermore, the percent of people over the age of 65 will increaE,e to over 23% in the next 100 years, resul-ting in an older and smaller work force.
In the United States, 40% of infertility can be accounted for by problems in the male. There~ore, WO 92/1019g 7$ ~ U 9 ;; PCT/US 91/0X986.-semen analysis should be an early diagnostic step forinvestigating fertility. In evaluating male factor infertility, tests have been developed to assess the quality of a mammalian spe~ sample. Examples include monitoring sperm morphology and motility and assessment of the ability of sperm to penetrate zona free hamster ; ova and undergo an acrosomF- reaction. In addition, antisperm antibodies can be detected in semen, and ; maternal sera. Post-coital examination of the female partner has been used to evaluate the ability of sperm to reach and survive in the cervical mucus.
Evaluating a female for infertility can be co-mplex. Examination of the fallopian tubes is an important step in mammalian fextility evaluation due to the evidence of increase of pelvic inflammatory disease. Currently, a hysterosalpolingogram (HSG) is the procedure of choice to examine the patency of the fallopian tubes. In addition to HSG, hysteroscopy, the direct examination of the uterus by a fiber optic device, can identity endometrial polyps, submucous leiomyomas Mullerian anomalies, and other abnormalities within the uterus itself.
Another category of diagnostis~ procedures includes examination of ovarian function including ovuslation 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 tasting can be performed by measuring luteinizing hormone, a pituitary hor~one which induces ovulation after a mid-cycle surge. Finally, serum progesterone levels can be measured to assess the adequacy of the luteal pAase of the menstrual cycle.
In orcler to rule out inadequate ovarian progesterone effects in secretory endometrium, W0~2/10199 2 0 7 5 3 0 0 PCT/US91/08986 so-called luteal phase defects, the endometrium itself has been directly assessed by performing an endometrial biopsy three days before the ~uspected 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 ; 10 neoplasia of the endometrium.
Finally, 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. There are no current methods for establishing the definite diagnosis of endometriosis without resorti~g to direct visualization of the pelvis and abdominal cavity by operative laparoscopy. Therefore there i5 a pressing need for a ' relatively non-invasive method of imaging endometrial implants.
Mullerian anomalies such as unicornate and bicornate uteri can be associated with infertility and recurrent pregnancy 105s. Currently, the diaynosis of these abnormalities frequently requires operative procedures such as hysteroscopy and laparoscopy. A
non-invasive method of delineating the anatomic distribution of functional Mullerian Ppithelium and thus anatomy of these structures would bP clinically ;
useful.
In vitro fertilization (IVF) requires the removal of ova from a mammalian ovary, and exposure of these ~', .

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ova to sperm outside the body. Fertilization of each ovum requires that at least one living sperm penetrates the zona pellucida touter covering) of the ovum and fuses with the pronucleus. Once this has occurred and the ova are fertilized, they can be transferred to a uterus where they then can become implanted on the uterine wall. If implantation occurs, the pregnancy proceeds as if fertilization had occurred within the body. IVF has gained widespread professional and public acceptance. However, despite the ever increasing frequency and refinement of this procedure, IVF attempks most often do not result in pregnancy.
IVF pregnancy rat~s are currently only about 15 to 20 percent. For a variety of reasons, exposing the ova to sperm does not nacessarily result in fertilization.
More often, fertilized ova fail to implant in the uterus. The low success rate of IVF often leads to an excessive financial and psychological burden for the infertile couple.
other assisted reproductive technologies include two modifications of the IVF technique. The first is gamete intra-fallopian transfer (GIFT), the second is zygote intrafallopian transfer (ZIFT). In the GIFT
procedure, the retrieved oocyte and sperm are mixed together and placed ~ack 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.
Finally, hyper-stimulation protocols necessary to retrieve many oocytes from the donor women may have 35 deleterious effects on the endometrium itself and ~ ;
decrease the rates of implantation. Two basic 2B7~30~
WO92/10l99 ; ~ PCT/US91/08986 .,,; , . .~ , . .

procedures have been utilized to help overcome this pro~lem. 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 æygote 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. All of these techniques attempt to ; maximize the quality of the eggs, zygotes produced after ~ertilization and the receptivity of the end-ometrium. Any procedurP which would enhance the implantation rate above the standard 15 to 20% would have a markedly positive e~fect on any of these technologies.
Even if implantation occurs, pregnancy loss may occur. Pregnancy loss during the first six weeks has bean shown to occur in 35 to 50% of pregnancies confirmed by hCG analysis. Wilcox et al., "Incid~nce of Early Loss of Pregnancy" New Engl. J~ Med. (1988) 319:189-194. Furthermore, the chance of a successful live birth a~ter three consecutive abortions without a live birth may be as low as 40-50~. There is clear precedence for autoimmune pregnancy loss and infertility. It has been shown that patients who have general autoimmune diseases have a high incidence of reproductive failure. Gleicher et al., '~The Reproductive Autoimmune Failure Syndrome~" Am. J.
Ostet. Gynlecol. (1988) 159:223-227. There is also evidence oE autoimmune mediated recurrent pregnanoy loss in patients without clinically apparent autoimmune disease. Lockwood et al., Lancet 11:742-45, (1986);
and Lockwood et al., "The Prevalence and Biological Significance of Lupus Anticoagulent and Anticardiolipin 2 0 7:S 3 0 0 PCT/US91/0~986 ~-Antibodies in a General Obstetric Population," Am. J.
Obstet. Gynecol., 161:369~373 (1989).
The incidence of Down Syndrome (DS) is ; approximately 1/1000 live births. Adams et al., "Down's Syndrome: Recent Trends in the United States", JAMA (1981) 246:758-760. This incidence is expected to increase modestly over the next 10 years as the number of women becoming parents between 35 and 49 years of - age increases. Goodwin and Huether, "Revised Estimates - 10 and Projections of Down Syndrome Births in the United States, and the Effects of Prenatal Diagnosis Utilization, 1970-2002", Prenatal Diagnosis (1987) 7:261-271. However, by the year 2000 women over the age of 34 will still account for only 8.1% of all live births and 39% of DS cases. Thus even a fully subscribed to maternal age-based DS screening program would fail to identify 61% of DS cases. Goodwin and Huether (1987). Moreover, only a minority (20% - 30%) of women 35 years of age or older avail themselves of fetal karyotype analysis. Goodwin and Huether id.; and Hook et al., "Use of Cytogenetic Diagnosis in New York State", New Engl. J. Med. (1981) 305:1410-1413.
This inherent inefficiency and practical underutilization of DS scre~ning has prompted a concerted effort to improve the ability to detect the DS fetus. Lockwood et al., "A Sonographic Screening Method for Down's Syndrome", Am J. Obstet. Gynecol., 157:803-808 (1987). These efforts have included the development and expansion of maternal serum biochemical ~;~
markers and the evaluation of potential sonographic indicators of the DS fetus. The association of decreased maternal serum ~-fetoprotein (MSAFP) levels with fetal DS has now been well established. Ris~
deter~ination based on a combination of MSAFP value and maternal age allows for the detection of a third of DS
fetuses whlle necessitating karyotype analysis in 5% of :

WO92/l~l99 ? 0 7~ 3 ~ 0 PC~/US~l/08986 pregnant patients under 35. Wald et al., "Maternal Serum Screening for Down's Syndrome in Early Pregnancy", Brit. Med. J. (1988) 297:883-887.
Recently, Bogart and associates observed higher ; 5 than expected concentrations of the HCG ~ subunit in women bearing DS fetuses b~etween 8 and 25 weeks gestation. Bogart et al., "Abnormal Maternal Serum Chorionic Gonadotropin Levels in Pregnancies with Fetal Chromosome Abnormalities", Prenatal Diagnosis (1987) 7:623-630. Since HCG values generally plateau at between 18 and 25 weeks of gestation, an elevated HCG
value appears to be a reliable index af DS risk.
Maternal serum unconjugated estriol levels may also be marginally lower in DS pregnancies. Canick et al., "Low Second Trimester Maternal Serum Uncon~ugated Oestriol in Pregnancies with Down's Syndrome", Br. J.
Obstet. Gynaecol. (1988) 95:330-33; and Macri et al., "Maternal Serum Down Syndrome Screening: Unconjugated Estriol is not Useful", Am. J. Obstet. Gynecol. (1990) 162:672-673. However, DS risk assignment paradigms which employ maternal age with MSAFP, HCG and estriol values may identify 60% of DS fetuses while necessitating karyotype analysis in only 5% of pregnant women. Wald et al., Id. Clearly additional maternal biochemical markers for ~etal DS and other chromosomal abnormalities would have great utility.
Localization of 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 hPmoglobin and hematocrit, white blood cell count, urine and serum human chorionic gonadotropin (HCG~ pregnancy test, ultrasound including vaginal probe ultrasound, culdocentesis (a diagnostic procedure W0 92/l~l~9 2q7~0 PC~/U591/~8~861 to detect blood in the perit:oneum), curettage of the endometrium to rule out the presence of products of conception within the uterus, laparoscopy and finally in emergency cases, laparotomy.
Given the wide variability of patient presentations and the course of ectopic pregnancy, the accurate diagnosis of this disorder is sometimes difficult. In one study of three hundred women, approximately a third were seen more than once, and 11%
were seen more than twice before the correct diagnosis was made. In addition, in a recent study of deaths from ~ctopic pregnancy, more than half of the cases were misdiagnosed leading to fatal maternal outcome.
Clearly, accurate and rapid diagnosis and/or treatment of an ectopic pregnancy would be an important advancP
for the field of obstetrics and gynecology.
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 mètastases 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.
In the United States, commonly employed 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.
In addition to the high failure rates of some of these ; WO92/10t99 2~753~o PCT/US9l/0898 _g_ methods, a number of these methods have serious pote-ntial complications for the users. For example, in addition to metabolic changles induced by some oral contraceptives, there is a ]potentially increased risk of untoward cardiovascular ~effects and thromboembolisms in susceptible patients. Moreover many o~ 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.
Notwithstanding the work reported in this field, a need still exists for improved diagnostic and therapeutic applications in the field of mammalian reproduction.
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 monitorinq 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.

Summary of the Invention The present invention provides methods of diagnosing fertili~y problems, methods of enhancing fertility and methods of decreasing fertility potential.
Before discussing the various embodiments of the invention, it should first be noted that the inventor has determi.ned that F~N plays a significant role or roles in the mammalian reproductive process. While the inventor has developed certain theories xegardin~ the ~, , , ,., - , , ; , ' ~ ' WO92/l0199 ~ 7 5 ~ O o PCT/~91/08986 ~

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.
In this regard, and as is more fully explain~d below, 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. Consistent with this role, 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.
For example, the unique glycosylation properties -of FF~ 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.
on the other hand, the inventor has theorized that FFN might serve to inhibit inappropriate cell binding (e.g. tubal implantation) and thus promote fertilization or the pre-implantation transport of the blastocyst.

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WO92/101~9 2075~0 ~ PCT/US91/08~6 - Should FFN have a relatively reduced cell and ECM
binding affinity compared with adult forms, it could serve as a "lubricant" preventing injudicious binding of gametes, zygote or blastocyst. Specifically, seminal and Mullerian tract epithelial FFN secretion could prevent sperm binding to the endocervix, endometrium or tubal epithelium prior to fertiliæation, - thus enhancing the latter's probability of occurrence.
Similarly, tubal epithelial and ~ollicular FFN could act to prevent oocyte adherence to the tubal epithelium with consequent failure of fertilization or, in the ; event of fertilization, ectopic implantation. Finally the presence of endometrial glandular epithelial FFN
synthesis and secretion in the very early luteal phase (prior to 19 days of the menstrual cycle) could prevent i 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. In addition, reduced FFN
cell and ECM binding affinity together with increased proteolytic susceptibility could facilitate preterm delivery secondary to either contractions or inflammation. This latter view is consistent with the association of preterm delivery with placental separation (abruption) and preterm membrane rupture. `
Therefore, manipulation of FFN concentrations or fibronectin moiety synthesi~ at these various sites could serve to enhance or inhibit fertilization, implantation and the maintenance of placental and membrane structural integrity throughout gestation.
Also, depending upon whether the FFN acts as a binding agent or a "lubricant", its presence at different :Locations and at different times during the WO92/10199 2 0 7 5 3 ~ O PCT/US91/08986 reproductive process may have opposite effects. For example, if, as is presumab:Ly believed, FFN does indeed act as a "lubricant", its presence may facilitate the movement of sperm through the vagina, the cervix, the uterus, and ultimately to the fallopian tubes where fertiliæation occurs. Similarly, the presence of 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.
However, 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.
Hence, it is within the scope of the present invention to enhance reproductive potential both by enhancing and retarding FFN levels depending upon the type of tissue involved and the time of treatment relative to the reproductive process. As a general proposition, it is expected that increased FFN levels would enhance reproductive potential in in vlvo fertilization (including artificial semination) at all stages except at the time and location of implantation.
Methods of raising and lowering FFN levels are ~ -discussed below.
It is within the scope of the present invention to regulate FFM levels as a means of affecting reproductive potential regardless of whether the FFN
level is increased or decreased, and regardless of whether the FFN acts in all instances as a "lubricant"
or a binding agent.

- WO92/10199 2075~

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Thus, 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 dlfferent locations and/or times -- e.g.
retardation of cell binding in the Mullerian tract, and ` promotion of implantation in the uterus.
In the circumstances, the discussion below and disclosure of the various embodiments of the invention may imply or presuppose a theory of FFN function that may not be entirely consistent with a theory which is implied or presupposed with respect to another embodiment. It is, however, believed that one skilled in the art will appreciate that the different or even ; inconsistent theories which are here proposed are not intended to detract from the scope or significance of the present invention, but rather to promote a better understanding of the invention and the potential uses ' herein described.
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. Other types of tissue which may be sampled include, but are not limited to, serum, sperm, ova, endometrium, placenta, uterine fluid and follicular fluid.
It has, however, been demonstrated that FFN is present in siynificant quantities in normal semen and post-coital cervico-vaginal mucus samples as well as from follicle aspirates. Thus, the method of this invention provides information as to whether the mammal is producing FFN and in appropriate amounts for optimal fertilization potential. For example, the method .

~ WO92/10199 2 ~ 7 5 3 (3 ~ PCT/US91/~8986 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 - 5 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 lO 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 15 comparison of FFN concentrations to adult fibronectin 7 concentrations can also be utilized for assessing fertility potential.
; The invention further provides a novel infertility and recurrent abortion screening test comprising 20 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- ;
25 gnized. Thus, the method of this invention provides a tool for cliagnosing mammals with an autoimmune infertility.
This invention further provides a novel method for altering the fertility potential of a mammal by 30 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 35 pharmaceutically acceptable substances. In one embodiment of the present invention, the probability :

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WO92/10~99 2 0 7 s 3 0 0 PCT/US91/0898~

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. It has now been recognized that during normal mammalian pregnancy, FFN
has been localized in at least several locations including the i~plantation zone where tropAoblastic cells make contact and attach to the uterus and within the chorionic membrane where trophoblastic cells make direct contact with the maternal decidua. In addition, it has now been established that decidualized endometrial stromal cells and endometrial glands synthesize and secrete FFN in vitro. Thus 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.
Further provided by 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 hais ~ow 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. Therefore the inventian includes a method of regulating FFN synthesis by administering systemically or topically a therapeuticalLy effective amount of putative paracrine effectors which are known : ,:: -- , . . .
. .

~ -- wo s2/lnlss ' ' ' ';
~ o 7 ~ 3 o Q PCT/~'S91/08986 regulators of fibronectin synthesis such as transforming growth factor ~ tTFG-~). Ignotz et al., "Regulation of Fibronectin and Type I Collagen mRNA
Levels by Transforming Growth Factor ~", J. Biol.
Chem., 262:6443-6446 (1987).
Additionally, 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. Similarly, 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.
Further provided by this invention is a novel method of decreasing free FFN in a mammal comprising administering anti-FFN antibodies to said mammal.
Thus, by the method of this invention, anti-FFN
antibodies bind to FFN expressed by the mammal effectively decreasing the amount of available or free FFN regulating the abi~lity of the sperm to *ertilize the ova or the conception to implant.
Fuxther provided by this invention is a novel method of reducing the reproductive potential a mammal.
By the method of this invention, 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 success~ully migrate to the uterus so as to implant.

WO92/1~199 2 0 7 5 3 0 0 PCT/US91/0~986 :' Further provided by t:his invention is a novel method of detecting the presence of aneuploid fetuses (including trisomy 21) in the first and second trimesters of pregnancy by assaying concentrations of FFN in maternal plasma.
Further provided ~y this invention is a novel method of imaging trophoblastic tissue, M~llerian tract epithelia and endometreotic implants using magnetic resonance imaging (MRI). 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. In a preferred embodiment 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.
Further provided by this invention is a method of imaging ectopic or normally localized Mullerian epithelial cells. The method comprises administering to a mammal a labeled antibody, wherein the label is not radioactive. In a preferred embodiment the label has paramagnetic properties and is administered systemica:Lly or infused into the cervix and thence uterus, fallopian tubes and abdomen. For example, endometriosis could be identified in patients without resorting to operative diagno~tic procedures. In the case of imaging of the uterus or other organs, direct topical application may be appropriate. As the concentratlon of the labeled antibody increases, the - WO92/10199 2 ~ 7 5 3`0-~ PCT/US91/08986 greater the nonspecific binding becomes allowing visualization of larger areas of tissue to determine for instance organ abnormalities along the Mullerian tract.

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Brief Description of the Drawin~
Figure l 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 ~; lO figure.
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Detailed Description of the Invention "Fetal Fibronectin" (FFN) as used herein refers to a class of proteins which bind to FDC-6 and are produced by mammalian trophoblasts and Mullerian epithelial cells, and are found in other mammalian reproductive fluids, e.g., seminal fluid, vaginal secretions and ovarian follicular fluids. The immunological binding partner (FDC-6), although previously thought not to bind to normal human adult tissues, binds to FFN produced by certain normal reproductive tissues.
"FDC-6" as used herein refers to the monoclonal antibody defining the oncofetal structure of fibronectin as disclosed in U.S. Patent No. 4,894,326.
25 U.S. 4,894,326 is incorporated by reference as if fully set forth herein.
U.S. 4,894,326 disclose~ an IgGl monoclonal antibody (FDC-6) which defines a fibronectin structure or structures located between the "Hep-2" and the "Fib-Z" domains in the COOH-tPrminal region of fibronectins isolated from hepatoma, sarcoma, and fetal ~ibroblasts. It is disclssed that this antibody indicates the presance of two classes of human fibronectin. First, fibronectin from fetal connective ~

WO92/10199 2 ~ 7 5 ~ O O PCT/US91/08986 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. In contrast, 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. Since the structure defined by 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 Z5 be useful for diagnosing human cancer and for monitoring and implementing various cancer treatments.
The mono~lonal antibody FDC-6, a product of hybridoma cell li~e 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 FOC-6 was established following immunization of mice with fibronectin isolat2d from human hepatoma cells. The hybridoma ATCC No. HB9018 was selected by WO92/10199 0 ~ ~
2 ~ 7 ~ 3 PCT/US91/08986 positive reactivity of its antibody (FDC~6) with - fibronectins from hepatoma, fibrosarcoma, and fetal fibroblasts and by negative reactivity with fibronectin from plasma. Other antibod:ies recognizing trophoblastic and/or pregnancy-specific decidual ECM
components are expected to b~ useful in this invention.
It has now been found t:hat 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. The recombinant DNA technique of 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 - 35 substitutions, deletions and insertions distant from the FFN epitope which result in either enhanced or WO92/10199 20~5300 reduced cell binding affinity respectively and thus either promote or inhibit respectively fertilization, implankation and placentation.
Generally, in order to create a site specific mutation, 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 nucleotide bases complementary to and sufficient to ~nneal to the single-~tranded 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, so~e 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 usd consists of radiolabeling the synthetic oligodeoxyribonucleotide used in the mutagenesic; and hybridizing it to the transformed cells under-stringent conditions. Cells which contain the mutant vector will retain the radiolabeled probe and 207~300 22 ,~;
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.
In addition, the unique glycosylation pattern of FFN may alter its susceptibility to proteases. Given the anatomic distri~ution of FFN in pregnancy (see Ex.
` 15 l), 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.
In a preferred embodiment, 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 acti~ity. Specifically, 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. Matsuura et al., 3'An ~-N-acetylgalactosaminylation at the Threonine Residue of a Defined Peptide Sequence Creates the Oncofetal ! WO 92/10199 2 0 7 5 3 ~ O PCT/US91/08986 Peptide ~pitope in Human Fibronectin", J. Biol. Chem., 264:10472-10476 (1989).
- Trophoblast derived EC~ components can be extracted from the ECM deposited by trophoblasts in culture. Similarly, 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.
Once 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 ~anipulations to regulate FFN synthesis, as described in detail below.
In another aspect of this invention, 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 ~5 female mam~al of the same species, comprising the steps of pro~iding at least one sample of semen or -- WO92/lO199 2 a 7 5 3 b o Pcr,us~l,089~6, 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 a~sisted reproductive technologies in more precisely optimizing attempted fertilization. These ~ethods 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 .
'. ,.

W~92/~0199 2 0 7 5 3 0 ~ PCT/US9l/08~86 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.
Further provided by this invention is a method to image potentially pathological trophoblastic tissue as well as physiological or pathological M~llerian tract epithelium in a mammal. By the method of this invention, 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 ~- 15 antibody couples to FFN and is imaged by an appropriate medical imaging device such as MRI. The use of radi-olabeled antibody subjects the patient's ov~ries, fallopian, endocervical and perhaps normal intrauterine `
pregnancies to potentially damaging levels of 20 radioactivity. Thus, nonradiolabeled antibodies are t the method of choice for imaging and treating tissue expressing FFN. In a preferred embodiment, MRI is used as the imaging method.
Conveniently, FDC-6 is a suitable antibody for use 25 in this invention. Other FFN antibodies prepared as described above are also expected to be suitable for use in the invention.
Examples of trophoblastic tissue to be Iocalized include but are not limited to ectopic pregnancies, 30 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. In addition, endometriosis or 35 M~llerian tract anomalies can be similarly imaged.

WO92/10199 2 0 7 ~ 3 Q ~ PCT/US91/0~86 --Suitable labels include but are not limited to paramagnetic labels such as microspheres with paramagnetic impurities or ]proteins with paramagnetic ligands.
- 5 In another aspect of this invention, 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.
Conveniently, the presence of autoantibodies that bind to FFN can be assayed by ~asic 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. 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 in~ertility are believed due to an autoimmune process ~`
leading to endogenous production of anti-FFN
antibodies. In one aspect, this autoimmune process is believed to prevent the proper biological function o~
FFN, causiny sperm aggregation, impeding fertilization and/or implantation or gamete transport and playing a role in repetitive miscarriage later in pregnancy.

- W092/10l99 -27- PCT/US91/~8986 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.
In another aspect of this invention, a method of `~ altering the probability that gametes will fertilize or that a conceptus will become implanted in a mammalian uterus is provided. By the method of this invention, FFN is infused into the uterine cavlty 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.
To decrease the probability of implantation, 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. Further, 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. For example, 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 amou~t of FFN
which when introduced onto the surfa~e 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

W~92/10199 2~7~ ~ ~
PCr/US91/08~6f.-concentrations in human re]productive fluids fall in ~-this range.
Since FFN is also present in sperm, semen and follicular fluid, it is be:Lieved to play a critical role in fertilization. Therefore therapeutic regime~s 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 t-eatment of a variety of conditions. It has been shown that the endocrine function of an ovary could be markedly changed by an intrauterine infusion. Helmer et al.
"Intrauterine Infusion of Highly Enriched Bovine Trophoblast Protein-1 Complex Exerts an Antiluteolytic Effect to Extend Corpus Luteum Life Span in Cyclic Cattle," J. Reprod. Fertil., 87:89-101 (1989). It has besn shown that rat uteri which received an ;;
intrauterine injection of luteinizing releasing hormone had a significantly increased rate of impl~ntation compared to uteri which had no injection. Jones, "Blastocyst Attachment in the Ovariectomized Rat Treated With an Intrauterine Injection of Luteinizing Hormone-releasing Hormone (LRH) r ~I Acta Endocrinol.
(Copenh), 103:266-8 (1983). In addition to ~he use of solutions, there are references citing use of gels which are instilled intracervically to facilitate labor and delivery. See e.g., Ekman et al., "Intracervical Instillation of PGE2-gel in Patients with Missed Abortion or Intrauterine Fetal Death," Arch Gynecol., 233241-245 (1983). Finally, an intrauterine vehicle WO92/10199 2 ~ 7 5 3 U O ~ P~T/US91/08~86 such as a slow release capsule either similar to those currently existing on the m;rket or modified to ~ facilitate slower release O:e a pharmacologic agent ; which might either enhance or decrease the synthesis of FFN could be utilized.
Other compounds includ:ing putative stromal-epithelial paracrine effectors such as TGF-~, 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, vitrone~tin, 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 concept:us will implant in the uterus.
In another aspect of this invention, a method of regulating FFN synthesis in a mammal is provided comprising aclministering ~o said mammal a pharmaceutically acceptable compound which regulates FFN synthes;is by mam~alian M~llerian epithelial cells and other reproductive tract tissue in appropriate amounts.

: . , WO92/101~9 2 0 7 5 3 Q ~ PCT/~'S91/0898~i -Compounds which regulal:e FFN synthesis by mammalian endometrial gland~; in culture can be selected ~ from the group consisting of estrogen, estrogen - analogues and progesterone i.nhibitors 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 ~rom 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 m~thod of contragestion can also be achieved, as previously described, by introducing estrogen, estrogen analogues and progesterone inhibitors and antagonists to the 15 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 o~
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.
In another aspect of this invention, a method of decreasing t:he reproductive potential of a mammal is provided. E~y the method of this invention, the reprodu~tive potential of the mammal is decreased by administering to the mammal a F~N antigen with or without adjuvant in an amount sufficient to raise antibodies to FFN, whereby the probability that :, WO92/10199 2 0 7 ~ 3 ~ 0 ~ PCT/US91/08986 gametes will meet and fertilize or a conceptus will migrate to the uterus is decreased.
Since 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.
In addition, since 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 i~plantation by the blastocyst or otherwise induce sterilization.
In addition, if a blastocyst were to initiate implantation, 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.
Generally the protein can be dissolved at between about l to 50 ~g/ml in sterile saline or saline with 0.4 mg aluminum hydroxide per ml as a vehicle. Generally 0.5 to l.O 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 antikodies from developing against portions of FFN in common with normal adult plasma or cellular fibronectin.

WO92/10199 2 0 ~ ~ 3 Q ~ PCT/US91/089~6 -~7 There iC7i precedence for i~munizing patients and animals against various products of pregnancy to induce contraception. For example, 88 subjects which were immunized with an hCG base vaccine have been investigated. Kharat et al , "Analysis of Menstrual Records of Women Immunized with Anti-hCG Vaccines Inducing Antibodies Partially Cross-Reactive with hLH,"
Contraception 41:293-9 (1990). In animals, it has been shown that antibodies made against pig zona pellucida could induce contraception in mares. Liu et al., "Contraception in Mares Heteroimmunized with Pig Zonae Pellucidae", J. Reprod. Fertil., 85:19-29 (1989).
Finally, in a study in dogs, it has been shown that contraception could be induced by immunizing dogs against gonadotropin releasing hormone. Gonzalez et al., "Immunological Approaches to Contraception in Dogs," J. Reprod. Fertil. Suppl. 39:189-198 (1989). -`
In another aspect of this invention, a method of detecting the presence of chromosomally abnormal conceptuses during mammalian gestation is providPd, 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.
Given the elevated concentrations of FFN that the inventor has measured in the media of primary trophoblast cultures and detected in th~
placental-endometrial interface of normal pregnancies, neonatal cord plasma and in the amniotic fluid and plasma of healthy pregnant women, measurement of maternal plasma FFN would be expected to reflect synthesis ~y the trophoblast or fetal tissue. Given that aneuploid trophoblasts have been shown to produce WO 92/101~9 2 ~ 7 5 3 0 0 PCT/US91/08~86 --3 1_ .

abnormal quantities of HCG and other trophoblast specific products, the production of FFN by aneuploid conceptuses is anticipated to be markedly abnormal and this abnormality is expected to be reflected in maternal plasma FFN concentrations.

Example ~
Immunohistochemical Studies of Placentae, Membranes and Beniqn and Maliqnant Reproductive 10 Tract Tissues obtained from Non-Preqnant Women 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 ectop~c preqnancy specimens with associate uterine decidual specimens were also obtained. Fetal tissue was obtained following midtrimester termin-ations. In addition, to account the inventor's observation of the occasional but consistent presence of cervico--vaginal FFN in healthy non-pregnant patients, the presence and the localization of proteins recognized by FFN specific antibodies in non-qestational reproductive tract tissues (benign and neoplastic) was evaluated. Immunoperoxidase staining was performed accordi~g to the manufacturer's instructionis, using an avidin-biotin peroxidase detection system (Vector Laboratories, Burlingame, CA~.

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WO92~10199 i P~/US91/0~9~6 , --~ 2~300 .

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).
5 ~m sections from formalin on Bouin's-fixed and paraffin embedded tissue were placed on glass slides previously coated with a film of 1% poly-d-lysine, 30-70,000 molecular weight (Sigma), dried at temperatures no greater than 60C and stored at room temperature until u ed. Routine immunoperoxidase staining was carried out.
The results showed that in pregnancy tissues, there was diffuse immunohistochemical staining for total fibronectin (tFN) throughout the decidua and villous stroma as well as the syncytiotrophoblast basement membrane and surface. There was highly specific intense staining for FFN on the ECM of the decidua basalis in proximity to extravillous anchoring trophoblasts and trophoblast cell columns of the pla~ental-decidual junction. This pattern of FFN
staining was seen in both term and first trimester specimens and in the tubal-trophoblast junction of ectopic pregnancies. The villi, decidua distant from the utero- placental junction, uterine decidua from ectopic gestations, and maternal blood vessels were consistently negative for FFN. Although the source of FFN in the tissues in which FFN is found is not clear, likely possibilitles are that FFN is secreted by the extravillous trophoblasts derived from the cytotrophoblastic shelf located in the decidua basalis and/or the decidual cells. In view of the acknowledged importance of cell-cell interactions in regulating the differentiat:ed phenotype of either or both cell types, WO92/10199 7 5 3 ~ ~ PCT/US91/08986 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.
Analyses of membran~ preparations revealed an analogous distribution of FFN in the chorionic ECM.
This site would appear to be ontogenically analogous to the localization of FFN in the placental-decidual junction since the chorion is a remnant of the trophoblastic shelf. Patients with severe chori-oamnionitis displayed an apparent reduction in FFN
staining, perhaps as a consequence of proteolysis.
Again, there was diffuse staining for tFN throughout the amnion and chorion ECM.
In fetal tissues, 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. However, it is unlikely that 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.
The results obtained with Mullerian tract tissue are shown in Table I which compares the immunohistochemical distribution of FFN in non-gestational reproductive tract tissue and reproductive tract malignancies. In benign tis~ues FFN
was localized to the juxta-luminal cytoplasm and secretions in some cervical, endometrial, and tubal epithelial specimens. It was similàrly localized to the epithelium of a mucinous cystadenoma of the ovary (Table I). Thus juxta-luminal cytoplasmic localization ~092/10199 2 0 7~3 0V PCT/US91/OB98i6 -~6-in Mullerian epithelia stands in contrast to the findings in placentae and membranes where FFN was restricted to the ECM.

TABLE I
DISTRIBUTION OF FFN IN REPRODUCTIVE TRACT TISSUES
A) Benign Tissues:
1) Normal vagina: no staining for FFN epitope, however, diffuse stromal staining for total fibronectin (tFN).
2) Normal cervix: positive discrete juxta-luminal endocervical gland cytoplasmic staining for FFN in some patients' specimens, however diffuse stromal ECM staining for tFN.

3) Tubo-ovarian complex: no staining for FFN, but diffuse staining of stromal ECM for tFN.

4) 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.

5) Normal premenopausal endometrium:
a) Proliferative Phase: No FFN staining, but di*fuse staining of stromal ECM for tFN.
b? Secretory Phase positive discrete juxta-luminal endometrial gland cytoplasmic staining, but only in tissues obtained from some patients in the early luteal pha~ie.

6) Menopausal endometrium: no staining for FFN, diffuse staining of stromal ECM for tFN.

7) Endometriosis o~ the colon: intense speci~ic staining ~or FFN in the juxta luminal cytoplasm of the endometrial glands with dif~use staining of stromal ECM and histiocytes for tFN;

.

WO92/10199 2 0 7 5 3 ~ O pcr/us9l/o8986 B) Maliqnancies:

8) Endometrial carcinomas and endometrial hyperplasia: no ~taining for FFN, diffuse staining of stromal ECM for tFN.

9) 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.
lO) Squamous cervical carcinomas (moderate and poorly differentiated) and an endocervical adenocarcinoma: no staining for FFN, diffuse staining of stromal ECM for tFN.
ll) Mixed M~llerian tumor of the uterus: no staining for FFN, diffuse staining of stromal ECM for tFN.
12) 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.
I3) Ovarian lymphoma: no staining for FFN, whereas a normal lymph node demonstrated trace intracytoplasmic staining for FFN an~
tFN in macrophages the (the latter may represent a peroxidase artifact). ~ -14) Dysgerminoma: no staining for FFN.
l~) A Papillary serous cystadenocarcinoma: no staining for FFN but diffuse staining of stromal ECM for tFN.

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W092/10l99 2 07 ~ 3 ~ o Example ?
Human Trophoblasts in Cult re Synthesize Fetal Fibronectin De Novo 5ELISA assays were performed to measure the concentration of FFN in trophoblast media and cell extract. ELISA assays were performed using ROMC~E~K~
according to the manufacturer's instructions (Adeza ~iomedicals, Sunnyvale, CA).
10Human cytotrophoblasts were purified as previously described~ Kliman et al., "Purification, Characterization and in vitro Differentiation of Cytotrophoblasts from Human Term Placentae", Endocrinol. 118: 1567-1582 (1986). The cytotrophoblasts were cultured in Dulbecco's Modified Eagles' Medium (DMEM) containing 25 mM ~lucose and 25 mM HEPES (DMEMHG) supplemented with gentamicin (50 ~g/ml), glutamine (4 mM), and 20% (v/v) heat-inactivated fetal calf serum. For preparation of cell extracts, cells were washed with phosphate buffered saline, scraped from the culture dish, and total cellular protein was extracted with an S~S-didechoate buffer.
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 negati~e villous staining for FFN in the placenta. A small cruantity of FFN was present intracellularly a~ter 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 vivc?, are induced in culture to produce significant FFN. It WO92/10199 2 0 7 ~ 3 O ~ PCT/US91/08986 was also determined that cultured trophoblasts secrete FFN. Although very little FFN could be measured in conditioned media from the first 24 h of culture, during the time interval from 24 to 48 h the media concentration of FFN averaged 4.5 ~g/ml.
This result indicated trophoblast secretion of newly synthesized FFN into the culture media. Based on ELISA, lO0 percent of trophoblast fibronectin FFN
contains the oncofetal domain, and is therefore reactive with FDC-6.

Example 3 Immunoassay Quantitation of FFN
Concentrations in Sperm. Follicular Fluid Samples of semen obtained from specimens sent to the Mount Sinai Perinatal Laboratory for routine semen analysis were analyzed for FFN concentrations by immunoassay according to the manufacturer's instructions (Romcheck~ Adeza Biomedical). Very high concentrations were found in semen, 77 - 648 ~g/ml, including the semen of an aspermic vasectomized male.
Subsequent studies suggested tight binding of FFN to sperm since washed sperm retained relatively high concentrations of FFN (95 - 1040 ng/ml). Residual follirular 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 FYN and total fibronectin, respectively, were observed. Thus follicular fluid from mature follicles appear to contain bot:h adult and oncofetal forms.
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WO92/10199 2~7~3~ - P~ S91/~89~6,~-Example 4 Analyses of FFN Secretion by Primary Cultures of_Endometrial Epithelial and Stromal Cells Glands were isolated f;rom biopsies of proliferative endometrium and cultured on collagen I
under polarizing conditions according to a modification of the method described by Schatz et al, "Culture of Endometrial Cells Under Polarizing Conditions", i 10 Differentiation, 42:184-190 (1990) while stroma was isolated from proliferative and secretory phase biopsies and cultured on plastic. 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.
Stromal cells were derived from a specimen of secretory endometrium and grown to confluence on plastic culture dishes in Basal Media (BM: Serum free DMEM ~ Ham's F12, 1/1) + 10% stripped calf serum (SCS).
At confluence within 15 days of plating, the experimental period was initiated by adding in BM + 10 SCS, either lOnM estradiol (E2), 1 ~M
Medroxyprogesterone acetate (MPA), E2 + MPA, or BM +

10% SCS with 0.1% ethanol (the vehicle control). The cultures were maintained in a 37C 95% air 5% C02 incubator. The medium was replenished every 4 - 5 days with the coxresponding experimental or control medium. -The conditioned medium was concentrated and assayed for levels of Prolactin (PRL~, (Amersham RIA) total fibronectin (Total Fibronectin Assay0, Ade~a 8iomedical) and (Romcheck~, Adeza Biomedical) FFNo At 17 days, media were collected and the cells harvested by scraping with rubber "policeman" into ice cold Hanks Balanced Sa-lt Solution (HBSS) containing a cocktail of v ~J

protease inhibitors (PI: soybean trypsin inhibitor, pepstatin, leupeptin, aprotinin, PMSF, all at a final concentration of lO ~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. lA) was detected in control or E2-supplemented medium, whereas MPA elicited only a small increase in PRL levels. However, 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. Both total fibronectin (Fig. lB) and FFN
tFig. lC) were readily detected in the stromal cell conditioned medium. Estradiol produced the most striking effect of any of the treatments, reducing levels of both total fi~ronectin and FFN to about half that of the corresponding controls by 5 day~, and to about 90% by 13 days. This result confirms that the stromal cells possess functional estrogen receptors.
Although MPA also inhibited outputs of fibronectin and FFN, adding E2 + ~PA to the cultures unaccountably elevated levels of total fibronectin to control values, but inhibited FFN midway between E2 and MPA alone. In contrast to the significant levals of fibronectin and FFN measured in the conditioned madium, neither protein was detected in the harvested cell pellets.

.'`:
''"''''''''' ' -. ~ .

W092/10199 2~753:~Q PCT/US91/08986 -~2-Isolated endometrial glands were distributed among 16 Millicell (Millipore) chambers containing collagen I
gels in BM + ITS++2% SCS and~ grown under polarizing conditions. These were divided into control (+ 0.1%
ethanol) and experimental (lOnM E2 + l ~M MPA) groups and, placed in a 37C 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. After an additional 7 day incubation, the medium from the B subgroup was collected and the cells harvested as above. The collected media were analyzed for FFN and total fibronectin contant and the cell pellets were assayed for protein content (after subtracting collagen blank values (<8% of total cell protein)), FFN and total fibronectin content.
In contrast to the results in stromal cell cultures (proliferative phase endometrial epithelial cells cultured under polarizing conditions on collagen (see Table II) release of FFN into the media and it appears that; l) FFN îs the primary and perhaps only fibronectin released by these primary gland cultures;
and that 2) estradiol plus MPA inhibits this production (or shuts off FFN synthesis completely).

v~ ~J U~
WO~2/1~199 PCT/US91/08986 TABLE II
PRODUCTION OF FFN BY PRIMARY
ENDOMETRIAL GLANDULAR_CULTURES OBTAINED
FROM THE PROL~FERATIVE PHASE
Experimental Conditions ~in ng/ml of media/mg cell protein) ._____ - ~ _ . . _ Control E2 + MPA
. . _ Days/Culture [FFN] [tFN] [FFN] [tFN] 0-6*
: _ .... .
(A) 0-7* 722 587 < 5 3.2 j~.
. . . .__. . _ . ._.
(B) 0-7* 864 602 < 5 5.7 . .... ___ . . ..
(B) 7-12* 1893 1836 < 5 _ 6 0 _ : ~
. Uncond Medla** 5 0 < 5 0.0 ~ .

~, Thus, the endometrial glandular epithelial cells from proliferative phase human endometrium release FFN, but virtually no adult fibronectin, into the media.
This xelease was completely inhibited by E2 + MPA. :.
' ,~

' - ' ' .

Claims (20)

Claims

1. A method of enhancing the reproductive potential of a mammal, comprising regulating the amount of fetal fibronectin in at least one preselected tissue of the mammal.

2. The method according to claim 1, further comprising the steps of measuring the amount of fetal fibronectin in at least one first preselected type of tissue, and causing an increase in fetal fibronectin in at least one second preselected type of tissue if the measurement indicates that the amount of fetal fibronectin present in the first tissue is below a predetermined level.

3. The method according to claim 2, wherein the at least one first and second type of tissue is selected from the group consisting of serum, semen, sperm, ovum, endometrium, placenta, uterine fluid and follicular fluid.

4. The method according to claim 1, wherein the amount of fibronectin is increased in the tissue by direct infusion of fetal fibronectin to the tissue.

5. The method according to claim 1, wherein the amount of fetal fibronectin is increased by administering to the mammal a pharmaceutically acceptable substance that increases endogenous fetal fibronectin synthesis.

6. The method according to claim 5, wherein the pharmaceutically acceptable substance is selected from the group consisting of progesterone, progesterone analogues and estrogen antagonists.

7. The method according to claim 1, wherein reproductive potential is enhanced by enhancing the ability of a conceptus to implant in the uterus, and including the step of causing the amount of fetal fibronectin in the uterine cavity to be decreased at about the time the uterus will be contacted with the conceptus.

8. The method according to claim 1, wherein the reproductive potential is enhanced by raising the level of fetal fibronectin in the reproductive tract, thereby inhibiting the binding of sperm, ova or blastocysts to Mullerian epithelia.

9. A method of determining the reproductive potential of a mammal, comprising the steps of measuring the level of fetal fibronectin in at least one preselected type of tissue of the mammal, and comparing the measured level of fetal fibronectin with a predetermined level.

10. The method according to claim 9, further comprising the steps of contacting the tissue or fluid with an appropriately labelled antibody specific to fetal fibronectin, allowing the antibody to bind with the fetal fibronectin present in the tissue, removing unbound antibody, and measuring the amount of the tissue-bound antibody, comparing the amount of bound antibody with said predetermined level.

11. The method according to claim 9, wherein the at least one type of tissue is selected from the group consisting of serum, semen, sperm, ovum, endometrium, placenta, uterine fluid and follicular fluid.

12. A method of assaying for infertility in a mammal comprising the steps of obtaining a serum sample from the mammal and measuring the amount of antibodies specific for fetal fibronectin in the serum sample.

13. A method of regulating the reproductive potential of a mammal comprising regulating the amount of fetal fibronectin in the mammal.

14. The method according to claim 13 further comprising the step of administering to the mammal an amount of fetal fibronectin sufficient to cause the production of fetal fibronectin antibodies wherein the antibodies obtain a titer sufficient to decrease fertility.

15. The method according to claim 13 comprising administering to the mammal a pharmaceutically acceptable substance that inhibits fetal fibronectin synthesis.

16. The method according to claim 15 wherein the pharmaceutically acceptable substance is selected from the group consisting of estrogen, estrogen analogues, fetal fibronectin specific antisense oligoribonucleotide inhibitors, and progesterone antagonists.

17. A method of decreasing the reproduction potential of a mammal comprising regulating the amount of fetal fibronectin in the mammal.

18. The method according to claim 17 comprising the step of impeding the ability of a conceptus to implant in the uterus, and including the step of causing the amount of fetal fibronectin in the uterine cavity to be increased at about the time the uterus will be contacted with the conceptus.

19. The method of claim 17, wherein the reproductive potential is decreased by lowering the level of fetal fibronectin in the female reproductive tract, thereby causing the sperm to bind to reproductive tract epithelia.

20. A method of contragestion comprising regulating the amount of fetal fibronectin at the site of implanted fetal tissue.