WO2009042127A2

WO2009042127A2 - Compositions and methods for rescuing fertility

Info

Publication number: WO2009042127A2
Application number: PCT/US2008/011035
Authority: WO
Inventors: Jonathan L. Tilly; Kaisa Selesniemi
Original assignee: The General Hospital Corporation
Priority date: 2007-09-24
Filing date: 2008-09-23
Publication date: 2009-04-02
Also published as: WO2009042127A3

Abstract

As described below, the present invention features compositions and methods for maintaining or increasing fertility (e.g., fertility in an aging female) or promoting neonatal health or survival.

Description

COMPOSITIONS AND METHODS FOR RESCUING FERTILITY

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.: 60/995, 190, filed September 24, 2007, the entire contents of which are incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY

SPONSORED RESEARCH This work was supported by the following grants from the National Institutes of

Health, Grant Nos: R37-012279. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Increasing numbers of women are delaying conception into their thirties and forties. Recent studies indicate that more than 20 percent of women in the United States now have their first child after age 35. As women age, their fertility declines. Reductions in fertility are generally observed in women over the age of 35, and infertility increases dramatically in women over the age of forty, as does the risk of miscarriage. Rising infertility and miscarriage rates are likely due to a number of factors, including declining egg quality. Decreases in implantation rates and pregnancy rates are also seen in older women undergoing assisted reproductive techniques. Maternal aging appears to be the single most important factor in predicting fertility outcome. Current reproductive therapies are not sufficient to enable many infertile women to conceive. Thus, improved methods for rescuing fertility and enhancing egg health in women are required.

SUMMARY OF THE INVENTION

As described below, the present invention features compositions and methods for maintaining or increasing fertility or promoting neonatal health or survival. In a first aspect, the invention features a method of improving reproductive capacity in a female subject (e.g., a subject between 35 and 50 years of age) in need thereof, the method comprising administering to the subject an effective amount of a cellular composition comprising a bone marrow derived cell, thereby improving reproductive capacity in the subject relative to a corresponding untreated control subject. In one embodiment, the method increases the subject's ability to conceive or carry a pregnancy to term relative to an untreated control subject. In another embodiment, the method reduces the risk of miscarriage or still birth relative to an untreated control subject. In a related aspect, the invention features, a method of promoting the health or survival of a neonate born to a female subject, the method comprising administering to the subject an effective amount of a cellular composition comprising a bone marrow derived cell prior to the conception of the neonate, thereby promoting neonatal health or survival. In one embodiment, the method reduces the risk of a genetic or other disorder in the neonate relative to a neonate born to an untreated control subject. In another embodiment, the method increases birth weight or APGAR score in the neonate relative to a neonate born to an untreated control subject, hi yet another embodiment, the method increases egg quality in the female subject.

In another aspect, the invention features a method of maintaining fertility in a female subject at risk of developing an age-related reduction in fertility, the method comprising administering to the subject an effective amount of a cellular composition comprising a bone marrow derived cell, thereby increasing fertility in the subject relative to a corresponding untreated control subject.

In yet another aspect, the invention features a method of increasing fertility in a female subject having an age-related reduction in fertility, the method comprising administering to the subject an effective amount of a cellular composition comprising a bone marrow derived cell, thereby increasing fertility in the subject relative to a corresponding untreated control subject.

In still another aspect, the invention features a method of maintaining fertility in a female subject in need thereof. The method involves harvesting a bone marrow derived cell from the subject; storing the bone marrow derived cell; and re- administering to the subject an effective amount of a cellular composition comprising the bone marrow derived cell, thereby maintaining fertility in the subject. hi another aspect, the invention features a method of treating or preventing infertility in a female subject. The method involves harvesting a bone marrow derived cell from a reproductively fit female subject; storing the bone marrow derived cell; and re- administering the bone marrow derived cell to the subject prior to, during, or after a reduction in reproductive fitness, thereby treating or preventing fertility in the subject relative to a corresponding untreated control subject.

In various embodiments of the above aspects, the method further involves the step of identifying the subject as having or at risk of developing reduced reproductive capacity. In various embodiments of the above aspects, the bone marrow is harvested from the subject prior to a reduction in reproductive capacity (e.g., between the ages of 15 and 35). In other embodiments of the above aspects, the bone marrow is harvested from the subject between the ages of about 21 and 30. In other embodiments of the above aspects, the bone marrow is re-administered to the subject between the ages of about 27 and 50 or between the ages of about 31 and 45. In yet other embodiments of the above aspects, the cellular composition contains between about 10³ and 10⁷ bone marrow derived cells (e.g., between about 15 x 10⁶ and 30 x 10⁶ cells). In various embodiments of the above aspects, the subject is administered one or more infusions comprising a bone marrow derived cell. In one embodiment, the infusion is administered to the subject annually or biannually. In various embodiments of the above aspects, the subject receives a first infusion at about the age of 30 to 35 and continues to receive annual or biannual infusions until the age at which conception is attempted or occurs.

In another aspect, the invention features a method of increasing reproductive capacity in an aging female subject, the method comprising administering to the subject an effective amount of a cellular composition comprising a bone marrow derived cell thereby increasing reproductive capacity in the subject relative to an untreated control subject.

In yet another aspect, the invention features a method of promoting the health or survival of a neonate born to an aging female subject, the method comprising administering to the subject an effective amount of a cellular composition comprising a bone marrow derived cell prior to conception of the neonate, thereby promoting the health or survival of the neonate relative to a neonate born to an untreated control subject. In various embodiments of the above aspects, the method reduces the risk of a genetic disorder in the neonate, increases birth weight or APGAR score in the neonate, or increases egg quality in the maternal subject. In another aspect, the invention features a pharmaceutical composition containing an effective amount of an isolated female bone marrow derived cell labeled for use in the treatment or prevention of an age-related reduction in fertility. In one embodiment, the composition comprises between about 10³ and 10⁷ cells. In another aspect, the invention features a kit comprising an effective amount of a female bone marrow derived cell and directions for use of the kit in the treatment or prevention of age-related infertility.

The invention provides therapeutic methods and compositions for the treatment of infertility, particularly in aging women, and for enhancing neonatal health or survival. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions

By "bone marrow derived cell" is meant a cell harvested from the bone marrow of a subject, or a progenitor or progeny cell thereof.

By "cellular composition" is meant a pharmaceutical composition that contains at least one or more cells. Preferably, the cellular composition contains an effective amount.

By "egg quality" is meant the health of an oocyte. Healthy oocytes are generally free from meiotic, mitotic, and/or genetic defects that would adversely effect a pregnancy, fetus, or neonate.

By "an effective amount" is meant the amount of an agent of the invention needed to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active agent(s) used to practice the present invention for therapeutic treatment of infertility or to increase reproductive capacity varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

By "fertility" is meant the ability of a subject to conceive. By "increasing fertility" is meant increasing the probability that a pregnancy will result in a sexually active female subject.

By "harvest" is meant obtain a cell from a subject using any method known in the art. By "storing" is meant holding under conditions that preserve the viability of a cell. Storage may be for a period of weeks, months, or years. Methods of storing viable bone marrow derived cells are known in the art, and include cryopreservation and culture. By "improve" or "promote" is meant any positive change. Change is determined relative to a corresponding control condition. For example, an increase in fertility is determined relative to the fertility in a corresponding control subject of similar age and/or health. An increase may be by at least about 10%, 20%, 30%, 40%, 50%, 75%, or even by as much as 100%. By "reproductive capacity" is meant the ability of a female subject to conceive a pregnancy, carry the pregnancy to term, and/or deliver a healthy neonate.

By "risk" is meant having a propensity to develop a condition. Risk can be determined on the basis of a genetic predisposition, or by evaluating one or more diagnostic parameters, such as age, health, hormonal production, the presence or absence of menses, and/or oocyte quality.

By "an age-related reduction in fertility" is meant a decrease in the ability to conceive a pregnancy. For example, a reduction in fertility may be detected by an increase in the amount of time needed for a sexually active woman to conceive.

By "obtaining" is meant harvesting, synthesizing, purchasing, or otherwise acquiring an agent.

By "reproductive fitness" is meant having the ability to conceive and carry a pregnancy to term. Women who are reproductively fit typically conceive within several months of commencing sexual activity in the absence of contraception. Women having a reduction in reproductive fitness typically do not conceive a pregnancy within six months, nine months, or twelve months of sexual activity in the absence of contraception.

By "reduces the risk of miscarriage" is meant reduces the propensity of a subject to fail to bring a pregnancy to term. Such a reduction is measured relative to the risk in a corresponding untreated control subject of similar age and physical condition. A reduction may be by at least about 5%, 10%, 20%, 30%, 40%, 50%, 75%, or even by as much as 100%.

By "subject" is meant a mammal, including, but not limited to, a human or non- human mammal, such as a bovine, equine, canine, ovine, or feline. As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition. untreated control subject

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram showing the experimental strategy used to obtain the results described herein. Figures 2A-2H are graphs that summarize the results obtained following bone marrow (BM) infusion into female mice of varying ages. The graphs show the breakdown of individual pregnancies in vehicle- and BM-infused female mice of different age groups, expressed as total number of pups delivered in a given pregnancy (white bars) and the number of those pups that survived to weaning at 21 days. Maternal age groups (in days) are as follows: A, 300-349; B, 350-399; C, 400-449; D, 450-499; E, 500-549; F, 550-599; G, 600+. Note that asterisks designate mice that had to be euthanized due to health complications or that died of natural causes. Panels C and D are highlighted to reflect the age period during which fertility of vehicle-infused mice declines most dramatically. Many age-matched BM-infused females remained fertile during this period and a high incidence of their offspring survived to weaning, as reflected in Figure 2H, which shows the total number of offspring born (white bars) and the total number that survived to weaning (black bars) in vehicle- and BM-infused females between 400-499 days of age (see panels C and D for individual mouse data).

Figure 3 is a graph showing that the total number of offspring that were born and that survived to weaning was greater in BM-infused females than in control females infused with vehicle (VH). Differences between BM and VH-infused females were greatest in females between 400-499 days of age. DETAILED DESCRIPTION OF THE INVENTION

The invention features compositions and methods that are useful for treating or preventing female infertility or promoting neonatal health or survival. The invention is based, at least in part, on the discovery that female fertility and the postnatal survival or health of her progeny can be increased by the administration of bone marrow-derived cells to the female subject at a time prior to the conception of such progeny.

Female Fertility and Aging.

Although female fertility declines with age, increasing numbers of women are postponing childbearing to years when their reproductive potential is suboptimal. Infertility in women increases from 11% to 87% between 35 and 45 years of age. Menopause for women occurs at approximately 50 years of age and is driven by declining ovarian follicle numbers. No clinical strategies are currently available to delay or prevent menopause in women. Three recent studies in mice showed that postnatal renewal of follicles occurs, and that the number of follicles does not start declining until early adulthood. Bone marrow has been shown to contain cells that form oocytes within immature follicles in chemotoxin-conditioned adult female mice. Recent studies show that BM-derived stem cells can differentiate into germ cells in male mice and men, and that bone marrow transplants into young female mice with chemotoxin induced infertility restores fertility and fecundity in the recipient. The results reported herein are the first to show that bone marrow-derived cells can sustain ovarian function and fertility in aging female subjects.

The invention features compositions and methods that are useful for restoring or increasing fertility in aging subjects, as well as enhancing the neonatal health and/or survival of progeny conceived by the subjects, relative to the health and/or survival of untreated control subjects. In one embodiment, the increased survival of the aging female subjects' progeny is related to an increase in oocyte quality. Improvements in oocyte quality include, but are not limited to, a reduced incidence of aneuploidy, reduced meiotic errors, increased fertilization rates, improved preimplantation embryonic developmental potential, and increased postnatal survival of offspring derived from said oocytes.

The present invention provides methods of treating female infertility and/or fertility-related disorders or symptoms thereof, including reductions in fertility normally associated with female aging. The invention involves administering a therapeutically effective amount of a pharmaceutical composition comprising a cellular composition described herein to a subject (e.g., a mammal such as a human female). Thus, one embodiment is a method of treating a subject suffering from or susceptible to infertility, or a fertility disorder or symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of a cellular composition herein sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.

The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a cellular composition described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the cellular compositions herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human female. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for reduced fertility associated with advancing age, or an infertility disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like). The compounds herein may be also used in the treatment of any other disorders in which infertility or a reduction in oocyte quality or ovarian function may be implicated.

In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to reduced fertility or a fertility disorder or symptoms thereof associated with normal aging, premature or normal menopause, reduced ovarian function, reduced oocyte quality, in which the subject has been administered a therapeutic amount of a cellular composition herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.

Cellular Compositions

Compositions of the invention include pharmaceutical compositions comprising bone marrow derived cells and a pharmaceutically acceptable carrier. Administration can be autologous or heterologous. Preferably, cells are obtained from one subject and administered to the same subject. For example, bone marrow derived cells are obtained from a subject during the course of her reproductive life. In one embodiment, bone marrow is harvested at any time following puberty and prior to menopause, for example, between the ages of about 15 and about 50. In another embodiment, bone marrow is harvested from a subject during the peak of the subject's reproductive potential, for example, between the ages of about 18 and about 30. In other embodiments, bone marrow is harvested from a subject at the age of 24, 25, 26, 27, 28, 29, 30, 31, and 32. The bone marrow derived cells are subsequently administered to the subject to maintain, improve, or restore her fertility. In one approach, bone marrow derived cells are administered to a subject at risk of a reduction in fertility. For example, a bone marrow derived cell is administered to a subject between 30 and 40 years of age to prevent a loss or reduction in fertility. In one embodiment, a bone marrow derived cell is administered to a 30, 31, 32, 33, 34, or 35 year old subject to maintain her fertility, to prevent a reduction in fertility (e.g., an age-related reduction in fertility), or to promote the health or survival of prospective progeny. In another embodiment, the cell is administered at least about one, two, three, four, five, six or seven years prior to conception of prospective progeny. In another approach, a bone marrow derived cell is administered to a subject diagnosed as having reduced fertility to increase fertility or to preserve remaining fertility. For example, a bone marrow derived cell is administered to a subject between the ages of 34 and 55 to increase or preserve remaining fertility or to delay menopause. In one embodiment, the cell is administered to a 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45-year-old woman diagnosed as having reduced fertility. Bone marrow derived cells of the invention or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, intrauterine injection, or parenteral administration. When administering a therapeutic or prophylactic composition of the present invention (e.g., a cellular composition), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

Compositions of the invention can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cells utilized in practicing the present invention in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the bone marrow cells, their progenitors, or their progeny.

The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.

Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form).

A method to potentially increase cell survival when introducing the cells into a subject in need thereof is to incorporate bone marrow cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) of interest into a biopolymer or synthetic polymer. Depending on the subject's condition, the site of injection might prove inhospitable for cell seeding and growth because of scarring or other impediments. Examples of biopolymer include, but are not limited to, cells mixed with fibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycans. This could be constructed with or without included expansion or differentiation factors. Additionally, these could be in suspension, but residence time at sites subjected to flow would be nominal. Another alternative is a three-dimensional gel with cells entrapped within the interstices of the cell biopolymer admixture. Again, expansion or differentiation factors could be included with the cells. These could be deployed by injection via various routes described herein.

Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the bone marrow derived cells or their progeny or progenitors as described in the present invention. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

One consideration concerning the therapeutic or prophylactic use of bone marrow derived cells of the invention is the quantity of cells necessary to achieve an optimal effect. In current human studies of autologous mononuclear peripheral blood cells, empirical doses ranging from 1 to 4 x 10⁷ cells have been used with encouraging results. However, different scenarios may require optimization of the amount of cells injected into a tissue of interest. Thus, the quantity of cells to be administered will vary for the subject being treated. In a preferred embodiment, between about 10⁴ to 10⁸, more preferably between about 10⁵ to 10⁷, and still more preferably, about 1 x 10⁶, 3 x 10⁶' 5 x 10⁶' 1 x 10⁷, 3 x 10⁷ , or 5 x 10⁷ cells of the invention can be administered to a human subject.

Fewer cells can be administered, if the cells are administered directly to the abdomen or to the ovary. Preferably, between 10² to 10⁶, more preferably 10³ to 10⁵, and still more preferably, 10⁴ cells can be administered to a human subject. However, the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including their size, age, sex, weight, and condition of the particular patient. As few as 100-1000 cells can be administered for certain desired applications among selected patients. Therefore, dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art. Cells are administered in an amount that is effective to preserve, maintain, enhance, or restore fertility. For some patients, a single infusion may be sufficient to achieve this purpose. Typically, cellular compositions of the invention are provided in one or more infusions administered over the course of between one and ten years. In one embodiment, a cellular composition of the invention is administered to a subject every 3, 6, 9 or 12 months. In another embodiment, the administration continues over the course of between 1 and 10 years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 years). Preferably, the administration is made every

While the specific examples described herein recite the use of bone marrow derived cells, one skilled in the art will appreciate that such cells encompass a heterogenous population of cell types, any of which may be useful in the methods of the invention, hi one embodiment, bone marrow derived cells or peripheral blood derived stem cells of the invention are used. In other embodiments, a cellular composition of the invention comprises a purified population of female germline stem cells. Those skilled in the art can readily determine the percentage of BM- derived stem cells that can differentiate into germ cells or can determine the percentage of female germline stem cells in a population using various well-known methods, such as fluorescence activated cell sorting (FACS). Preferable ranges of purity in populations comprising bone marrow-derived or female germline stem cells are about 50 to about 55%, about 55 to about 60%, and about 65 to about 70%. More preferably the purity is about 70 to about 75%, about 75 to about 80%, about 80 to about 85%; and still more preferably the purity is about 85 to about 90%, about 90 to about 95%, and about 95 to about 100%. Purity of bone marrow-derived or female germline stem cells can be determined according to the genetic marker profile within a population. Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage).

The skilled artisan can readily determine the number of cells and optional additives, vehicles, and/or carrier in compositions and to be administered in methods of the invention. Typically, any additives (in addition to the active stem cell(s) and/or agent(s)) are present in an amount of 0.001 to 50 % (weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, still more preferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and still more preferably about 0.05 to about 5 wt %. Of course, for any composition to be administered to an animal or human, and for any particular method of administration, it is preferred to determine therefore: toxicity, such as by determining the lethal dose (LD) and LD₅₀ in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations can be ascertained without undue experimentation.

Combination Therapies

Cellular compositions of the invention may be administered together with any reproductive therapy known to the skilled artisan, including but not limited to, in vitro fertilization.

Kits

The invention provides kits for the treatment or prevention of infertility, particularly infertility related aging. In another embodiment, the kit provides for the promotion of neonatal health or survival. In one embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of a cell isolated using the methods described herein (e.g., a bone marrow derived cell) in unit dosage form, hi some embodiments, the kit comprises a sterile container that contains a therapeutic or prophylactic cellular composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

If desired a cell of the invention is provided together with instructions for administering the cell to a subject having or at risk of developing infertility or a reduction in fertility associated with normal aging or menopause. The instructions will generally include information about the use of the composition for the treatment or prevention of infertility. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of infertility or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES The experiments described below, were used to determine whether bone marrow infusions into non-conditioned, aging subjects could prevent or rescue age-related decline in fertility and fecundity; to determine whether donor-derived BM cells can contribute to the functional germ line of the recipient; and, to determine the effects of bone marrow infusions on the postnatal survival of the offspring.

Example 1 : Age-related reductions in female fertility were prevented, delayed, or reversed by adult bone marrow infusions

Previous work has shown that bone-marrow transplants or blood transfusions result in donor-derived eggs appearing in the ovaries of chemically sterilized female mice. The present study examines the impact of infused bone-marrow cells harvested from young adult female donors on fertility in aging female mice without any prior chemotherapy/radiotherapy exposure. Wild-type female mice at 8 months of age were given once-monthly tail vein injections of vehicle (VEHj_nf, n=12) or 2-3X10⁷ BM-derived mononuclear cells (BMj_nf, n=12) harvested from young adult (3 months of age) donor females with "ubiquitous" transgenic expression of GFP driven by the chicken β-actin promoter. GFP-expressing mice were obtained from Jackson Laboratory, strain C57BL/6-Tg(ACTB-EGFP)10sb/J. Males were introduced into the females' cages when the mice reached 9 months of age. Treated and untreated control females were mated. Pregnancy rate and offspring survival to weaning (day 21) was monitored before the next mating attempt. Mating trials were conducted from 300 until about 600 days of age, when all females had become infertile. Between 300-349 days of age, comparable numbers of VEHj_nf and BMj_nf females achieved pregnancies with birth of offspring (Figure 1). As the age of the mice increased, increased fertility was observed in the treated group relative to the control group. In subsequent mating trials at least 2 and up to 4 additional mice in the BMj_nf group consistently achieved pregnancies and delivered offspring when compared with age-matched control mice (VEHj_nf counterparts).

Post-natal development may be compromised due to reductions in egg quality associated with increased maternal age. To assess whether offspring born to aged females survived and developed postnatally offspring survival to weaning was analyzed. In mothers of advanced age (between 400-499 days of age), only 37.5% of the 32 pups born to control VEHj_nf females survived to weaning. In contrast, more than 75% of the pups born to BMj_nf females survived to weaning, and none exhibited any overt health complications. No differences in pup survival were observed in younger mothers. Comparable survival to day 21 was observed for offspring born to 300-399 day-old mothers infused with VEH or BM.

Figure 1 provides a breakdown of individual pregnancies in vehicle- and BM- infused (BMj_nf) female mice of different age groups. Results are expressed as total pups delivered in a given pregnancy (white bars) and the number of those pups that survived to weaning at day 21 (black bars). The greatest differences between BM-infused and control mice are shown in Figures 1C and ID. Between 400 and 499 days age period during which fertility of vehicle-infused mice declines most dramatically, whereas many age-matched BMj_nf females remain fertile and exhibit a very high incidence of offspring survival to weaning. This latter point is reflected in panel H, which shows the total number of offspring born (white bars) and that survived to weaning (black bars) in VEH,_nf and BM_inf females between 400-499 days of age (see C and D for individual mouse data). The last successful pregnancy of VEH- and BM-infused females was attained at 597 and 633 days of age, respectively. The total number of offspring born to BMinf females was higher than controls (198 vs 130, respectively; P = 0.12).

Offspring survival to weaning was similar in both groups until maternal age of 399 days, at which time offspring survival to day 21 was higher in BMinf females of 400-499 days of age compared to age-matched VEH-infused controls (78 ± 12% vs 38 ±15%, P < 0.05). Although not statistically significant, offspring survival to weaning was also higher for 500-599 day-old females receiving BMinf than for VEH-infused mice (39± 15% vs 14 ± 8%).

Although pup survival to weaning decreased in pregnancies achieved by BM,_nf females after 500 days of age, 100% of the offspring delivered by two BM,_nf females survived to weaning. Since age-associated deterioration of egg quality is known to profoundly impact on pregnancy outcome, these findings indicate that offspring survival - a marker for egg quality - is vastly improved in aging females by infusions of BM- derived cells.

In sum, age-related loss of female fertility can be prevented, delayed, or reversed by infusions with cells derived from adult bone marrow (Figures 2 and 3). Since the same females retained the ability to become pregnant and deliver offspring over the course of the mating trials, bone marrow infusions are effective at preventing infertility in aging females. Postnatal survival of the offspring delivered by BMinf dams between 400-499 days of age was dramatically improved through as-yet unknown mechanisms. The ability of BMinf to sustain fertility with age appears independent of germline cells in the transplants since all offspring were host-derived.

The results described above were carried out using the following materials and methods.

Animals

Wild-type C57B1/6 female mice were obtained from Taconic (Albany, NY) as retired breeders. Wild-type C57BL/6 male mice and transgenic (Tg) β-actin-promoter- driven-EGFP mice (stock 003291) were originally obtained from Jackson Laboratories (Bar Harbor, Maine). All animal husbandry and procedures were approved by the institutional animal care and use committee of Massachusetts General Hospital. Bone Marrow Infusions

BM cells were isolated by crushing the tibia and femur of young female EGFP- Tg mice. After removal of red blood cells, 15-30x106 mononuclear cells were infused into non-conditioned recipients via lateral tail vein starting at 8-months of age. Parallel infusions into control animals were performed with VEH. Infusions were repeated monthly for 15 months.

Mating Trials and Offspring Survival Starting at 9-months of age, female mice receiving monthly injections of vehicle or BM cells were mated with wild-type C57B1/6 males. The number of pregnancies with pups delivered per female, and the number of offspring as well as offspring survival to weaning (day 21) were monitored. The next mating attempt was performed after weaning of offspring from the prior pregnancy. The genotype of each offspring was determined by PCR amplification of genomic DNA using primers specific to EGFP.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A method of improving reproductive capacity in a female subject in need thereof, the method comprising administering to the subject an effective amount of a cellular composition comprising a bone marrow derived cell, thereby improving reproductive capacity in the subject relative to a corresponding untreated control subject.

2. The method of claim 1, wherein the method increases the subject's ability to conceive or carry a pregnancy to term relative to an untreated control subject.

3. The method of claim 1, wherein the method reduces the risk of miscarriage or still birth relative to an untreated control subject.

4. A method of promoting the health or survival of a neonate born to a female subject, the method comprising administering to the subject an effective amount of a cellular composition comprising a bone marrow derived cell prior to the conception of the neonate, thereby promoting neonatal health or survival.

5. The method of claim 4, wherein the method reduces the risk of a genetic or other disorder in the neonate relative to a neonate born to an untreated control subject.

6. The method of claim 4, wherein the method increases birth weight or APGAR score in the neonate relative to a neonate born to an untreated control subject.

7. The method of any one of claims 1-4, wherein the method increases egg quality in the female subject.

8. A method of maintaining fertility in a female subject at risk of developing an age-related reduction in fertility, the method comprising administering to the subject an effective amount of a cellular composition comprising a bone marrow derived cell, thereby increasing fertility in the subject relative to a corresponding untreated control subject.

9. A method of increasing fertility in a female subject having an age-related reduction in fertility, the method comprising administering to the subject an effective amount of a cellular composition comprising a bone marrow derived cell, thereby increasing fertility in the subject relative to a corresponding untreated control subject.

10. A method of maintaining fertility in a female subject in need thereof, the method comprising

(a) harvesting a bone marrow derived cell from the subject;

(b) storing the bone marrow derived cell; and (c) re-administering to the subject an effective amount of a cellular composition comprising the bone marrow derived cell, thereby maintaining fertility in the subject.

11. A method of treating or preventing infertility in a female subject, the method comprising (a) harvesting a bone marrow derived cell from a reproductively fit female subject;

(b) storing the bone marrow derived cell; and

(c) re-administering the bone marrow derived cell to the subject prior to, during, or after a reduction in reproductive fitness, thereby treating or preventing fertility in the subject relative to a corresponding untreated control subject.

12. The method of any one of claims 1-11, wherein the method further comprises the step of identifying the subject as having or at risk of developing reduced reproductive capacity.

13. The method of any one of claims 1-11, wherein the bone marrow is harvested from the subject between the ages of 15 and 35.

14. The method of claim 13, wherein the bone marrow is harvested from the subject between the ages of about 21 and 30.

15. The method of any one of claims 1-11, wherein the bone marrow is re- administered to the subject between the ages of about 27 and 50.

16. The method of any one of claims 1-11, wherein the bone marrow is re- administered to the subject between the ages of about 31 and 45.

17. The method of any one of claims 1-11, wherein the cellular composition comprises between about 10³ and 10⁷ bone marrow derived cells.

18. The method of claim 9, wherein the cellular composition comprises between about 15 x 10⁶ and 30 x 10⁶ cells.

19. The method of any one of claims 1 -3, wherein the subject is administered one or more infusions comprising a bone marrow derived cell.

20. The method of claim 20, wherein the infusion is administered to the subject annually or biannually.

21. The method of claim 20, wherein the subject receives a first infusion at about the age of 30 to 35.

22. The method of claim 20, wherein the subject receives annual or biannual infusions until the age at which conception is attempted or occurs.

23. A method of increasing reproductive capacity in an aging female subject, the method comprising administering to the subject an effective amount of a cellular composition comprising a bone marrow derived cell thereby increasing reproductive capacity in the subject relative to an untreated control subject.

24. A method of promoting the health or survival of a neonate born to an aging female subject, the method comprising administering to the subject an effective amount of a cellular composition comprising a bone marrow derived cell prior to conception of the neonate, thereby promoting the health or survival of the neonate relative to a neonate born to an untreated control subject.

25. The method of claim 24, wherein the method reduces the risk of a genetic disorder in the neonate.

26. The method of claim 24, wherein the method increases birth weight or APGAR score in the neonate.

27. The method of claim 23 or 24, wherein the method increases egg quality in the maternal subject.

28. The method of any one of claims 1-9, wherein the method further involves the step of harvesting a bone marrow derived cell from the subject.

29. A pharmaceutical composition comprising an effective amount of an isolated female bone marrow derived cell labeled for use in the treatment or prevention of an age-related reduction in fertility.

30. The pharmaceutical composition of claim 29, wherein the composition comprises between about 10³ and 10⁷ cells.

31. A kit comprising an effective amount of a female bone marrow derived cell and directions for use of the kit in the treatment or prevention of age-related infertility.

32. The kit of claim 31 , wherein the instructions are for using the kit in the method of any one of claims 1-27.