Chimeric ovarian follicles-based therapy for treatment of female infertility

Abstract

The present invention relates to methods for reviving aged oocytes by generating chimeric follicles using young somatic cells. Chimeric follicles as defined herein can be used to treat infertility or increase fertility in a female subject.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to Singapore application No. 10202205146T filed on May 17, 2022, the contents of which are hereby incorporated by reference in their entirety for all purposes. Field of the Invention

[0003] The present invention belongs to the field of cell biology. In particular, the present invention relates to a method for rejuvenating aged oocytes. The present invention further discloses a method for treating infertility or improving fertility by using young somatic cells to produce reconstituted chimeric follicles, the reconstituted chimeric follicles comprising aged oocytes and young somatic cells. Background of the Invention

[0005] The mammalian ovary contains many ovarian follicles, which shelter and nourish oocytes during development. The mammalian ovarian follicles contain oocytes and somatic cells or non-germ cells around them, including granulosa cells (GC) and theca cells (theca cell). These somatic cells are the smallest functional units that maintain the reproductive capacity of women, and they grow and develop together with oocytes. Importantly, somatic cells, especially GC, can nourish the developing oocytes by a specialized structure called transzonal projection (transzonalprojection, TZP), and provide energy, nutrients and microenvironment for the development and growth of oocytes. These allow oocytes to undergo maturation with the ability to be fertilized, thereby producing viable embryos. In addition, GC produces the female hormone estrogen, which is crucial for female reproductive health.

[0006] A recent report states that infertility affects one in six people worldwide, and age-related decline in female fertility is a serious global social and clinical problem. Maternal aging has a deleterious effect on fertility achieved either spontaneously (naturally) or via ART / in vitro fertilization (IVF) due to the progressive loss of ovarian follicle quantity and quality with age and limitations of assisted reproductive technology (ART). Maternal aging is associated with somatic cell senescence or apoptosis, leading to ovarian follicular atresia.

[0007] To improve fertility in female subjects, an existing method to rejuvenate aged oocytes is mitochondrial transfer, which works by replacing damaged mitochondria in aged oocytes from recipient female subjects with healthy mitochondria from donor female subjects. This approach is controversial and raises safety concerns because donor mitochondrial DNA is transferred to the developing embryo.

[0008] Therefore, there is a need to develop new methods to rejuvenate aged oocytes for the prevention and treatment of infertility, which improve the number and competence of oocytes without transferring donor DNA to recipient oocytes.

[0009] Overview

[0010] In one aspect, an in vitro method of rejuvenating an aged oocyte is provided, comprising combining an aged oocyte with a donor ovarian follicle that does not contain an oocyte to generate a chimeric ovarian follicle.

[0011] In another aspect, a chimeric ovarian follicle is provided, comprising an aged oocyte and a donor somatic cell.

[0012] In another aspect, a method of treating infertility or improving fertility in a female subject in need thereof is provided, comprising:

[0013] a) obtaining ovarian follicles from a subject and isolating aged oocytes from the ovarian follicles;

[0014] b) obtaining ovarian follicles from a donor and removing oocytes from the donor ovarian follicles;

[0015] c) transplanting aged oocytes from a) together with oocyte-free ovarian follicles from b) to generate chimeric ovarian follicles;

[0016] d) culturing the chimeric ovarian follicles to generate mature chimeric ovarian follicles; and

[0017] e) i) implanting chimeric ovarian follicles into female subjects to treat infertility or enhance fertility; or

[0018] ii) ovulating mature oocytes from mature chimeric ovarian follicles and isolating mature oocytes for in vitro fertilization.

[0019] definition

[0020] As used herein, the term "rejuvenating" in the context of oocytes refers to the process of restoring the microenvironment of an aged oocyte to promote the development, growth and maturation of the oocyte so that the oocyte has the potential to develop into a viable embryo.

[0021] The term "folliculogenesis" refers to the development and maturation of ovarian follicles from the primitive stage to the primary stage, the secondary stage, the early antral (preantral) stage and the antral stage. Ovarian follicles include oocytes and somatic cells. These somatic cells may include, but are not limited to, cumulus cells, granulosa cells and theca cells. The ovarian follicles of each stage may be characterized by the presence of one or more specific cell types and / or phenotypes. For example, the ovarian follicles of the primitive stage may be characterized by the presence of oocytes surrounded by pre-granulosa cells, the ovarian follicles of the primary stage may include oocytes surrounded by a single layer of granulosa cells, and the ovarian follicles of the secondary stage may include oocytes surrounded by more than one layer of granulosa cells. The ovarian follicles of the secondary stage mature into the preantral stage, which includes oocytes, more than one layer of granulosa cells and theca cells. The preantral stage ovarian follicles subsequently progress to the antral stage, which is characterized by forming an antrum surrounding immature oocytes in the somatic cells that include more than one layer of granulosa cells and theca cells. The ovarian follicles in the antral stage refer to preovulatory follicles or mature follicles, and the immature oocytes in the preovulatory follicles may be fully grown germinal vesicle (GV) stage oocytes.

[0022] As used herein, the term "chimeric ovarian follicle" refers to a follicle comprising an oocyte obtained from an ovarian follicle of a first subject and a somatic cell obtained from an ovarian follicle of a second subject. The first subject may also be referred to as a "recipient," and the second subject may also be referred to as a "donor." The terms "chimeric ovarian follicle," "chimeric follicle," "reconstructed chimeric ovarian follicle," and "reconstructed chimeric follicle" are used interchangeably in this context.

[0023] The term "somatic cell" refers to a non-reproductive cell. In the context of the present invention, "somatic cell" refers to a non-reproductive somatic cell that surrounds an oocyte during folliculogenesis in an ovarian follicle. These cells may include, but are not limited to, pre-granulosa cells, cumulus cells, granulosa cells, and theca cells. For example, in the primitive stage of folliculogenesis, somatic cells comprise pre-granulosa cells, in the primary and secondary stages, somatic cells comprise granulosa cells, and in the early antral (pre-antral) stage and antral stage, somatic cells comprise granulosa cells and theca cells. These cells around the oocyte undergo cell division during oocyte development.

[0024] As used herein, the term "aging" with respect to ovarian follicles refers to ovarian follicles whose quality has deteriorated. One measure of ovarian follicle quality is the ability of ovarian follicles to develop and mature during folliculogenesis. Thus, aging ovarian follicles are ovarian follicles that are not able or less able to develop into mature ovarian follicles to induce ovulation of oocytes. Aged ovarian follicles contain aging oocytes and somatic cells that are less able or incapable of promoting oocyte development.

[0025] As used herein, the term "young" with respect to ovarian follicles refers to ovarian follicles that are capable of developing into mature follicles during folliculogenesis.

[0026] "Mature ovarian follicle" refers to an ovarian follicle in the antral stage of follicular development, which contains granulosa cells and fully grown germinal follicle stage oocytes. The terms "mature ovarian follicle" and "preovulatory ovarian follicle" are used interchangeably in this context.

[0027] A "fully grown germinal vesicle stage oocyte" refers to an oocyte that is immature and has the capacity to undergo maturation.

[0028] "Mature oocyte" refers to an oocyte or egg that has undergone meiotic maturation and has the ability to undergo fertilization at metaphase II (MII) arrest. The terms "mature oocyte", "ovulated oocyte" and "MII oocyte" are used interchangeably in this context.

[0029] As used herein, the term "competence" refers to the ability of an oocyte to develop, grow, and mature for fertilization that produces a viable embryo. A viable embryo can progress in the uterus and develop into a fetus.

[0030] The term "subject" refers to a human or non-human mammal. Examples of such mammals include, but are not limited to, primates, mice, rats, guinea pigs, rabbits, and dogs.

[0031] The term "infertility" refers to a disorder of the female reproductive system characterized by the inability or reduced ability of the oocyte to be fertilized by a male germ cell, or the inability or reduced ability of a fertilized oocyte to grow and develop into a viable embryo.

[0032] As used herein, the term "fertile" refers to the ability of an oocyte to be fertilized by a male germ cell or the ability of a fertilized oocyte to grow and develop into a viable embryo.

[0033] As used herein, in the context of the concentration of a component and the percentage of a compound, the term "about" generally refers to + / -5% of the value, + / -4% of the value, more generally + / -3% of the value, more generally + / -2% of the value, even more generally + / -1% of the value, and even more generally + / -0.5% of the value. Throughout the present disclosure, certain embodiments may be disclosed in the form of a range. It should be understood that the description in the form of a range is only for convenience and brevity and should not be interpreted as a rigid limitation on the scope of the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values ​​within the range. For example, the description of a range (such as 1 to 6) should be considered to have specifically disclosed sub-ranges (such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc.) and individual numbers (e.g., 1, 2, 3, 4, 5 and 6) within the range. This applies regardless of the width of the range. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0036] Figure 1 Shows the development of ovarian follicles, the maturation of oocytes during folliculogenesis, and the interactions between oocytes and somatic cells within ovarian follicles. Figure 1 A shows an overview of follicular development, oocyte maturation, and early embryonic development. Figure 1 B shows the physical interaction between the oocyte and granulosa cells within an ovarian follicle during folliculogenesis. The follicle contains somatic or non-germline granulosa cells that nourish the oocyte through a specialized structure called the trans-zone projection (TZP).

[0037] Figure 2 Showing the size and mass of young and aged ovarian follicles in three-dimensional (3D) culture. Figure 2 A shows that aged follicles grow more slowly than young follicles, and Figure 2 B shows the reduction in size of aged follicles compared with the size of young follicles. Figure 2 C shows that aged follicles are more susceptible to atresia during in vitro culture when compared to young follicles. When cultured and encapsulated in a hydrogel of alginate reconstructed basement membrane (rBM) interpenetrating networks (Alg-rBM IPNs), young follicles maintain their 3D structure with GC proliferation and sinus formation (white arrows). In contrast, aged follicles experience increased atresia during in vitro culture ( Figure 2C, right). A follicle is considered atretic if the contact between the oocyte (white arrowhead) and the granulosa cell is disrupted, resulting in the release of the oocyte from the follicle (upper right), or if the follicle contains an apoptotic or dead oocyte (lower right). Figure 2 D shows that the mortality rate is significantly higher in old follicles compared with young follicles.

[0038] Figure 3 showed generation of chimeric follicles to rejuvenate aged oocytes. Figure 3 A shows the method of generating chimeric follicles by implanting aged oocytes into young follicles. Figure 3 B shows that the generated chimeric follicles developed and grew to a size of 300 μm in diameter, and Figure 3 C shows the generated chimeric follicle progressing in development and developing to an antrum stage chimeric follicle (evidenced by the formation of an antrum). Figure 3 D shows the maturation rate of oocytes in chimeric follicles containing young oocytes and young GCs (YY).

[0039] Figure 4 showed that chimeric follicles generated by transplanting aged oocytes in young follicles improved the quality of these aged oocytes. Figure 4 A shows three criteria used to determine the quality of aged oocytes, namely spindle morphology, chromosome segregation and mitochondrial distribution in oocytes. Figure 4 B shows that culturing aged oocytes in young follicles within chimeric follicles (AY) significantly restored meiotic maturation of aged oocytes to a level similar to that of young oocytes grown in young follicles within chimeric follicles (YY). Figure 4 C shows representative live cell images of meiotic spindles and chromosomes in mature oocytes from chimeric follicles containing aged oocytes and young somatic cells (AY) and chimeric follicles containing aged oocytes and aged somatic cells (AA). Figure 4 The left panel of C shows an example of chromosome misalignment in an aged oocyte from an AA-reconstructed mosaic follicle, and Figure 4 C The image in the middle panel shows an example of an abnormal spindle in an aged oocyte from an AA-reconstructed chimeric follicle. Figure 4 C The image in the right panel shows an example of normal spindle morphology (white arrows) and chromosome alignment (white arrowheads) in an aged oocyte from an AY reconstructed chimeric follicle. Figure 4 D shows the quantification of the percentage of chromosome misalignment on the metaphase plate in AA-reconstructed and AY-reconstructed mosaic follicles. Figure 4E shows spindle abnormalities in oocytes from AA-reconstructed mosaic follicles and AY-reconstructed mosaic follicles. Disorganized spindles and misplaced chromosomes were present in aged oocytes from AA-reconstructed mosaic follicles, while the occurrence of spindle and chromosome arrangement abnormalities was significantly reduced in oocytes from AY-reconstructed mosaic follicles. Figure 4 F shows confocal microscopy images of mitochondrial distribution patterns in oocytes from reconstructed chimeric follicles containing 1) young oocytes and young granulosa cells (YY), 2) aged oocytes and aged granulosa cells (AA), and 3) aged oocytes and young granulosa cells (AY). Mitochondria were detected by using MitoTracker Red CMXRos. Figure 4 G shows the average size of mitochondrial clusters in oocytes from 1) YY reconstructed chimeric follicles, 2) AA reconstructed chimeric follicles, and 3) AY reconstructed chimeric follicles. The size of mitochondrial clusters in oocytes from AY reconstructed chimeric follicles was significantly reduced compared to oocytes from AA reconstructed chimeric follicles.

[0040] Figure 5 Showing the effect of young GCs on aneuploidy in aged oocytes. Figure 5 A shows 3D reconstruction of chromosomes and kinetochores in oocytes from AA-reconstructed chimeric follicles and AY-reconstructed chimeric follicles. Figure 5 B shows that the percentage of chromosomal abnormalities was significantly reduced in mosaic follicles compared with senescent follicles.

[0041] Figure 6 Showing the influence of young GCs on embryonic development. Figure 6 A Schematic diagram showing the process of rejuvenation of aged oocytes by young GCs to embryonic development of fertilized eggs. Figure 6 B shows that the percentage of blastocyst formation is higher in aged oocytes (AY) grown with young GCs compared to aged oocytes (AA) grown with aged GCs. Figure 6 C shows immunofluorescence images of blastocysts generated from 1) YY reconstructed chimeric follicles, 2) AA reconstructed chimeric follicles, and 3) AY reconstructed chimeric follicles. Figure 6 D shows that the size and total cell number of blastocysts developed from AY chimeric follicles are significantly improved compared to the size and total cell number of blastocysts developed from AA chimeric follicles. Figure 6 E shows that oocytes from AY reconstructed chimeric follicles produced significantly higher numbers of live pups compared to AA reconstructed chimeric follicles. Representative images of surrogate mothers transplanted with AY and AA reconstructed chimeric follicles and pups from AY and AA reconstructed chimeric follicles are shown in Figure 6Left panel of E. Two pups were born after 20 2-cell stage embryos derived from AY reconstructed chimeric follicles were transferred into surrogate mothers, whereas no pups were born after 20 AA 2-cell stage embryos derived from AA chimeric follicles were transferred. Figure 6 The middle panel of E shows the live birth rate of pups after transfer of 2-cell embryos from YY, AA, and AY oocytes into surrogate mothers. Figure 6 The right panel of E shows day 1 and day 16 pups generated from oocytes obtained from YY and AY chimeric follicles.

[0042] Figure 7 Displayed using MTS-mCherry-GFP 1-10 Possible mitochondrial transport from granulosa cells to oocytes in chimeric follicles in transgenic mice. Figure 7 A shows the experimental design to study mitochondrial transport within chimeric follicles using transgenic MTS-mCherry-GFP expressing mitochondria-targeted mCherry. 1-10 Mouse somatic cells and unlabeled oocytes from wild-type mice were generated. Figure 7 B shows the absence of mitochondrial transport from surrounding somatic cells to the oocyte in chimeric follicles.

[0043] Detailed description of the invention

[0044] The present invention relates to a follicular somatic cell-based therapy for treating female infertility.

[0045] In a first aspect, the invention relates to an in vitro method of rejuvenating aged oocytes comprising combining aged oocytes with donor ovarian follicles that do not contain oocytes to generate chimeric ovarian follicles.

[0046] In one example, aging oocytes are obtained from the ovarian follicles of the subject. The ovarian follicles can be aging ovarian follicles whose quality has deteriorated. The quality of the ovarian follicles can be determined by one or more characteristics or phenotypes. Examples of characteristics or phenotypes that can be used to determine the quality or age of ovarian follicles include but are not limited to the formation of trans-zone projections (TZPs), the ability of somatic cells to promote oocyte development, the progression of ovarian follicles to the antral stage, and the number of mature oocytes produced in each ovulatory cycle.

[0047] In one example, aged ovarian follicles may have poorly formed TZPs relative to young ovarian follicles, delay or failure of ovarian follicle development to mature ovarian follicles or antral stages, slower growth rates, a reduction in the number of TZPs and mature oocytes produced per ovulatory cycle, and increased apoptosis or atresia rates, including somatic cells that may not undergo active division or may undergo less active division and may have increased apoptosis relative to somatic cells of young ovarian follicles, or a combination thereof. Aged oocytes of the present invention may be obtained from ovarian follicles of subjects suffering from infertility or reduced fertility.

[0048] In some instances, the subject may suffer from infertility or reduced fertility due to advanced age. In other instances, the subject may suffer from infertility or reduced fertility due to reasons other than advanced age, such as obesity and / or conditions including but not limited to premature ovarian failure, polycystic ovary syndrome, endometriosis and uterine fibroids. In the context of fertility, it is generally understood that advanced age refers to the actual age of increase. In one example, the subject may have an actual age of about 35 years old or greater than 35 years old. For example, a female subject may be about 35 years old, 40 years old, 45 years old, 50 years old, 55 years old, 60 years old, 65 years old or 70 years old.

[0049] It should be understood that the causes of infertility are not exhaustive and may include any cause that results in the inability of an oocyte to be fertilized by a male germ cell or the inability of a fertilized oocyte to grow and develop into a viable embryo.

[0050] Aged oocyte can be the oocyte that has experienced quality deterioration.A measure of oocyte quality is the ability of oocyte to develop and mature during folliculogenesis.Therefore, aged oocyte refers to the oocyte with insufficient ability or lower ability to develop and mature during folliculogenesis.The phenotype that can be used to determine that oocyte develops into the ability of mature oocyte can include but not limited to the morphology of spindle, the arrangement of chromosome, the distribution of mitochondria, chromosome cohesion and ATP level.Aged oocyte can show spindle abnormality, chromosome misplacement, abnormal mitochondrial distribution, chromosome cohesion change and ATP level change.As is well known in the art, spindle abnormality comprises the spindle with the asymmetric shape exceeding two spindle poles, chromosome is not arranged in the center of oocyte, mitochondria gather in cytoplasmic compartment, chromosome cohesion does not exist or reduces with respect to young oocyte, and ATP level reduces with respect to young oocyte.

[0051] The aging ovarian follicle from which aging oocytes are obtained may comprise somatic cells that are less able or incapable of promoting oocyte development. The inability of somatic cells to promote oocyte development or the reduction in the ability to promote oocyte development may be attributed to the deterioration of somatic cell quality. The ability of somatic cells to promote oocyte development may be determined by one or more of reactive oxidative stress levels, mitochondrial membrane potential, DNA damage and apoptosis.

[0052] In one example, somatic cells that are less capable or incapable of promoting oocyte development, relative to somatic cells that are capable of promoting oocyte development, are characterized by one or more characteristics or phenotypes selected from the group consisting of: higher levels of reactive oxidative stress, lower mitochondrial membrane potential, increased DNA damage and apoptosis, reduced ATP levels, absence or reduction of cohesion, or a combination thereof.

[0053] In the methods of the present invention, aged oocytes obtained from aged ovarian follicles are combined with donor ovarian follicles that do not contain oocytes.

[0054] In one example, the donor ovarian follicle can be a young follicle obtained from a fertile subject. The fertile subject can have a chronological age of about 35 years or less. The subject can be about 35 years old, about 30 years old, about 25 years old, or about 20 years old. In another example, the subject does not suffer from a disease or condition associated with infertility.

[0055] Donor ovarian follicles can be identified by one or more characteristics or phenotypes including, but not limited to, well-formed trans-zone projections (TZPs), somatic cells capable of contributing to oocyte development, the somatic cells are actively dividing and have no or low mortality compared to aged ovarian follicles, and young ovarian follicles that can progress to the antral stage during folliculogenesis or progress to the antral stage within an expected time period.

[0056] Relative to somatic cells that are less capable or incapable of promoting oocyte development, the somatic cells of the donor ovarian follicles that are capable of promoting oocyte development have one or more predetermined phenotypes selected from the group consisting of: lower levels of reactive oxidative stress, higher mitochondrial membrane potential, less DNA damage and apoptosis, increased ATP levels, increased cohesion, or a combination thereof.

[0057] In one example, the aged ovarian follicle from which the aged oocyte is obtained is selected from the group consisting of primordial follicles, secondary follicles, early antral follicles, and antral follicles. It is generally understood that primordial follicles are ovarian follicles in the primordial stage of folliculogenesis, secondary follicles are ovarian follicles in the secondary stage of folliculogenesis, early antral follicles are ovarian follicles in the early antral stage of folliculogenesis, and antral follicles are in the antral stage of folliculogenesis.

[0058] The ovarian follicles of the present invention can be obtained from a subject by means known in the art. In one example, the ovarian follicles can be obtained by enzymatic digestion or manual dissection or both. The ovarian follicles can be obtained from the ovaries obtained from the subject by manually dissecting the ovaries using a needle and incubating the ovaries in a culture medium containing an enzyme to separate the ovarian follicles. The enzyme can be collagenase. In another example, the ovarian follicles can be obtained by using a manual dissection for defollicleing of the ovaries obtained from the subject. Similarly, the oocyte can be obtained or removed from the ovarian follicles obtained from the subject by means known in the art. The oocyte can be obtained or removed from the ovarian follicles manually or by enzymatic digestion. In one example, the oocyte can be obtained or removed from the ovarian follicles by incubating the oocyte in a culture medium containing an enzyme capable of digesting the cells surrounding the oocyte. The enzyme can be trypsin or collagenase. In another example, the oocyte can be obtained or removed from the ovarian follicles by manual means including mouthpipetting. In one example, the subject is a female subject.

[0059] Aged oocytes obtained from aged ovarian follicles are combined with donor ovarian follicles that do not contain oocytes to generate chimeric ovarian follicles. Aged oocytes obtained from aged ovarian follicles are manually combined with donor ovarian follicles that do not contain oocytes to generate chimeric ovarian follicles. In one example, aged oocytes are transplanted into ovarian follicles that do not contain oocytes to generate chimeric follicles using a mouth pipette by picking up oocytes and releasing oocytes into ovarian follicles.

[0060] In one example, follicles isolated from subjects and donors and chimeric ovarian follicles are cultured as three-dimensional cultures. In one example, chimeric ovarian follicles are cultured in alginate gel or Alg-rBM IPN. In one example, Alg-rBM IPN contains alginate and basement membrane protein. In one example, the concentration of alginate gel is about 0.1% to 0.5%. The concentration of alginate gel can be about 0.1%, about 0.2%, about 0.3%, about 0.4% or about 0.5%. In a preferred example, the concentration of alginate gel is about 0.3%.

[0061] In one example, ovarian follicles from a subject and donor ovarian follicles are cultured in a first culture medium, and chimeric ovarian follicles are cultured in a second culture medium. The culture medium may include, but is not limited to, α-minimum essential medium (αMEM), follicle-stimulating hormone (FSH), bovine serum albumin (BSA), fetal bovine serum (FBS), ITS, fetuin, and oocyte-derived growth factors and combinations thereof. In one example, the first culture medium includes αMEM, FSH, BSA, ITS, and fetuin. In one example, the second culture medium includes αMEM, FSH, BSA, ITS, fetuin, and growth differentiation factor 9 (GDF9). In another example, chimeric follicles may be cultured in a culture medium comprising αMEM, FSH, BSA, ITS, fetuin, and growth differentiation factor 9 (GDF9). In another example, chimeric follicles may be cultured in a culture medium comprising αMEM, FSH, BSA, and ITS. In another example, ovarian follicles may be cultured in a culture medium comprising αMEM, FSH, BSA, and ITS.

[0062] In one example, the chimeric ovarian follicle can be cultured in the second culture medium to generate a mature chimeric ovarian follicle, and can be induced to ovulate a mature oocyte from a mature chimeric ovarian follicle. In one example, the mature chimeric ovarian follicle comprises a fully grown germinal vesicle (GV) stage oocyte and a somatic cell. The fully grown germinal vesicle stage oocyte should be understood as an immature oocyte. It should be generally understood that immature oocytes cannot be fertilized, and will need to undergo meiotic maturation to form mature oocytes. In one example, human chorionic gonadotropin (hCG) can be used to induce mature oocytes for ovulation. The mature oocyte of ovulation can then be fertilized by male germ cells and / or can be used for assisted reproductive technology, which includes in vitro fertilization and intracytoplasmic sperm injection (intracytoplasmicsperminjection).

[0063] In one example, the mature chimeric ovarian follicle is an antral stage chimeric follicle.

[0064] In one example, the somatic cells include granulosa cells.

[0065] One or more predetermined phenotypes may be used to determine the quality of mature chimeric ovarian follicles. One or more predetermined phenotypes may include, but are not limited to, the presence of fully grown GV oocytes, the formation of an antrum, and combinations thereof. In one example, mature chimeric ovarian follicles may be characterized by the presence of fully grown GV oocytes and the formation of an antrum.

[0066] In one example, the aged oocyte is a mammalian oocyte, and the donor ovarian follicle is a mammalian ovarian follicle. In a preferred example, the mammalian oocyte is a human or rodent oocyte. In another preferred example, the mammalian donor ovarian follicle is a human or rodent ovarian follicle.

[0067] In another aspect, the present invention relates to a chimeric ovarian follicle comprising an aged oocyte and a donor somatic cell. Without being bound by theory, the donor somatic cell in the chimeric ovarian follicle rejuvenates the aged oocyte, allowing the aged oocyte to develop and mature.

[0068] In one example, aged oocytes can be obtained from aged ovarian follicles containing somatic cells that are less capable or incapable of promoting oocyte development. In one example, donor somatic cells are obtained from young ovarian follicles containing somatic cells that can promote oocyte development, wherein oocytes have been removed. Aged oocytes are combined with young ovarian follicles that do not contain oocytes to generate chimeric ovarian follicles.

[0069] In one example, the donor somatic cells include granulosa cells.

[0070] In another aspect, the present invention relates to a method for treating infertility or improving fertility in a female subject in need thereof, comprising:

[0071] a) obtaining ovarian follicles from a subject and isolating aged oocytes from the ovarian follicles;

[0072] b) obtaining ovarian follicles from a donor and removing oocytes from the donor ovarian follicles;

[0073] c) transplanting aged oocytes from a) together with oocyte-free ovarian follicles from b) to generate chimeric ovarian follicles;

[0074] d) culturing the chimeric ovarian follicles to generate mature chimeric ovarian follicles; and

[0075] e) i) implanting chimeric ovarian follicles into female subjects to treat infertility or enhance fertility; or

[0076] ii) ovulating mature oocytes from mature chimeric ovarian follicles and isolating mature oocytes for in vitro fertilization.

[0077] In one example, the female subject may be infertile, less infertile, or may suffer from a disorder related to fertility. Disorders related to fertility may include, but are not limited to, early menopause and abnormal oocyte maturation.

[0078] The mature chimeric ovarian follicles can be implanted into a female subject via means known in the art. In some examples, the quality of the mature chimeric ovarian follicles can be determined prior to implantation into a female subject.

[0079] In one example, mature oocytes can be ovulated by contacting mature chimeric ovarian follicles with hCG. Mature oocytes can be isolated via means known in the art and can subsequently be used for in vitro fertilization. In one example, oocytes are isolated manually or by enzymatic digestion.

[0080] In one example, the female subject is a female mammalian subject. In one example, the mammalian subject is a human or a rodent.

[0081] In one aspect, provided herein is a use of a chimeric ovarian follicle in the preparation of a medicament for treating infertility or improving fertility in a female subject in need thereof, wherein the chimeric ovarian follicle comprises an oocyte from the female subject and a donor somatic cell.

[0082] A chimeric ovarian follicle comprising an oocyte from a female subject and a donor somatic cell, the chimeric ovarian follicle being used for treatment.

[0083] The present invention described illustratively herein can be suitably implemented in the absence of any one or more elements, one or more restrictions not explicitly disclosed herein. Therefore, for example, the terms "comprise", "include", "contain" etc. should be interpreted broadly and without limitation. In addition, the terms and expressions adopted herein are used in descriptive and non-restrictive terms, and the use of these terms and expressions is not intended to exclude any equivalents of the features shown and described or parts thereof, but it should be recognized that various modifications within the scope of the invention claimed are possible. Therefore, it should be understood that, although the present invention has been specifically disclosed by preferred embodiments and optional features, those skilled in the art may adopt the modifications and changes of the present invention embodied in the preferred embodiments disclosed herein and the optional features, and it is believed that such modifications and changes are within the scope of the invention.

[0084] The invention has been described broadly and generically herein. Each narrower class and sub-general grouping falling within the generic disclosure also forms a part of the invention. This includes the generic description of the invention with any proviso or negative limitation removing any subject matter from the generic class, whether or not the removed subject matter is expressly stated herein.

[0085] Other embodiments are within the accompanying claims and non-limiting examples.In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0086] Experimental Section

[0087] The present invention will be further described in more detail by referring to specific examples, non-limiting examples and comparative examples, which should not be construed as limiting the scope of the present invention in any way.

[0088] Materials and methods

[0089] Isolation, encapsulation and culture of follicles

[0090] Ovaries were dissected from female mice at 2-3 months of age (young) or 14-18 months of age (aging). Ovaries were broken into small pieces and incubated in dissecting medium for 30-40 minutes to obtain individual follicles by using a 26G needle, the dissecting medium containing L-15 (Thermo Scientific, No. 11415064) with 1% fetal bovine serum (FBS) and 100U / ml penicillin / streptomycin and supplemented with 2mg / ml collagenase (SCR103, Sigma) and 10U / ml DNA enzyme (D4263, Sigma). Secondary follicles were then separated and placed in a maintenance medium containing αMEM (Glutamax, Thermo Scientific No. 32561102) with 5%FBS and 100U / ml penicillin-streptomycin for 2 hours, and then encapsulated. Each follicle was individually encapsulated in 4.5 μL alginate-rBM IPN beads. After a single wash with maintenance medium, the IPN beads were placed in 96-well plates or 24-well plates. Follicles were then cultured in growth medium containing a 1:1 mixture of αMEM Glutamax and F-12Glutamax, enriched with 5% FBS, 100mIU / ml follicle stimulating hormone (FSH, Sigma), 5μg / ml insulin, 5μg / ml transferrin and 5μg / ml selenium. Follicle cultures were maintained at 37°C, with half of the growth medium renewed every other day.

[0091] Generation of reconstructed chimeric follicles (RCF)

[0092] To prepare oocytes for transplantation, growing oocytes were denuded from secondary follicles using 0.25% trypsin treatment or repeated mouth pipetting with a thin oocyte-sized glass pipette. Denuded oocytes were placed in maintenance medium containing αMEM (Glutamax, Thermo Scientific No. 32561102) and 5% FBS and 100U / ml penicillin-streptomycin until use.

[0093] In order to generate RCF, by 27G needle tip being gently dragged over culture dish surface to make it bend.First, this curved needle tip is used for anchoring and stabilizing the r-follicle designated for transplantation, prevents the undesirable movement during RCF formation process.Subsequently, pierce follicle gently with the glass mouth pipette of thin oocyte size, and carefully aspirate oocyte, leave empty oocyte bag in follicle.Use the glass mouth pipette of identical thin oocyte size to pick up naked oocyte rapidly, and be released into follicle bag gently.The same method is used for next follicle.After generating 3-10 RCF, these RCF are encapsulated in alginate reconstructed basement membrane (rBM) interpenetrating network (Alg-rBM IPN) beads immediately, and cultivate in the growth medium supplemented with 100ng / ml GDF9.On the second day, replace half of culture medium with the fresh growth medium without GDF9.

[0094] Oocyte maturation, fertilization and embryo culture

[0095] Follicles were extracted from Alg-rBM IPN beads using 10 IU / mL alginate lyase (A1603, Sigma). Oocytes cultured in follicles were exposed and matured in maturation medium (αMEM with 10% FBS, 1.5 IU / ml human chorionic gonadotropin [hCG], 10 ng / ml epidermal growth factor [EGF], and 10 mIU / ml FSH) at 37°C in 5% CO2 in air for 16 hours.

[0096] In vitro matured oocytes were used for in vitro fertilization (IVF). To prepare for IVF, caudae epididymides from two 3-4 month old ICR male mice were placed in 2 drops (100 μL / drop) of mineral oil. Pre-incubation medium (KYD-002-05-EX, Cosmo Bio) was cut to release sperm, and then capacitation was performed at 37 ° C and 5% CO2 for 1 hour. MII oocytes were then placed in 100 μL mHTF medium (KYD-008-02-EX-X5, Cosmo Bio) at 37 ° C and 5% CO2 for 30 minutes, and then fertilized with 3 μL sperm suspension. After 6 hours of fertilization, the zygotes were washed 3 times with mHTF and cultured overnight in 50 μl mHTF. Then, the resulting 2-cell stage embryos were transferred to continuous single culture medium (Continuous Single Culture Media) (CSCM-C, Fujifilm No. 90165), and they were cultured at 37 ° C for 4 days, or they were transferred to pseudo-pregnant female mice.

[0097] Embryo transfer

[0098] Female B6C3HF2 mice (3-6 months old) were used as surrogate mothers in this study. Recipient females (0.5 dpc pseudo-pregnant mice) were anesthetized with 2.5% Avertin. Animals were checked for loss of pedal reflex and sprayed with ethanol. A small incision was then made along the dorsal midline of the skin and muscle layer on the left side of the animal. A drop of epinephrine was placed on the muscle layer before incision to prevent excessive bleeding. The ovarian fat pad was clamped with forceps and pulled out through the incision, carrying with it the ovary, fallopian tube, and the upper part of the left uterine horn. Under a stereomicroscope with a fiber optic light source, a tear was created at the ovarian sac to expose the infundibulum of the fallopian tube and prepare for fallopian tube transfer. The tip of the transfer pipette (loaded with HEPES buffered medium and embryos flanked by "bubbles") was inserted into the infundibulum, and the embryo was blown into the fallopian tube with a mouth pipette. The bubbles in the ampulla indicate a successful transfer. The uterus, fallopian tube, and ovary were returned to their original position in the body cavity. The muscle and skin layers were sutured. The previous steps were repeated to transfer additional embryos to the right oviduct. A total of 18 to 20 two-cell stage embryos were transferred into one female recipient.

[0099] Immunofluorescence

[0100] Oocytes, follicles or embryos were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 40 minutes and then incubated in membrane permeabilization solution (0.5% Triton X-100) for 40 minutes. After blocking overnight with 10% bovine serum albumin in PBS, samples were incubated with primary antibodies at 4°C overnight, followed by incubation with secondary antibodies at room temperature for 1-3 hours.

[0101] Confocal microscopy

[0102] For live follicles, images were acquired with a Zeiss LSM710 confocal microscope with a 10x or 20x objective. For live oocytes, images were acquired with a Zeiss LSM980 microscope using a 40x C-Apochromat 1.2-numerical aperture water immersion objective. Follicles and oocytes were maintained at 37°C with 5% CO2 during imaging. For fixed follicles or oocytes, images were acquired with a Zeiss LSM980 microscope and processed after acquisition using ZEN (Zeiss).

[0103] result

[0104] Example 1

[0105] Ovarian follicle development

[0106] During folliculogenesis, ovarian follicles develop from the primitive stage to the primary stage, secondary stage, and antral stage. Somatic cells including pre-granulosa cells, granulosa cells, and theca cells promote the development of oocytes within the ovarian follicles, and the ovarian follicles grow and mature in the antral stage. The ovarian follicles in the antral stage contain fully grown germinal follicle oocytes that mature after meiotic maturation. The mature oocytes are then released from the ovarian follicles for fertilization ( Figure 1 A). The somatic cells of the ovarian follicle include granulosa cells, cumulus cells, and theca cells, and the ovarian follicle grows and develops together with the oocyte. These somatic cells nourish the oocyte throughout development through a specialized structure called the transzone projection (TZP) ( Figure 1 B).

[0107] The effects of aging on the quality of ovarian follicles were studied. Follicles were cultured in three-dimensional culture, which allowed the quantification of quality attributes between young and aged follicles, namely size and mortality of follicles obtained from young and aged mice. Young follicles grew at a slower rate and had a larger diameter ( 1.3 × 10 cm) after 6 days of culture in three-dimensional culture compared to aged follicles. Figure 2 A and Figure 2 B). During in vitro culture, young follicles maintain a three-dimensional structure with GC proliferation and antrum formation, whereas aged follicles are more susceptible to atresia than young follicles. Premature release of oocytes from atretic follicles has been observed due to disruption of contact between oocytes and granulosa cells or apoptotic or dead oocytes in atretic follicles ( Figure 2 C). In addition, the mortality rate of aged follicles in 3D culture was significantly higher compared with that of young follicles ( Figure 2 D).

[0108] Overall, this suggests that young follicles are of higher quality than old follicles.

[0109] Example 2

[0110] Generation of reconstructed chimeric follicles

[0111] Reconstructed chimeric ovarian follicles were generated to rejuvenate aged oocytes by implanting aged oocytes into young follicles that did not contain oocytes ( Figure 3 A).

[0112] The results confirmed high-quality reconstructed chimeric follicles, which exhibited actively dividing somatic cells and well-formed TZP and ZP, and these reconstructed chimeric follicles were able to progress to the antral stage. These reconstructed chimeric follicles containing young granulosa cells and aged oocytes grew to their maximum potential size of 300 μm in diameter after one week of three-dimensional culture, indicating high-quality ovarian follicles in the preovulatory state ( Figure 3B). Live cell images show that reconstructed chimeric follicles containing aged oocytes and young granulosa cells develop antrums and maintain actively dividing cells, and develop TZP ( Figure 3 C). The number of mature oocytes (MII oocytes) was counted, and the results showed that about 96% of mature oocytes were released from the reconstructed chimeric ovarian follicles containing young oocytes and young somatic cells (YY) during ovulation ( Figure 3 D).

[0113] Example 3

[0114] Restoration of vitality of aged oocytes

[0115] The chimeric follicle technique improves the quality of aging oocytes, such as Figure 4 The maturation rate of oocytes was measured by the three criteria shown in A, including spindle morphology, chromosome segregation, and mitochondrial distribution. In addition, the maturation rate of oocytes was calculated. The maturation rate of oocytes in reconstructed chimeric follicles containing aged oocytes and young granulosa cells (AY) was significantly increased compared with chimeric follicles containing aged oocytes and aged granulosa cells (AA) ( Figure 4 B). The quality of aged oocytes in AY-reconstructed chimeric follicles was significantly higher than that in AA-reconstructed chimeric follicles ( Figure 4 C) Aged oocytes in AA reconstructed mosaic follicles exhibited chromosome misalignment and spindle abnormalities, whereas reconstructed AY mosaic follicles exhibited normal spindle morphology and normal chromosome arrangement.

[0116] The percentages of spindle abnormality and chromosome misalignment were calculated. Compared with oocytes from AA-reconstructed mosaic follicles, oocytes from AY-reconstructed mosaic follicles had more chromosome misalignment ( Figure 4 D) and spindle abnormalities ( Figure 4 E) was significantly reduced. When compared with YY reconstructed chimeric follicles or AA reconstructed chimeric follicles, mitochondrial distribution was improved in AY reconstructed chimeric follicles ( Figure 4 F). The average size of mitochondrial clusters in oocytes from reconstructed chimeric follicles was calculated, and the size of mitochondrial clusters in oocytes from AY reconstructed chimeric follicles was significantly reduced compared with that in oocytes from AA reconstructed chimeric follicles ( Figure 4 G) These findings suggest that the quality of aged oocytes in AY-reconstructed chimeric follicles is significantly higher than that of aged oocytes in AA-reconstructed chimeric follicles and that young granulosa cells improve the quality of aged oocytes.

[0117] Example 4

[0118] Effects of young granulosa cells on aneuploidy of aged oocytes

[0119] One of the main reasons for the increased risk of miscarriage in aging female subjects is the increased rate of aneuploidy in their eggs. The effect of rejuvenating aging oocytes with young granulosa cells on aneuploidy was studied in mice. Euploid mouse eggs contain 20 pairs of chromosomes and therefore have 40 centromeres. Immunofluorescence analysis showed that aging oocytes in AY reconstructed mosaic follicles contained 20 pairs of chromosomes and 40 centromeres, while oocytes in AA reconstructed mosaic follicles contained abnormal numbers of chromosomes and centromeres ( Figure 5 A). The percentage of chromosomal abnormalities was calculated, and the results showed that young GCs reduced the percentage of chromosomal abnormalities in aged oocytes compared to aged oocytes grown with aged GCs ( Figure 5 B).

[0120] Example 5

[0121] Effects of young granulosa cells on embryonic development

[0122] Because young GCs reduce aneuploidy in aged oocytes, the effects of young follicular somatic cell environments on embryonic development were investigated. To evaluate whether young follicular somatic cell environments promote embryonic development, in vitro fertilization was performed and the early embryonic development stages of zygotes were monitored ( Figure 6 A). The results demonstrated that aged oocytes grown with young GCs in AY reconstructed chimeric follicles had a significantly higher percentage of blastocyst formation rate compared to aged oocytes grown with aged GCs in AA reconstructed chimeric follicles ( Figure 6 B). In addition, the percentage of blastema formation in aged oocytes in AY-reconstructed chimeric follicles was significantly higher compared with that in aged oocytes in AA-reconstructed chimeric follicles ( Figure 6 C and Figure 6 D). Subsequently, the live birth rates of pups generated by YY, AY, and AA reconstructed chimeric follicles were calculated. The results showed that the live birth rate of pups generated by AY reconstructed chimeric follicles was significantly increased compared to the live birth rate of pups generated by AA reconstructed chimeric follicles ( Figure 6 E).

[0123] Example 6

[0124] Mitochondrial transport from somatic cells to oocytes

[0125] Existing methods of rejuvenating aged oocytes by mitochondrial transfer, which replaces damaged mitochondria in an egg with healthy mitochondria from another egg of a female donor, are controversial because the developing embryo contains a third unknown donor mitochondrial DNA and poses a safety issue. Therefore, the transport of mitochondria (mtDNA) from surrounding somatic cells to oocytes was studied. MTS-mCherry transgenic mice expressing mitochondrial-targeted mCherry were used to clearly identify the origin of mitochondria. Wild-type oocytes were implanted into follicles isolated from MTS-mCherry transgenic mice ( Figure 7 A). After 5 days of culture, oocytes were isolated from the reconstructed chimeric follicles and the oocyte tissue was stained with anti-mCherry antibody to determine the presence of mCherry-positive mitochondria. The results clearly showed that mCherry-positive mitochondria were absent in wild-type oocytes after co-culture with MTS-mCherry somatic cells ( Figure 7 B) Aged oocytes grown with young granulosa cells contained no DNA from another female subject, confirming that mitochondrial DNA was not transferred to the developing embryo.

[0126] Equivalent solution

[0127] The aforementioned embodiments are presented for the purpose of illustrating the present invention, and should not be construed as imposing any limitation on the scope of the present invention. It will be apparent that many modifications and changes can be made to the specific embodiments of the present invention described above and shown in the embodiments without departing from the basic principles of the present invention. All such modifications and changes are intended to be encompassed by the application.

Claims

1. An in vitro method for rejuvenating aged oocytes comprising combining said aged oocytes with donor ovarian follicles that do not contain oocytes to generate chimeric ovarian follicles.

2. The in vitro method according to claim 1, wherein the aged oocytes are obtained from aged ovarian follicles comprising somatic cells that are less capable or incapable of contributing to oocyte development.

3. The in vitro method of claim 2, wherein the somatic cells that are less capable or incapable of promoting oocyte development are characterized by one or more phenotypes selected from the group consisting of: higher levels of reactive oxidative stress, lower mitochondrial membrane potential, increased DNA damage and apoptosis, reduced ATP levels, absence or reduction of cohesion, or a combination thereof, relative to somatic cells that are capable of promoting oocyte development.

4. The in vitro method according to any one of claims 1 to 3, wherein the donor ovarian follicle is a young follicle.

5. The in vitro method according to any one of claims 1 to 4, wherein the donor ovarian follicles comprise somatic cells capable of promoting oocyte development.

6. The in vitro method of claim 5, wherein the somatic cells capable of promoting oocyte development have one or more predetermined phenotypes selected from the group consisting of: lower reactive oxidative stress levels, higher mitochondrial membrane potential, less DNA damage and apoptosis, increased ATP levels, increased cohesion, or a combination thereof, relative to somatic cells that are less capable or incapable of promoting oocyte development.

7. The in vitro method according to any one of claims 1 to 6, further comprising culturing the chimeric ovarian follicles to generate mature chimeric ovarian follicles and inducing ovulation of mature oocytes from the mature chimeric ovarian follicles.

8. The in vitro method of claim 7, wherein the mature chimeric ovarian follicle is an antral stage chimeric follicle.

9. The in vitro method according to claim 7 or 8, wherein the mature chimeric ovarian follicles comprise fully grown germinal vesicle (GV) stage oocytes and somatic cells.

10. The in vitro method according to any one of claims 3 to 9, wherein the somatic cells comprise granulosa cells.

11. The in vitro method according to any one of claims 1 to 10, wherein the aged oocyte is a mammalian oocyte and the donor ovarian follicle is a mammalian ovarian follicle.

12. The in vitro method of claim 11, wherein the mammalian oocyte is a human or rodent oocyte, and wherein the mammalian donor ovarian follicle is a human or rodent ovarian follicle.

13. A chimeric ovarian follicle comprising an aged oocyte and a donor somatic cell.

14. The chimeric ovarian follicle of claim 13, wherein the aged oocyte is obtained from an aged ovarian follicle comprising somatic cells that are less capable or incapable of contributing to oocyte development.

15. The chimeric ovarian follicle according to claim 13 or 14, wherein the donor somatic cell is obtained from an ovarian follicle comprising somatic cells capable of promoting oocyte development.

16. The chimeric ovarian follicle of claim 15, wherein the donor ovarian follicle is a young follicle.

17. The chimeric ovarian follicle of any one of claims 13-16, wherein the somatic cell is a granulosa cell.

18. A method for treating infertility or improving fertility in a female subject in need thereof, comprising: a) obtaining ovarian follicles from the female subject and isolating aged oocytes from the ovarian follicles; b) obtaining ovarian follicles from a donor and removing oocytes from said donor ovarian follicles; c) transplanting said aged oocyte from a) together with said ovarian follicle without oocyte from b) to generate chimeric ovarian follicles; d) culturing the chimeric ovarian follicles to generate mature chimeric ovarian follicles; as well as e) i) implanting the chimeric ovarian follicle into the female subject to treat infertility or improve fertility; or ii) ovulating a mature oocyte from said mature chimeric ovarian follicle and isolating said mature oocyte for in vitro fertilization.

19. The method of claim 18, wherein the female subject is a female mammalian subject.

20. The method of claim 19, wherein the mammalian subject is a human or a rodent.

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