WO1994009805A2

WO1994009805A2 - Method of treating recurrent miscarriage and improving the efficacy of assisted reproductive techniques using placental protein 14

Info

Publication number: WO1994009805A2
Application number: PCT/CA1993/000449
Authority: WO
Inventors: Anthony E. Bolton; Tin-Chiu Li
Original assignee: Intermune Life Sciences, Inc.
Priority date: 1992-11-03
Filing date: 1993-11-03
Publication date: 1994-05-11
Also published as: AU5332694A; ZA938202B; WO1994009805A3; CA2124694A1; JPH07502755A; EP0627931A1; IL107469A0

Abstract

A method of preventing miscarriage in a female by administering to the female a therapeutically effective amount of an active substance selected from the group consisting of PP14, derivatives of PP14, muteins of PP14, fragments of PP14, and subunits of PP14, so as to prevent miscarriage. A method of testing a female for the possibility of future miscarriage by measuring the level of PP14 in the female, and comparing the measured level to the level of PP14 present in normal fertile women. A method of improving the efficacy of assisted reproductive techniques such as artificial insemination, in-vitro fertilization, or gamete intra-fallopian transfer, by adding an effective amount of PP14 to a sample comprising an embryo, fertilized or unfertilized egg, mixture of eggs and sperm, or sperm alone, prior to, concurrently with, or following introduction of the sample into the patient.

Description

METHOD OF TREATING RECURRENT MISCARRIAGE AND

IMPROVING THE EFFICACY OF ASSISTED REPRODUCTIVE

TECHNIQUES USING PLACENTAL PROTEIN 14

BACKGROUND OF THE INVENTION ^ 1 • Field of the Invention:

The present invention relates to inhibitors of immune cell proliferation and function, and more particularly to the use of Placental Protein 14 (PP14) to treat recurrent miscarriage. The invention also relates to methods for 10 improving the efficacy of assisted reproductive techniques, using PP14. 2. Background of the Invention:

The human immune system functions to protect the organism from infection and from foreign antigens by

15 cellular and humoral mechanisms. The immune system consists of a complex organization of many types of lymphocytes, and macrophage or other antigen-presenting cells . These agents regulate each other by means of multiple cell-cell interactions and by elaborating soluble

20 factors, including lymphokines and antibodies, that have autocrine, paracrine, and endocrine effects on immune cells. Disorders of the regulation of this system may result in the uncontrolled proliferation of immune cells

V and eventually to malignancy, uncontrolled response to

25 foreign antigens or organisms leading to allergic or

1

SUBSTITUTE SHEET inflammatory diseases, aberrant immune responses directed against host cells leading to organ damage and dysfunction, or generalized suppression of the immune response leading to severe and recurrent infections. Interleukin 1 (IL-1) is a peptide cytokine secreted by a variety of cell types including accessory cells of the immune system, and the antigen presenting cells. Interleukin 1 has a variety of functions including an involvement in the activation of immune system T-cells. Cells secreting IL-1 include monocytes present in the circulating blood, macrophages found in interstitial fluid, and dendritic cells.

It now appears established that IL-1 is a central mediator of inflammatory reactions and is important in pathogenesis of chronic inflammatory diseases, of which rheumatoid arthritis (RA) is one example. Evidence of this function of IL-1 has been derived from a variety of experimental approaches and may be summarized as follows:

1. Prostaglandins and leukotrienes are mediators of inflammatory reactions; hence non-steroidal anti- inflammatory drugs, which inhibit cyclooxygenase and prostaglandin synthesis, are useful therapeutically in such conditions. IL-1 mobilizes free arachidonate, the precursor of prostaglandins and leucotrienes, by activating phospholipase, and also induces cyclooxygenase.

2. IL-1 stimulates binding of T-cells to endothelial cells, thought to be the first step in their influx into joints.

3. Injection of recombinant IL-1 into joints causes an influx of inflammatory cells, followed by a loss of proteoglycan from the cartilage.

Treatment of allergies and autoimmune diseases has been based on modalities which are toxic to immune cells, that inhibit production of antibodies, or inhibit the effects of mediators of the immune response, such as

SUBSTITUTE SHEET histamine. Over the past several years many soluble lymphokines which regulate the immune system have been characterized. Drugs which allow manipulation of the production or function of such factors would be of use in the treatment of autoimmune diseases and perhaps in the treatment of diseases resulting from the uncontrolled proliferation of immune cells.

It is now becoming widely accepted that IL-1 (both IL- 1 alpha and IL-1 beta) are important mediators of inflammatory responses. IL-1 appears to directly cause cartilage breakdown in knee joints, and may be central in the pathogenesis of rheumatoid arthritis. An inhibitor may, therefore, be of importance in treatment of this disease. Placental Protein 14 (PP14) is a natural product present at elevated levels in the peripheral circulation early in pregnancy, peaking around week 9-10. There are reports in the literature that there is a marked improvement in some sufferers of rheumatoid arthritis in the first trimester of pregnancy. Similar reports of an improvement in patients with chronic asthma during the first trimester of pregnancy can also be found in the published literature. Chronic asthma is an inflammatory disease, and although IL-1 has not yet been implicated in its etiology, this remains a possibility.

Pregnancy is a normal state in which at least one aspect of the immune response--reaction to foreign antigens--is suppressed with regard to paternal antigens expressed by the fetus. In this regard, certain women suffer from recurrent miscarriage, which is generally defined as the loss of three or more consecutive pregnancies before the 20th week of gestation. Recurrent miscarriage may arise as a result of problems associated with implantation of the fertilized egg in the uterus. It

SUBSTITUTE SHEET is believed that the failure to implant successfully may somehow relate to immune system phenomena.

Women may suffer from other reproductive problems or defects which make it difficult or impossible to become pregnant and/or to implant and carry a fetus to term. Various assisted reproductive techniques have been developed, such as artificial insemination (Al) , in-vitro fertilization (IVF) , and gamete intra-fallopian transfer (GIFT) , which permit women having such problems or defects to reproduce successfully. The techniques are also commonly employed to increase the fertility of other animals. Assisted reproductive techniques do not always succeed, and it would therefore be desirable to improve the efficacy of such techniques.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that women who experience recurrent miscarriage frequently exhibit greatly reduced levels of PP14. Thus, a method of preventing miscarriage is provided according to the present invention, which comprises administering to a female an active substance selected from the group consisting of

PP14, derivatives of PP14, muteins of PP14, fragments of

PP14, and subunits of PP14, in an amount effective to prevent future miscarriage. A variety of these natural and recombinant forms of PP14 may be used according to the invention, with natural PP14 being the preferred material.

Normal, fertile women have been found to exhibit a concentration of PP14 in flushings from the uterine lumen of about 95.1 ng/ml +/- 7.5 ng/ml (means +/- SD after logarithmic transformation, n=8) . The actual luminal concentration of PP14 is much higher, as the concentration of PP14 is diluted in the flushings by washing fluid.

Accordingly, the invention preferably contemplates the administration of PP14 in an effective amount so as to

SUBSTITUTE SHEET provide a concentration of PP14 which exhibits in-vitro activity when subjected to the testing procedures described in the present application. In this regard, administration of PP14 in an amount ranging from about 0.1 μg/ml to about 5 mg/ml, and preferably from about 5 μg/ml to about 5 mg/ml, is contemplated by the invention.

The effective amount of PP14 utilized will depend on such factors as the level of PP14 already present in the female, the form of PP14 utilized, and method and site of administration, the stage of the female's menstrual cycle, and other factors known to those of ordinary skill in the art.

While the invention is particularly intended for the prevention of miscarriage in females who suffer from recurrent miscarriage, the method of treatment of the invention is also believed to be useful to prevent miscarriage in females who have not experienced recurrent miscarriage.

The Placental Protein 14 (PP14) material may be administered to the female by any appropriate route, including intravenous injection, intramuscular injection, oral administration, intra-vaginal administration, intra- cervical administration, intra-uterine administration, intra-tubal administration, topical administration, rectal administration, and inhalation. In addition, the PP14 may be delivered to the site of treatment by means of a controlled-release delivery system, or by means of a gel, such that the PP14 does not wash out of the site of administration too easily. The PP14 active substance may be administered in admixture with a pharmaceutically acceptable carrier.

According to a second aspect of the invention, a method of testing a female for the possibility of future miscarriage is provided, which comprises:

SUBSTITUTE SHEET (a) measuring the level of PP14 present in a predetermined body fluid or tissue sample of a female; and

(b) comparing the level of PP14 present in the predetermined body fluid or tissue sample of the female to the level of PP14 present in a comparable body fluid or tissue sample of a normal, fertile female suggests the possibility of future miscarriage.

As PP14 is synthesized in the endometrial glands and secreted into the uterine lumen, a preferred site in which to measure level of PP14 is the uterine lumen. A preferred method according to the invention for measuring PP14 in the uterine lumen is by flushing the uterus with a saline solution, collecting the resulting washings, and subsequently determining the concentration of PP14 at this site using radioimmunoassay or other techniques. Measurement of the endometrial production of PP14 is known to vary depending on the particular stage of the menstrual cycle; thus, the day of collection of the washings should be standardized. A preferred day of collection of the endometrial washings is the seventh day after the midcycle luteinizing hormone (LH) peak in the female, which date closely proximates the time of normal implantation.

The above-discussed technique of collecting endometrial washings is preferably utilized to detect the level of PP14 present in the uterine lumen of the female. The particular process for measuring PP14 concentration in the body fluid or tissue sample may be any known assay, including those selected from the group consisting of radioimmunoassay, ELISA assays, and immunoblotting assays. A third aspect of the invention relates to a method of improving the efficacy of assisted reproductive techniques, such as artificial insemination (Al) , in-vitro fertilization (IVF) , and gamete intra-fallopian transfer (GIFT) , by adding an effective amount of PP14 to any sample (embryo, fertilized or unfertilized egg, mixture of eggs

SUBSTITUTE SHEET and sperm, or sperm alone) that is to be introduced into the uterus in order to induce pregnancy.

PP14 is found in relatively high concentrations in seminal plasma. However, sperm used for artificial insemination (Al) and other known assisted reproductive techniques are frequently washed in order to remove prostaglandins present in the seminal plasma before introducing them into the uterus in the Al procedure. This conventional washing step results in the removal of PP14 from the seminal plasma. Thus, the invention contemplates the addition of PP14 to sperm before the Al procedure, preferably by including PP14 in the medium used to dilute washed sperm prior to insemination.

In any assisted reproductive technique, the addition of extra medium into the uterus along with gametes inevitably dilutes the normal PP14 concentration present in the uterus. The invention thus contemplates adding an effective amount of PP14 to the gamete medium or the like, so as to provide a PP14 concentration having in-vitro activity when subjected to the testing procedures described below. In general, adding PP14 to the sample in an amount ranging from about 0.1 μg/ml to about 5 mg/ml, and preferably from about 5 μg/ml to about 5 mg/ml, is contemplated by the invention. The PP14 may be added to the embryo, fertilized or unfertilized egg, mixture of eggs and sperm, or sperm alone prior to, concurrently with, or following introduction of the sample into the patient.

DETAILED DESCRIPTION OF THE INVENTION Of the range of proteins which are known to be associated with the pregnancy state, one has been found to exhibit an immunosuppressive activity in a variety of in- vitro tests. Further investigation of the mode of action of this peptide, PP14, has indicated that it inhibits IL-1 production by peripheral white blood cells (containing both

SUBSTITUTE SHEET T-lymphocytes and monocytes) after stimulation. The concentration at which PP14 is active appears to be the levels at which it is found normally during pregnancy. The time course of this inhibition of IL-1 production closely relates to immunosuppressive activity of the molecule, indicating that its primary effect is on monocytes rather than other immune system cells.

A variety of proteins are expressed at high levels during pregnancy. One of these, PP14, is a major secretory protein of decidual tissue, where it comprises about 10% of the total soluble protein. As increased levels of PP14 appear early in a normal pregnancy, PP14 is believed to be associated with the process of implantation and in maintaining the early conceptus, which may be particularly prone to immune rejection by the maternal immune system.

The present invention is based on the discovery that PP14 is commonly present in greatly reduced levels in women who experience recurrent miscarriage. The invention therefore contemplates a method of preventing miscarriage by administering to a female an amount of PP14 effective to prevent future miscarriage.

The PP14 active substance utilized in the inventive method may be obtained from a variety of sources, including mammalian placenta, mammalian blood, amniotic fluid, seminal plasma, cells in tissue culture, decidual cells, decidual organs, endometrial cells, endometrial organs, as well as recombinant protein sources, including sources containing eukaryotic cells or prokaryotic cells engineered to express PP14, muteins of PP14, fragments of PP14 or subunits of PP14.

Useful forms of PP14 for purposes of the present invention therefore include natural and recombinant forms of PP14 itself, as well as derivatives, muteins, fragments, and subunits of PP14, provided that the desired therapeutic activity of the substance (prevention of miscarriage) is

SUBSTITUTE SHEET maintained. Natural PP14 is the preferred material utilized according to the invention.

Natural PP14 is a glycoprotein comprising about 17.5% carbohydrate content. The details of this carbohydrate content are not presently known; however, PP14 binds strongly to the lectin concanavalin-A, which is known to have an affinity for terminal alpha-D-mannosyl and alpha-D- glucosyl residues, as well as to wheat germ agglutinin, which has an affinity for N-acetyl-beta-D-glucosaminyl residues. The presence of the latter is further evidenced by the reduction of the interaction of PP14 with specific antibodies caused by treatment of PP14 with the enzyme beta-N-acetyl glucosaminidase, which removes these residues. The nucleotide and amino acid sequence of PP14 cDNA, as deduced by Julkunen et al . , is shown in accompanying Sequence Description (SEQ ID N0:1) . The entire disclosure of Julkunen et al. , "Complete Amino Acid Sequence of Human Placental Protein 14: A Progesterone-Regulated Uterine Protein Homologous to 3-lactoglobulins," Proc. Natl. Acad. Sci. USA. Vol. 85, pages 8845-8849, December 1988, is incorporated herein by reference.

It will be understood that the precise chemical structure of PP14 will depend on a number of factors. For example, since ionizable amino and carboxyl groups are present in the molecule, a particular protein may be obtained as an acidic or basic salt, or in neutral form. All forms of PP14 which retain their therapeutic activity for purposes of the instant invention are intended to be within the scope of the definition of "PP14".

It should be noted that the N-terminal amino acid sequence of PP14 shows substantial sequence homology with certain animal 3-lactoglobulins, but the biological activities of these proteins are not well understood.

SUBSTITUTE SHEE"^ Furthermore, there is some sequence homology between PP14 and human serum retinol binding protein.

The term "recombinant" used herein refers to PP14 produced by recombinant DNA techniques wherein the gene coding for the PP14 is cloned by known recombinant DNA technology. For example, the human gene for PP14 may be inserted into a suitable DNA vector, such as a bacterial plasmid, and the plasmid used to transform a suitable host. The gene is then expressed in the host to produce the recombinant protein. The transformed host may be prokaryotic or eukaryotic, including mammalian, yeast, Aspergillus and insect cells. One preferred embodiment employs bacterial cells as the host.

Therapeutically useful derivatives of PP14 may be prepared by augmenting the primary amino acid sequence of the protein PP14 with at least one additional molecule selected from the group consisting of glucose moieties, lipids, phosphate groups, acetyl groups, hydroxyl groups, saccharides, methyl groups, propyl groups, amino acids, and polymeric molecules. Augmentation may be accomplished through post-translational processing systems of the producing host, or it may be carried out in-vitro. Both techniques are well-known in the art.

Referring to the Sequence Description of PP14, it should be noted that the peptide includes three potential glycosylation sites at amino acid residues 28-30, 63-65, and 85-87. Glycosylation is a process of forming a protein derivative, wherein a portion of the protein's amino acid sequence is augmented by a sugar moiety. It will therefore be understood that therapeutically useful derivatives of PP14 may be prepared by addition of one or more sugar residues to the protein, or alternatively by removal of some or all of the sugar residues from the sites of glycosylation on the PP14 molecule.

10

SUBSTITUTE SHEET Other therapeutically useful derivatives of PP14 may be formed by modifying at least one amino acid residue of PP14 by oxidation, reduction, or other derivatization processes known in the art. Muteins of PP14 which do not destroy the activity of the protein may be used as the active treating substance of the instance invention. Muteins may be prepared by modification of the primary structure of the protein itself, by deletion, addition, or alteration of the amino acid incorporated into the sequence during translation. For example, at least one cysteine residue of PP14 may be replaced with a conservative amino acid, in order to eliminate sites of undesirable intramolecular disulfide bond formation. Crosslinking is undesirable if it changes the conformation of PP14 so as to render the protein essentially inactive for purposes of treating recurrent miscarriage. Also, it may be desirable to replace a methionine which is not essential to bioactivity with a conservative amino acid. As referred to herein, a conservative amino acid alteration is defined as one which does not significantly adversely affect biological activity and involves substitutions of the amino acid.

The conservative amino acid that may be substituted for cysteine and methionine include at least: serine, alanine, glycine, valine, threonine, leucine, isoleucine, and tyrosine. More preferably they include serine and alanine. Most preferably, cysteine may be replaced with serine and methionine replaced with alanine.

Placental Protein 14 (PP14) is believed to exist in nature as a dimer of two identical, non-covalently linked protein subunits. Accordingly, since each subunit is believed to have the amino acid sequence shown in SEQ ID NO:l, a subunit of PP14 could be used as the therapeutic active substance for treating recurrent miscarriage according to the instant invention. The invention also

11

SUBSTITUTE SHEET encompasses use of subunits of PP14 that are covalently linked, either naturally or by artificial techniques known in the art.

In addition, it is contemplated that fragments of PP14 would be useful for treating recurrent miscarriage, provided that such fragments retained their therapeutic activity. Referring to SEQ ID N0:1, the protein fragment defined by amino acid residues 63 through 160 is believed to be a therapeutically active fragment of PP14 for purposes of the invention. This fragment includes four cysteine amino acids, which are believed to have biological activity.

The fragment defined by amino acid residues 80 through 105 is also believed to be therapeutically active for purposes of the invention. The fragment defined by residues 80-105 is believed to be therapeutically active because: it is a linear sequence not involving disulfide bridges; the fragment contains a glycosylation site (at residues 85-87) ; the fragment has two tyrosine residues which, because of their phenyl side chains, may be involved in receptor interaction; and the double lysine sequence at residues 80-81 have double amino acid group activity, suggesting potential receptor activity.

The above-described forms of PP14 are used in an effective therapeutic amount, which will vary depending on the level of PP14 already present in the female, the side and the method of administration, the form of PP14 utilized, the stage of the menstrual cycle of the female, and other factors understood to those having ordinary skill in the art.

In general, one of ordinary skill in the art will be able to arrive at therapeutically effective dosages of PP14 for treatment of miscarriage, based on the range of concentrations of PP14 which has been discovered to provide suitable in-vitro activity. As shown in Example 4 below,

12

SUBSTITUTE H normal, fertile women have been found to exhibit a concentration of PP14 in flushings from the uterine lumen of about 95.1 ng/ml +/- 7.5 ng/ml (means +/- SD after logarithmic transformation, n=8) . The actual luminal concentration of PP14 is much higher, as the concentration of PP14 is diluted in the flushings by the washing fluid. Accordingly, the invention preferably contemplates the administration of PP14 in an effective amount so as to provide a concentration of PP14 which exhibits in-vitro activity when subjected to the testing procedures described below. In this regard, administration of PP14 in an amount ranging from about 0.1 μg/ml to about 5 mg/ml, and preferably from about 5 μg/ml to about 5 mg/ml, is contemplated by the invention to prevent miscarriage. The Placental Protein 14 (PP14) material may be administered to the female by any appropriate route, including intravenous injection, intramuscular injection, oral administration, intra-vaginal administration, intra- cervical administration, intra-uterine administration, intra-tubal administration, topical administration, rectal administration, and inhalation. As previously noted, the PP14 may be delivered to the site of treatment by means of a controlled-release delivery system, or by means of a gel, such that the PP14 does not wash out of the site of administration too easily. Suitable controlled release drug delivery systems and gels are well-known in the pharmaceutical arts. The PP14 active substance may be administered in admixture with one or more pharmaceutically acceptable carriers well-known in the art. The invention is preferably intended for the prevention of miscarriage in females who suffer from recurrent miscarriage; however, the method of treatment of the invention is also believed to be useful to prevent miscarriage in females who have not previously had a

13

SUBSTITUTE SHEET miscarriage. Also, the method of the invention is intended for the treatment of humans and other animals.

(a) measuring the level of PP14 present in a predetermined body fluid of tissue sample of a female; and

(b) comparing the level of PP14 present in the predetermined body fluid or tissue sample of the female to the level of PP14 present in the comparable body fluid or tissue sample of a normal, fertile female, wherein a level of PP14 below that present in a normal, fertile female suggests the possibility of future miscarriage.

As PP14 is synthesized in the endometrial glands and secreted into the uterine lumen, a preferred site to which to measure levels of PP14 is the uterine lumen. PP14 levels present in other body fluids or tissues could be measured and compared with corresponding samples from fertile individuals. A preferred method according to the invention for measuring PP14 in the uterine lumen is by flushing the uterus with a saline solution, collecting the resulting washings, and subsequently determining the concentration of

PP14 at this site using radioimmunoassay or other techniques.

Measurement of the endometrial production of PP14 is known to vary depending upon the particular stage of the menstrual cycle; thus, the day of collection of the washings should be standardized. A preferred day of collection of the endometrial washings is the seventh day after the midcycle luteinizing hormone (LH) peak in the female, which date closely proximates the time of normal implantation in healthy individuals.

The above-discussed technique of collecting endometrial washings is preferably utilized to detect the

14

SUBSTITUTE SHEET level of PP14 present in the uterine lumen of the female . The particular process for measuring PP14 concentration in the body fluid for tissue sample may be carried out using any known assay, including those selected from the group consisting of radioimmunoassay, ELISA assays, and immunoblotting assays. As normal, fertile women exhibit a concentration of PP14 in flushings from the uterine lumen of about 95.1 ng/ml +/- 7.5 ng/ml, levels below this are believed to indicate the possibility of future miscarriage.

It will be understood that the above-described method of testing for the possibility of future miscarriage may be employed in conjunction with other techniques for assessing fertility. For example, hysterosalpingogram, laparoscopy, or hysteroscopy techniques well-known in the art may be used in conjunction with the inventive testing method, to provide a complete prognosis.

A third aspect of the invention relates to a method of improving the efficacy of assisted reproductive techniques, such as artificial insemination (Al) , in-vitro fertilization (IVF) , and gamete intra-fallopian transfer

(GIFT) , by adding an effective amount of PP14 to any sample

(embryo, fertilized or unfertilized egg, mixture of eggs and sperm, or sperm alone) that is to be introduced into the uterus in order to induce a pregnancy.

A description of assisted reproductive techniques is found in Tan et al . , Infertility: Your Questions Answered

(McGraw-Hill Book Co., 1991) , the disclosure of which is incorporated herein by reference. In-vitro fertilization is a technique whereby one or more mature eggs (oocytes) are removed from the woman's ovary, fertilized outside of the body by sperm either from the husband or from a donor, then transferred back into the uterus. The fertilized eggs which are transferred into the uterus are also called "pre-embryos" . A similar process is

15

SUBSTITUTE SHEET embryo transfer (ET) , in which embryos are formed outside of the uterus then transferred into the uterus. An "embryo" is generally defined as the state of a fertilized egg two or more weeks after fertilization. Gamete intra-fallopian transfer (GIFT) is a treatment for women who have patent (not blocked) fallopian tubes. The first few steps of GIFT are similar to those in IVF, namely, stimulation of the ovary with drugs, monitoring follicular growth, administration of human chorionic gonadotrophin (hCG) to induce maturation of the eggs, and collection of the eggs. After collection of the eggs, the maturity of the eggs is observed, then sperm and eggs are separately drawn into a catheter and deposited into the patient's fallopian tubes. The resulting pre-embryo then moves down the fallopian tubes and into the uterus where implantation occurs. Thus, the primary distinction between GIFT and IVF is that in GIFT, fertilization occurs within a fallopian tube, whereas in IVF fertilization occurs in a laboratory. Direct intra-peritoneal insemination (DIPI) involves stimulating the patient's ovaries with drugs, observing the development of the follicles, administering hCG to induce egg maturation, then injecting a washed sample of semen through the top of the vagina into the Pouch of Douglas. Peritoneal oocyte and sperm transfer (POST) is a similar treatment in which eggs and washed sperm are both transferred into the Pouch of Douglas proximate to the opening of the fallopian tubes.

Zygote intra-fallopian tube transfer (ZIFT) , also known as pronuclear stage tubal transfer (PROST) , is an in- vitro fertilization process wherein fertilized eggs are transferred into the fallopian tubes by laparoscopy when the pronuclei form (at about 18 hours following fertilization) . Tubal embryo transfer (TET) , also known as tubal embryo stage transfer (TEST) , is a similar process;

16

SUBSTITUTE however, transfer of the fertilized eggs into the fallopian tubes is not carried out until the pre-embryo has divided into two or more cells.

In direct oocyte transfer (DOT) , eggs are collected according to IVF, then the eggs are incubated for a few hours prior to the addition of sperm. Eggs are transferred into the uterus using IVF once sperm are seen binding to the eggs.

Trans-uterine fallopian transfer (TUFT) involves the transfer of a pre-embryo into the fallopian tube using a catheterizing instrument.

Artificial insemination (Al) is a procedure in which sperm is introduced into the patient's genital tract by artificial means rather than by intercourse. Insemination may be either into the cervix or into the uterine cavity itself (intra-uterine insemination, IUI) .

Unwashed semen is generally not employed in IUI because the protective cervical mucus barrier is bypassed in this process, and unwashed sperm would create a risk of infection or allergic reactions. Also, prostaglandins present in the semen may cause contractions of the uterus which may be painful and cause ejection of semen from the uterus.

Washing of the sperm is carried out by repeated extractions in appropriate solvents, followed by centrifugation to recover the sperm. Alternatively, the semen may be mixed with a cleaning solvent, and the healthy sperm permitted to swim to the surface of the solvent where they are collected for use in the Al or IVF process. PP14 is found in relatively high concentrations in seminal plasma. However, as discussed above, sperm used for artificial insemination (Al) and other assisted reproductive techniques are frequently washed prior to use in order to remove prostaglandins present in the seminal plasma. The invention therefore contemplates the addition

17

SUBSTITUTE SHEET of PP14 to sperm which has been washed, to restore PP14 to levels which exhibit in-vitro activity.

For example, before sperm is introduced into a patient by Al, PP14 may be added to the sperm sample, preferably by including PP14 in the medium used to dilute the washed sperm prior to insemination. Similarly, PP14 may be added to washed sperm before use in other assisted reproductive techniques, in order to replace PP14 which is lost during the conventional washing stage. As discussed above, fertile women have been found to exhibit a concentration of PP14 in flushings from the uterine lumen of about 95.1 ng/ml +/- 7.5 ng/ml. Moreover, in any assisted reproductive technique, whether or not washed sperm are being employed, the addition of extra medium into the uterus along with the gametes inevitably dilutes the normal PP14 concentration present in the uterus. The invention thus contemplates adding an effective amount of PP14 to the gamete medium or the like, so as to provide a PP14 concentration having in-vitro activity when subjected to the testing procedures described below. In general, adding PP14 to the sample in an amount ranging from about 0.1 μg/ml to about 5 mg/ml, and preferably from about 5 μg/ml to about 5 mg/ml, is contemplated by the invention. It will be understood that the amount of PP14 added to the sample to be introduced into the patient to induce pregnancy will vary depending, for example, on the level of PP14 already present in the patient, the particular assisted reproductive technique employed, whether or not washed sperm are employed, the volume of the sample to be introduced into the patient, and other factors understood to those of ordinary skill in the art.

As discussed, the PP14 may be added to any sample that is to be introduced into the reproductive tract of the patient. For example, PP14 may be added to an embryo,

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SUBSTITUTE SHEET fertilized or unfertilized egg, mixture of eggs and sperm, or sperm alone, and may be added prior to, concurrently with, or following introduction of the sample into the patient . The following additional comments, and Examples 1-3 below, further describe the immuno-inhibitory properties of PP14. While not wishing to be bound by any particular theory, it is believed that the potential efficacy of PP14 in preventing miscarriage is related to the immuno- suppressive activity of natural PP14.

PP14 has been found to be a natural inhibitory regulator of the immune response; the protein is present in the tissues and bloodstream of pregnant women. The pregnancy-associated protein, PP14, has been found to inhibit IL-1 production by stimulated macrophages and to inhibit monocytes lymphokine secretion, IL-2 receptor expression and proliferation of mitogen or allogeneically stimulated lymphocytes.

PP14 has been found to inhibit mitogenic stimulation of proliferation and secretion of interferon and IL-2 by lymphocytes. A similar inhibition of allogeneic stimulation of lymphocytes has also been measured. These effects have been found to be accompanied by a reduction in the affinity of high affinity lymphocyte IL-2 receptors and an inhibition of the expression of functional IL-2 receptors.

PP14 suitable for purposes of the present invention is found in extracts of human decidual tissue; the material binds to the monoclonal antibody disclosed in accordance with the present invention. The activities observed from in-vitro test systems correlate with the PP14 content of tissue extracts and other preparations, as measured in a radioimmunoassay for PP14 that utilizes a polyclonal antibody. This radioimmunoassay is described in the publication by Anthony E. BOLTON et al. , entitled "The

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SUBSTITUTE SHEET Radioimmunoassay of Human Placental Protein 14 (PP14)", Clinica Chimica Acta, 135 (1983) 283-291. The PP14 suitable for purposes of the present invention has been observed to be functional in inhibition of the proliferation of mitogen and allogeneically stimulated peripheral blood mononuclear cells; the inhibition of the production and/or release of IL-1 by stimulated peripheral blood mononuclear cells; and the reduction in the affinity of binding of IL-2 to stimulated peripheral blood mononuclear cells.

Activation of the immune regulatory and proliferative capacity of lymphocytes is a complex process mediated by a number of lymphokines and requiring the cooperation of accessory cells. These cells secrete the lymphokine IL-1 in response to antigenic stimulation. This factor is required for the expression of the IL-2 receptor by lymphocytes which then respond mutagenically to IL-2.

PP14 inhibits the elaboration of IL-1 by activated or stimulated peripheral blood monocytes; this may account for many of the modulating activities of PP14. Similar suppressive effects are induced by crude decidual extracts; this activity is abolished by an antibody to PP14. Thus, PP14 has been discovered to have immunosuppressive activity related to its ability to decrease expression or secretion of IL-1 and possibly independent effects on lymphocyte secretion and proliferation.

Many human diseases result from an abnormal, unregulated proliferation of lymphocytes, or from an uncontrolled immune response directed against the patient's own cells or tissues. Some of these defects may result from elevated, unregulated secretion of lymphokines. An example is rheumatoid arthritis, in which IL-1 has been shown to activate synovial prostaglandin and leukotriene production, enhance T-cell binding to endothelia, and to reproduce several aspects of the condition in animal

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SUBSTITUTE SHEET models. Similar evidence exists for other chronic inflammatory diseases. PP14, an inhibitor of IL-1 production, is a potential treatment modality for such diseases, including syndromes such as asthma, which is characterized by excessive secretion of leukotrienes in the bronchial tree, and allergic dermatitis and inflammatory bowel diseases, which are characterized by chronic overproduction of mediators of inflammation.

Other autoimmune diseases, such as systemic lupus erythematosus, Sjogren's syndrome, and scleroderma are characterized by abnormal ratios of a variety of different types of T- and B-lymphocytes which may result from defective regulation of their proliferation. More extreme losses of regulation of growth may be accompanied by further genetic changes in the cells and result in overgrowth of a malignant clone of cells leading to a malignancy in the patient. Such malignant cells often still require autocrine or paracrine stimulation by a lymphokine growth factor in order to drive their proliferation.

The activation of lymphocytes, resulting in cell proliferation, is a complex response mediated by a number of peptide messengers, the cytokines. At a simplified level, T-lymphocytes are activated by a sequential process. First, there is a requirement for a cytokine Interleukin-1

(IL-1) secreted by accessory cells (e.g., cells of the monocyte/macrophage lineage) . In the presence of IL-1 the cytokine Interleukin-2 (IL-2) increases the expression of its own receptors, making the cell more responsive to IL-2 -- a positive feedback cycle. Thus, the proliferation of

T-cells requires the presence of both IL-1 and IL-2.

One approach to investigating the mode of action of lymphoproliferation with an inhibitor such as PP14 is to add an excess of cytokine along with the inhibitor and determine whether the suppression is reversed. One source

21

SUBSTITUTE SHEET of a crude mixture of cytokines is the supernatant taken from cultured activated lymphocytes. Such supernatants were demonstrated to reverse the suppressive action of PP14, indicating a mode of action relating to the secretion/activity of cytokines. The addition of recombinant IL-1 at a single dose significantly reduced the suppressive action of PP14 on lymphoproliferation, as shown in the following Table:

TABLE 1 This table shows the effect of the addition of 5U/ml of recombinant IL-1 on the suppression of tritiated thymidine uptake by stimulated lymphocytes. Decidual sample no. PP14 c ml %su ression of 3H-Tdr

DE A

DE B DE C DE D DE E

Mean (+/-S.D.)

These data indicate that PP14 may be operating via an IL-1-mediated mechanism.

To investigate this possibility further, peripheral blood mononuclear cells (a mixture primary of T-cells and monocytes) were activated using the mitogen PHA in the presence and absence of inhibitory amounts of PP14. The amount of IL-1 released into the supernatants of the cultured cells was measured after different times of culture. The results from the two experiments carried out are shown in the attached Figure, and the data given in

Table 2 below. It can be seen that PP14 significantly inhibited the release of IL-1 into the supernatant by activated cells. These data on IL-1 strongly suggest that

PP14 is acting at the IL-1 level of T-cell activation by inhibiting its synthesis/release.

22

SUBSTITUTE SHEET TABLE 2 This Table shows the effect of PP14 in decidual extracts on the release into cell supernatants of IL-1 by stimulating peripheral blood lymphocytes.

TABLE 2

Experiment 1 TIME (hours)

22.5 41 65 89

Unstimulated 0.2 0.2 0.1 0.1

Immunoabsorbed extract 1.583 1.266 1.232 1.196

Unabsorbed extract 0.406 0.216 0.212 0 . 2

% Suppression 75% 83 % 91? 83 ^!

Experiment 2

Unstimulated

Immunoabsorbed extract 1 . 789 2 . 554 2 . 709 2 . 807

Unabsorbed extract 0.7 1.162 1.630 1.967

% Suppression 61% 55% 40% 30%

NOTES

Unstimulated -- spontaneous release of IL-1 from unstimulated lymphocytes.

Immunoabsorbed extract -- IL-1 release from stimulated cells in the presence of a crude decidual extract from which PP14 had been specifically removed by monoclonal antibody immunoabsorption.

Unabsorbed extract -- IL-1 release from stimulated lymphocytes in the presence of a crude decidual extract containing 8.0 μg/ml of PP14.

23

SUBSTITUTE SHEET Percent suppression -- the suppression of IL-1 released into the cell culture supernatants by PP14 in decidual extracts, expressed as a percent of the release of

IL-1 in the presence of decidual extracts from which PP14 had been removed.

Individual results for each experiment are means of triplicate determinations-.

A monoclonal antibody which specifically binds to PP14 has been isolated and characterized. This monoclonal antibody is designated MAb 14/1/1, and was derived from hybridomal cell lines produced by fusion, using a polyethylene glycol method of spleen cells from mice immunized with crude extracts of deciduum with the myeloma cell line P3/NSl/l-Ag4-l, which is a standard myeloma cell line. Clones secreting anti-PP14 were selected using the radiolabelled PP14 used for radioimmunoassay (BOLTON et al. , 1983, supra) . Positive cultures were cloned three times by limiting dilution and those yielding the highest titers used to induce tumors in Balb/C mice. IgG was isolated from ascitic fluid from ion exchange chromatography or affinity chromatography on protein -A.

The specificity of the antibody was examined in two ways. First, the two-site immunoradiometric assay was set up using a polyclonal extracting antibody and the monoclonal as labelled antibody. Polyclonal antibody, raised against crude decidual tissue extract, was covalently linked to Sepharose-4B by standard procedures. This was incubated in excess in the presence of standard PP14 or the potentially cross-reacting protein for two hours at room temperature. Subsequently, radioiodinated monoclonal antibody was added, incubated a further two hours, and the solid-coupled antibody washed and counted for radioactivity. This represents a conventional 2-site immunoradiometric assay. The cross-reaction of various

24

SUBSTITUTE SHEET decidual/placenta proteins was investigated in this system.

The following gave less than 0.1% cross reaction: hPL, SPl, PP5, PP12, pregnancy-associated plasma protein-A (PAPP-A) , placental alkaline phosphatase, placental malic dehydrogenase, placental sphyngomyelinase, placental arylamidase, placental choline acetyltransferase, with only PP14 having any observed activity in this assay.

The other method involved investigating the binding to the monoclonal antibody of radioactively-labelled pure proteins. No significant binding of prolactin, hCG, or PAPP-A was detected. This antibody could be used in the assay, for example by a two-site immunoradiometric procedure, and purification of PP14. As previously mentioned, the details of the radioimmunoassay for PP14 are disclosed in the 1983 Bolton et al. article, identified above.

The following examples are given to illustrate the invention, but are not deemed to be limiting thereof.

25

SUBSTITUTE SHEET Example 1 Evidence that PP14 inhibits IL-1 release Method 1 - the mitogenic response of human lymphocytes Human peripheral blood lymphocytes proliferate in response to stimulation by the mitogen phytohaemagglutinin (PHA) , the proliferation being measured by the incorporation of tritiated thymidine into the DNA of the dividing cells. This is a standard test of lymphocyte responsiveness. PP14 inhibits lymphocyte proliferation in response to PHA stimulation. The addition of recombinant IL-1 partially reverses the inhibition caused by PP14.

The test system used in this example was to investigate the effect of recombinant IL-1 on the mitogen- induced stimulation of PP14-inhibited peripheral blood lymphocytes. The use of PP14-inhibited cells in experiments such as this unique methodology.

The PP14 inhibition of cells in this example was carried out as follows. Cells were treated with crude extracts of human decidual tissue in which the PP14 concentration was measured by radioimmunoassay. As a control for each treated cell preparation, a portion of the cells were treated with the same extract that had been immunoabsorbed using a monoclonal antibody of PP14 , MAb/PP14/l/l, to remove the PP14 in a highly specific manner. The difference between the activity of the cells treated with these two preparations represented the effect of PP14. Thus, the effects measured were those of PP14 , which specifically binds to this monoclonal antibody. Peripheral blood mononuclear cells were isolated from whole blood obtained from healthy donors by a standard density gradient centrifugation method. After washing in a physiological medium, the cells were resuspended at an appropriate concentration of viable cells and incubated in

26

SUBSTITUTE SHEET the presence of the PP14 preparation and the mitogen at a stimulatory concentration.

The effects of IL-1, or other test compound, were assessed by inclusion at appropriate concentrations in this incubation, including also the necessary controls. The cells were incubated for 72 hours at 37°_SC in an atmosphere of 5% carbon dioxide and 100% humidity under sterile conditions. Six hours prior to termination of the cultures, the cells were pulsed with 1 μCi tritiated thymidine, and on termination the cells were harvested automatically onto glass fiber filters.

The degree of lymphoproliferation was assessed by measuring the incorporation of tritiated thymidine into the harvested cells by liquid scintillation counting. Inhibition of tritiated thymidine uptake by PP14, and the effect of IL-1 were as follows:

% inhibition of tritiated thymidine uptake

Without IL-1 With 5U/ml IL-1

45.4 +/- 11.6 25.0 +/- 8.0 These are mean results from 15 different experiments, each performed in triplicate, using 5 different decidual extracts as" the source of PP14 and cells from 2 separate donors. The results are significantly different (p <0.0001), indicating a significant reversal of the inhibitory effect of PP14 on lymphoproliferation by IL-1.

Example 2 The effect of PP14 on the production of IL-1 by stimulated peripheral blood mononuclear cells. Peripheral blood mononuclear cells, as isolated by density gradient centrifugation, contain not only lymphocytes (the majority of cells present) but also monocyte/macrophages, which are necessary accessory cells

27

SUBSTITUTESHEET in the lymphoproliterative reaction described above. It is this latter cell type which synthesizes and secretes IL-1. These cells can be activated to secrete their specific products which include IL-1 by both mitogens (e.g., PHA) and lipopolysaccharide (LPS) .

The test system used in this example was to investigate the production of macrophage/monocyte products released from stimulated PP14-inhibited peripheral blood mononuclear cell preparations, measuring the products by commercially available immunoassay systems. The use of PP14-inhibition of cells was carried out as in Method 1 of Example 1 above, which again is unique methodology.

Peripheral blood mononuclear cells were prepared as under Method 1. Appropriate numbers of cells were incubated in the presence of the PP14 preparation and the stimulator for various times under the conditions described above. At the termination of the culture period the cells were harvested and the supernatants assayed for IL-1 and Tumour Necrosis Factor (TNF) , both known to be products of macrophage/monocyte cells. Typical results are summarized below.

Inhibition of IL-1 release by stimulated peripheral mononuclear cells by PP14 :

% inhibition of IL-1 release PHA stimulated cells LPS stimulated cells 82.5 67.0

These are mean results from 3 experiments.

This inhibition of IL-1 release from stimulated (using either PHA or LPS) mononuclear cells by PP14 is dose dependent, as shown in Figure 1, wherein • represents cells stimulated with mitogen (PHA) and o represents cells stimulated with LPS.

28

SUBSTITUTE SHEET Example 3 Comparison of IL-1 and TNF production by PP14- inhibited stimulation peripheral blood mononuclear cells. Cells stimulated with mitogen:

IL-1 produced TNF produced stimulated control 2.60 ng/ml 1043 pg/ml

+PP14 0.46 ng/ml 1474 pg/ml

(4 μg/ml)

Example 4 This example illustrates the measurement of PP14 in endometrial washings taken from normal, fertile women in comparison with endometrial washings taken from women having recurrent miscarriage.

Methodology:

PP14 is synthesized in the endometrial glands and secreted into the uterine lumen; therefore the most logical site in which to measure levels of PP14 in these women is in the lumen. Measurement of levels of PP14 in the uterine lumen was achieved by flushing the uterus with 10 ml of saline solution, and collecting the resulting endometrial washings. As it is known that endometrial production of PP14 varies at different stages of the menstrual cycle, the day of collection of the washings was standardized to be the 7th day after the midcycle LH peak in all of the women tested. The day of collection of the endometrial washings therefore was closely related to the normal time of implantation in the uterus.

All measurements of PP14 are expressed in ng/ml, measured using the radioimmunoassay technique discussed above.

SUBSTITUTE SHEET PP14 concentration in normal fertile women:

Initially, the concentration of PP14 present in flushings from the uterine lumen of normal, fertile women was measured. The concentration of PP14 in endometrial washings at day LH + 7 of the menstrual cycle in these fertile women was found to be 95.1 ng/ml +/- 7.5 ng/ml (mean +/- SD after logarithmic transformation, n=8) .

PP14 concentration in women with recurrent miscarriage: The concentration of PP14 in flushings from the uterine lumen of women who had experienced recurrent miscarriage was measured, using the technique discussed above. As defined herein, recurrent miscarriage means the loss of three or more consecutive pregnancies before 20 weeks of gestation.

The concentration of PP14 in the endometrial washings at day LH +7 of the menstrual cycle of 9 women with recurrent miscarriage was as follows: Patient No. Cone, of PP14 in Endometrial Washings (ng/ml)

1 nd

2 5.3

3 nd 4 nd

5 nd

6 nd

7 nd

8 nd 9 200 nd = not detectable in the assay, i.e., < 3 ng/ml

From these data it can be seen that only 1 of the 9 recurrent miscarriage patients had a PP14 level in the endometrial washings within the normal range.

30

SUBSTITUTE SHEET The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention and all such modifications are intended to be included within the scope of the claims.

31

SUB 26412XX

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Bolton, Anthony E.

Drizen, Alan

(ii) TITLE OF INVENTION: METHOD OF TREATING

RECURRENT MISCARRIAGE AND IMPROVING THE EFFICACY OF ASSISTED REPRODUCTIVE TECHNIQUES USING PLACENTAL PROTEIN 14

(iii) NUMBER OF SEQUENCES: 1

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: NATH, AMBERLY & ASSOCIATES

(B) STREET: 1835 K Street, N.W. , Suite 750

(C) CITY: Washington

(D) STATE: D.C.

(E) COUNTRY: U.S.A.

(F) ZIP: 20006

(v) COMPUTER READABLE FORM

(A) MEDIUM TYPE: Diskette, 3.5 inch, 360 Kb storage

(B) COMPUTER: IBM PC/XT/AT compatible

(C) OPERATING SYSTEM: MS-DOS 6.0

(D) SOFTWARE: Word Perfect 5.1 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Nath, Gary M.

(B) REGISTRATION NUMBER: 26,965

(C) REFERENCE/DOCKET NUMBER: 26412XX-PC (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202) 775-8383

(B) TELEFAX: (202) 775-8396 (2) INFORMATION FOR SEQ ID NO:1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 819 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double stranded

(D) TOPOLOGY: linear

(x) PUBLICATION INFORMATION:

(A) AUTHORS: Julkunen, Mervi

Seppala, Markku Janne, Olli A.

(B) TITLE: Complete Amino Acid

Sequence of Human Placental Protein 14 : a Progesterone-

32

SUBSTITUTE SHEET Regulated Uterine Protein

Homologous To

/3-Lactoglobulins

(C) JOURNAL: Proc. Natl. Acad. Sci. USA

(D) VOLUME: 85

(F) PAGES: 8845-8849

(G) DATE: DEC-1988

(K) RELEVANT RESIDUES IN SEQ ID NO:1: FROM 1 to 819

33

SUBSTITUTE SHEET

34

SUBSTITUTE SHEET 539

TGG TAC TTG CTG GAC TTG AAA CAG ATG GAA GAG 572

Trp Tyr Leu Leu Asp Leu Lys Gin Met Glu Glu 150

CCG TGC CGT TTC TAG CTCACCTCCG CCTCCAGGAA 607

Pro Cys Arg Phe AM 160 162

GACCAGACTC CCACCCTTCC ACACCTCCAG AGCAGTGGGA CTTCCTCCTG 657

CCCTTTCAAA GAATAACCAC AGCTCAGAAG ACGATGACGT GGTCATCTGT 707

GTCGCCATCC CCTTCCTGCT GCACACCTGC ACCATTGCCA TGGGGAGGCT 757

GCTCCCTGGG GGCAGAGTCT CTGGCAGAGG TTATTAATAA ACCCTTGGAG 807

CATGAAAAAA AA 819

35

SUBSTITUTE SHEET

Claims

WHAT IS CLAIMED IS:

1. A method of preventing miscarriage in a female by administering to said female a therapeutically effective amount of an active substance from the group consisting of PP14, derivatives of PP14, muteins of PP14, fragments of PP14, and subunits of PP14, so as to prevent miscarriage in the female.

2. The method of claim 1, wherein said derivatives of PP14 comprise PP14 augmented by at least one additional molecule selected from the group consisting of glucose moieties, lipids, phosphate groups, acetyl groups, hydroxyl groups, saccharides, methyl groups, propyl groups, amino acids, and polymeric molecules.

3. The method of claim 1, wherein said derivative of PP14 comprise PP14 having at least one amino acid residue that has been modified by oxidation or reduction.

4. The method of claim 1, wherein the PP14 comprises a dimer of two non-covalently linked protein subunits.

5. The method of claim 4, wherein at least one of said subunits is employed as the active substance administered for said treatment.

6. The method of claim 5, wherein the subunit has the nucleotide and amino acid sequence shown in SEQ ID NO:l.

7. The method of claim 1, wherein the PP14 comprises two covalently linked protein subunits, wherein each of the subunits has the nucleotide and amino acid sequence shown in SEQ ID NO:l.

8. The method of claim 1, wherein said fragment of PP14 has the nucleotide and amino acid sequence of residues 63 through 160 of SEQ ID NO:l.

9. The method of claim 1, wherein said fragment has the nucleotide and amino acid sequence of residues 80 through 105 of SEQ ID NO:l.

36

SUBSTITUTE SHEET

10. The method of claim 1, wherein said active substance is administered by a method selected from the group consisting of intravenous injection, intramuscular injection, oral administration, intra-vaginal administration, intra-cervical administration, intra- uterine administration, intra-tubal administration, topical administration, rectal administration, and inhalation.

11. The method of claim 1, wherein said active substance is obtained from a source selected from the group consisting of mammalian placenta, mammalian blood, amniotic fluid, seminal plasma, cells in tissue culture, decidual cells, decidual organs, endometrial cells, endometrial organs, and sources containing eukaryotic or prokaryotic cells engineered to express PP14, muteins of PP14, fragments of PP14 or subunits of PP14.

12. The method of claim 1, wherein said active substance is administered in admixture with a pharmaceutically acceptable carrier.

13. The method of claim 1, wherein the active substance is administered in an amount of from about 0.1 μg/ml to about 5 mg/ml.

14. The method of claim 1, wherein the active substance is natural PP14.

15. A method of testing a female for the possibility of future miscarriage, which comprises:

(a) measuring the level of PP14 present in a predetermined body fluid or tissue sample of a female; and

(b) comparing the level of PP14 present in the predetermined body fluid or tissue sample of the female to the level of PP14 present in a comparable body fluid or tissue sample of a normal, fertile female, wherein a level of PP14 below that present in ^' a normal, fertile female suggests the possibility of future miscarriage.

37

16. The method of claim 15, wherein step (a) comprises measuring the level of PP14 present in the uterine lumen of a female.

17. The method of claim 16, wherein the level of PP14 in the uterine lumen of the female is measured in step (a) by flushing the uterus with a saline solution, collecting an endometrial washing from the uterus, and measuring the amount of PP14 in the endometrial washing.

18. The method of claim 17, wherein the level of PP14 present in the flushings from the uterine lumen of the female is compared to a normal level of about 95.1 ng/ml +/- 7.5 ng/ml.

19. The method of claim 15, wherein the level of PP14 in the female is measured in step (a) by subjecting a body fluid or tissue sample from the female to an assay selected from the group consisting of radioimmunoassays, ELISA assays, and immunoblotting assays.

20. The method of claim 15, wherein the level of PP14 present in the predetermined body fluid or tissue sample of the female is measured on the seventh day after the midcycle luteinizing hormone peak in the female.

21. A method of improving the efficacy of an assisted reproductive technique, which comprises adding a therapeutically effective amount of PP14 to a sample that is to be introduced into the uterus of a female in order to induce a pregnancy.

22. The method of claim 21, wherein the assisted reproductive technique is artificial insemination.

23. The method of claim 21, wherein the assisted reproductive technique is in-vitro fertilization.

24. The method of claim 21, wherein the assisted reproductive technique is gamete intra-fallopian transfer.

25. The method of claim 21, wherein the sample comprises a fertilized or unfertilized egg.

38

SUBSTITUTE SHEET

26. The method of claim 21, wherein the sample comprises a mixture of sperm and at least one egg.

27. The method of claim 21, wherein the sample comprises sperm.

28. The method of claim 21, wherein the PP14 is added to the sample before the sample is introduced in a uterus.

29. The method of claim 21, wherein the assisted reproductive procedure is artificial insemination, the sample comprises sperm, and the PP14 is added to a medium used to dilute the sperm prior to insemination.

30. The method of claim 21, wherein the PP14 is administered in an amount of from about 0.1 μg/ml to about 5 mg/ml.

31. The method of claim 21, wherein the female is a human.

32. A method of preventing infertility by administering to a female a therapeutically effective amount of an active substance selected from the group consisting of PP14, derivatives of PP14, muteins of PP14, fragments of PP14, and subunits of PP14, so as to prevent miscarriage in the female.

33. The method of claim 21, wherein the PP14 inhibits the production of interleukin I to prevent infertility.

34. The method of claim 32, wherein said derivatives of PP14 comprise PP14 augmented by at least one additional molecule selected from the group consisting of glucose moieties, lipids, phosphate groups, acetyl groups, hydroxyl groups, saccharides, methyl groups, propyl groups, amino acids, and polymeric molecules.

35. The method of claim 32, wherein said derivatives of PP14 comprise PP14 having at least one amino acid residue that has been modified by oxidation or reduction.

36. The method of claim 32, wherein the PP14 comprises a dimer of two non-covalently linked protein subunits.

39

SUBSTITUTE SHEET

37. The method of claim 36, wherein at least one of said subunits is employed as the active substance administered for said treatment.

38. The method of claim 37, wherein the subunit has the nucleotide and amino acid sequence shown in SEQ ID NO:l.

39. The method of claim 32, wherein the PP14 comprises two covalently linked protein subunits, wherein each of the subunits has the nucleotide and amino acid sequence shown in SEQ ID NO:l.

40. The method of claim 32, wherein said fragment of PP14 has the nucleotide and amino acid sequence of residues 63 through 160 of SEQ ID NO:l.

41. The method of claim 32, wherein said fragment has the nucleotide and amino sequence of residues 80 through 105 of SEQ ID NO:l.

42. The method of claim 32, wherein said active substance is administered by a method selected from the group consisting of intravenous injection, intramuscular injection, oral administration, intra-vaginal administration, intra-cervical administration, intra- uterine administration, intra-tubal administration, topical administration, rectal administration, and inhalation.

43. The method of claim 32, wherein said active substance is obtained from a source selected from the group consisting of mammalian placenta, mammalian blood, amniotic fluid, seminal plasma, cells in tissue culture, decidual cells, decidual organs, endometrial cells, endometrial organs, and sources containing eukaryotic or prokaryotic cells engineered to express PP14, muteins of PP14, fragments of PP14 or subunits of PP14.

44. The method of claim 32, wherein said active substance is administered in admixture with a pharmaceutically acceptable carrier.

40

SUBSTITUTE SHEET

45. The method of claim 32, wherein the active substance is administered in an amount of from about 0.1 μg/ml to about 5 mg/ml.

46. The method of claim 32, wherein the active substance is natural PP14.

41

SUBSTITUTE SHEET