WO2020210741A1 - Embryo culture media supplement - Google Patents

Abstract

Described herein are improved supplemental compositions for use in methods of culturing embryos in media that have not previously been recognized as beneficial for embryo development. Also provided are methods and kits related to the same.

Description

EMBRYO CULTURE MEDIA SUPPLEMENT

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent

Application No. 62/833,505, filed April 12, 2019, the entire contents of which are incorporated herein by reference.

FIELD OF USE

[0002] The present application generally relates to methods and compositions for in vitro culture. In particular, the methods and compositions generally relate to culture media supplements and methods of their use that have not previously been recognized as beneficial for embryo development and in serum free medium.

BACKGROUND

[0003] Infertility generally refers to the inability to conceive after a period of unprotected sex. It is estimated that approximately 6% of women in the U.S. experience infertility. In the UK, some reports suggest that about 14% of couples experience fertility challenges. A number of factors can contribute to or be the primary cause of infertility, and can affect both genders. Factors can include, but are not limited to, DNA damage, genetics, health problems, toxins, immune system challenges, hormonal or endocrine issues, and infections (caused by viruses or other microorganisms).

[0004] In vitro fertilization (IVF) is one approach that can assist in achieving a successful pregnancy for a couple that otherwise may be infertile. IVF is a process by which an egg from a mother is fertilized by a sperm outside of the body, or in vitro. Despite its success, IVF methods stand to gain from improvements in technology and methodology In particular, improvements in the reproducibility of cell culture methods are needed to advance the preservation of the biological activities of cells that undergo cell processing activities, including embryo growth and development, cryopreservation, thawing, resuspension, expansion, and fertilization. [0005] Currently, many culture methods involve the use of a serum supplement such as Synthetic Serum Substitute (SSS), Plasmanate (PL), or maternal serum (MS). These supplements often contain a mixture of components derived from human donors such as human serum albumin and an aqueous lipoprotein fraction derived from human plasma (e.g., Fraction IV). Unfortunately, these supplements are prone to variations in quality and stability. Thus, there is a need for an alternative serum supplement that can avoid these deviations to provide reproducible improvements to cell culture methods and outcomes.

SUMMARY

[0006] This invention is predicated on the unexpected discovery that culturing embryos in a medium that is supplemented with a chemically defined supplement comprising recombinant human serum albumin, dextran, and one or more CD44 ligands leads to improved embryo development and healthier embryos. Accordingly, this disclosure generally provides supplemental compositions, culture media, kits, and methods of their use in culturing embryos, enhancing sperm survival, enhancing egg survival, enhancing

fertilization, and assessing a product for use in human Assisted Reproductive Technologies.

[0007] In some aspects, provided herein are supplemental compositions for cell culture comprising, consisting of, or consisting essentially of: (a) Human Serum Albumin (HSA); (b) dextran; and (c) one or more CD44 ligands. In some embodiments, the supplemental compositions further comprise, consist of, or consist essentially of insulin, epidermal growth factor (EGF), and/or platelet-derived growth factor (PDGF).

[0008] In some embodiments of the supplemental composition, the HSA is recombinant HSA (rHSA). In some embodiments, the rHSA is expressed in bacteria, yeast, insects, or plants. In some embodiments, the rHSA is expressed in yeast.

[0009] In some embodiments of the supplemental compositions, the CD44 ligand is selected from the group of matrix metalloproteinase, hyaluronic acid, osteopontin, serglycin, chondroitin, fibrin, P-selectin, basic Fibroblast Growth Factor (b-FGF), heparin binding epidermal growth factor (HB-EGF), collagen, fibronectin, and laminin. In some

embodiments, the collagen is selected from collagen I, collagen II, collagen III, collagen IV, and collagen V. In some embodiments of the supplemental compositions, the collagen is collagen IV. In some embodiments of the supplemental compositions, the CD44 ligand is fibronectin. In some embodiments of the supplemental compositions, the fibronectin is recombinant fibronectin. In some embodiments of the supplemental compositions, the laminin is selected from laminin-111, laminin-211, laminin-121, laminin-221, laminin-332, laminin-3 A32, laminin-3B32, laminin-311, laminin-3al 1, laminin-321, laminin-3 A21, laminin-411, laminin-421, laminin-511, laminin-521, laminin-213, laminin-423, laminin-522, and laminin-523.

[0010] In some embodiments of the supplemental compositions, the compositions further comprise, consist of, or consist essentially of one or more embryotrophic factors. In some embodiments of the supplemental compositions, the embryotrophic factor is an anti oxidant. In some embodiments of the supplemental compositions, the embryotrophic factor is conditioned media derived exosomes from mesenchymal stem cells.

[0011] In some aspects, provided herein are methods of culturing an embryo in vitro , comprising, consisting of, or consisting essentially of culturing the embryo in a medium supplemented with a composition comprising, consisting of, or consisting essentially of: (a) Human Serum Albumin (HSA); (b) dextran; and (c) one or more CD44 ligands. In some embodiments, the supplemental compositions further comprise, consist of, or consist essentially of insulin, epidermal growth factor (EGF), and/or platelet-derived growth factor (PDGF).

[0012] In some embodiments of the methods, the medium is a sequential medium or a medium that is replenished at least once during the culturing of the embryo. In some embodiments of the methods, the medium is a medium suitable for culturing a human embryo. In some embodiments of the methods, the medium is Continuous Single Culture™ medium, P-1 medium, G1 medium, G2 medium, Human Tubal Fluid (HTF) medium, modified HTF medium, Whitten’s medium, Ham’s F-10 medium, KSOM medium, Global Medium, or Single Step Medium™. In some embodiments, the medium does not contain serum. [0013] In some embodiments of the methods, the HSA is recombinant HSA (rHSA).

In some embodiments of the supplemental compositions, the rHSA is expressed in bacteria, yeast, insects, or plants. In some embodiments, the rHSA is expressed in yeast.

[0014] In some embodiments of the methods, at least one biological parent of the embryo is suffering from infertility or undergoing infertility treatment.

[0015] In some embodiments of the methods, the embryo is cultured for about 1 day to about 7 days. In some embodiments of the methods, the embryo is cultured for about 3 days to about 6 days. In some embodiments of the methods, the embryo is cultured until it reaches an 8-cell stage or a blastocyst stage.

[0016] In some embodiments of the methods, the methods further comprise, consist of, or consist essentially of implanting the embryo into a female patient.

[0017] In some aspects, provided herein are methods of assessing a product for use in human Assisted Reproductive Technologies, comprising, consisting of, or consisting essentially of: (a) culturing an embryo in a medium supplemented with a composition comprising, consisting of, or consisting essentially of: (a) Human Serum Albumin (HSA); (b) dextran; and (c) one or more CD44 ligands in the presence of the product; (b) evaluating the embryo during at least one stage of development; and (c) determining the acceptability of the product based on the evaluation. In some embodiments, the medium does not contain serum. In some embodiments, the supplemental compositions further comprise, consist of, or consist essentially of insulin, epidermal growth factor (EGF), and/or platelet-derived growth factor (PDGF).

[0018] In some embodiments of the methods, the HSA is recombinant HSA (rHSA).

In some embodiments of the supplemental compositions, the rHSA is expressed in bacteria, yeast, insects, or plants. In some embodiments, the rHSA is expressed in yeast.

[0019] In some embodiments of the methods, the embryo is a mouse embryo. In some embodiments of the methods, the embryo expresses a reporter gene. In some embodiments of the methods, the evaluation comprises evaluating the morphology of the embryo. In some embodiments of the methods, the evaluation comprises evaluating the expression of the reporter gene. In some embodiments, the medium does not contain serum. In some embodiments, the supplemental compositions further comprise, consist of, or consist essentially of insulin, epidermal growth factor (EGF), and/or platelet-derived growth factor (PDGF).

[0020] In some aspects, provided herein are methods of fertilizing an egg, comprising, consisting of, or consisting essentially of fertilizing the egg in a medium supplemented with a composition comprising, consisting of, or consisting essentially of: (a) Human Serum Albumin (HSA); (b) dextran; and (c) one or more CD44 ligands. In some embodiments, the egg is from a subject suffering from infertility or undergoing infertility treatment. In some embodiments, the medium does not contain serum. In some embodiments, the supplemental compositions further comprise, consist of, or consist essentially of insulin, epidermal growth factor (EGF), and/or platelet-derived growth factor (PDGF).

[0021] In some embodiments of the methods, the HSA is recombinant HSA (rHSA).

In some embodiments of the supplemental compositions, the rHSA is expressed in bacteria, yeast, insects, or plants. In some embodiments, the rHSA is expressed in yeast.

[0022] In some aspects, provided herein are methods of enhancing sperm survival, comprising culturing the sperm in a medium supplemented with a composition comprising, consisting of, or consisting essentially of: (a) Human Serum Albumin (HSA); (b) dextran; and (c) one or more CD44 ligands. In some embodiments, the sperm is from a subject suffering from infertility or undergoing infertility treatment. In some embodiments, the medium does not contain serum. In some embodiments, the supplemental compositions further comprise, consist of, or consist essentially of insulin, epidermal growth factor (EGF), and/or platelet-derived growth factor (PDGF).

[0023] In some embodiments of the methods, the HSA is recombinant HSA (rHSA).

In some embodiments of the supplemental compositions, the rHSA is expressed in bacteria, yeast, insects, or plants. In some embodiments, the rHSA is expressed in yeast.

[0024] In some embodiments of the methods, the concentration of recombinant HSA in the supplemented medium is about 1 mg/mL. In some embodiments of the methods, the concentration of dextran in the supplemented medium is about 2 mg/mL. In some

embodiments of the methods, the concentration of insulin in the supplemented medium is about 0.1. mg/L to about 3 mg/L. Insulin further may be included at a concentration of about 0.2 mg/L, about 0.3 mg/L, about 0.4 mg/L, about 0.5 mg/L, about 0.6 mg/L, about 0.7 mg/L, about 0.8 mg/L, about 0.9 mg/L, about 1.0 mg/L, about 1.1 mg/L, about 1.2 mg/L, about 1.3 mg/L, about 1.4 mg/L, about 1.5 mg/L, about 1.6 mg/L, about 1.7 mg/L, about 1.8 mg/L, about 1.9 mg/L, about 2.0 mg/L, about 2.1 mg/L, about 2.2 mg/L, about 2.3 mg/L, about 2.4 mg/L, about 2.5 mg/L, about 2.6 mg/L, about 2.7 mg/L, about 2.8 mg/L, about 2.9 mg/L, or about 3.0 mg/L. In certain embodiments, the product concentration of insulin is about 2 mg/L. In certain embodiments, the working concentration is about 0.2 mg/L. In certain embodiments, the product concentration is about 2 mg/L.

[0025] In some embodiments of the methods, the concentration of EGF in the supplemented medium is about 0 pg/L to about 12 pg/L. For product concentrations, EGF further may be included at a concentration of about 8.5 pg/L, about 8.6 pg/L, 8.7 pg/L, 8.8 pg/L, 8.9 pg/L, 9.0 pg/L, 9.1 pg/L, 9.2 pg/L, 9.3 pg/L, 9.4 pg/L, 9.5 pg/L, about 9.6 pg/L, about 9.7 pg/L, about 9.8 pg/L, about 9.9 pg/L, about 10.0 pg/L, about 10.1 pg/L, about 10.2 pg/L, about 10.3 pg/L, about 10.4 pg/L, about 10.5 pg/L, about 10.6 pg/L, about 10.7 pg/L, 10.8 pg/L, about 10.9 pg/L, about 11 pg/L, about 11.1 pg/L, about 11.2 pg/L, about 11.3 pg/L, about 11.4 pg/L, about 11.5 pg/L, about 11.6 pg/L, about 11.7 pg/L, about 11.8 pg/L, about 11.9 pg/L, or about 12.0 pg/L. For working concentrations, EGF further may be included at a concentration of about 0.1 pg/L, 0.2 pg/L, 0.3 pg/L, 0.4 pg/L, 0.5 pg/L, 0.6 pg/L, 0.7 pg/L, 0.8 pg/L, 0.9 pg/L, 1.0 pg/L, 1.1 pg/L, 1.2 pg/L, 1.3 pg/L, 1.4 pg/L. In certain embodiments, the product concentration of EGF may be about 10 pg/L. In certain embodiments, the working concentration of EGF may be about 1 pg/L.

[0026] In some embodiments of the methods, the concentration of PDGF in the supplemented medium is about 0.01 to about 0.4. For product concentrations, PDGF further may be included at a concentration of about 0.017 pg/L, about 0.018 pg/L, about 0.019 pg/L, about 0.020 pg/L, about 0.021 pg/L, about 0.022 pg/L, about 0.023 pg/L, about 0.024 pg/L, about 0.025 pg/L, about 0.026 pg/L, about 0.027 pg/L, about 0.028 pg/L, about 0.029 pg/L, about 0.030 pg/L, about 0.031 pg/L. For working concentrations, PDGF further may be included at a concentration of about 0.17 pg/L, about 0.18 pg/L, about 0.19 pg/L, about 0.20 pg/L, about 0.21 pg/L, about 0.22 pg/L, about 0.23 pg/L, about 0.24 pg/L, about 0.25 pg/L, about 0.26 pg/L, about 0.27 pg/L, about 0.28 pg/L, about 0.29 pg/L, about 0.30 pg/L, about 0.31 pg/L. In certain embodiments, the product concentration of PDGF may be about 0.24 pg/L. In certain embodiments, the working concentration of PDGF may be about 0.024 Pg/L.

[0027] In some aspects, provided herein are kits with a composition comprising, consisting of, or consisting essentially of: (a) Human Serum Albumin (HSA); (b) dextran; and (c) one or more CD44 ligands and instructions for use. In some embodiments, the compositions further comprise, consist of, or consist essentially of insulin, epidermal growth factor (EGF), and/or platelet-derived growth factor (PDGF). In some embodiments, the kits further comprise, consist of, or consist essentially of a culture medium suitable for use in culturing an embryo, fertilizing an egg, or culturing sperm.

[0028] In some embodiments of the kits, the HSA is recombinant HSA (rHSA). In some embodiments of the supplemental compositions, the rHSA is expressed in bacteria, yeast, insects, or plants. In some embodiments, the rHSA is expressed in yeast.

[0029] In some aspects, provided herein is a culture medium comprising, consisting of, or consisting essentially of: (a) a culture medium suitable for use in culturing an embryo, fertilizing an egg, or culturing sperm; (b) rHSA; (c) dextran; and (d) one or more CD44 ligands. In some embodiments of the medium, the HSA is recombinant HSA (rHSA). In some embodiments of the medium, the rHSA is expressed in bacteria, yeast, insects, or plants. In some embodiments, the rHSA is expressed in yeast.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. l is a graphical depiction of evaluations of collagen I and collagen IV and dextran.

[0031] FIG. 2 shows Enzyme Linked ImmunoSorbent Assay (ELISA) results for experimental growth factors and cytokines. [0032] FIG. 3 is a graphical depiction of a MEGA™ Assay evaluating nine experimental conditions at 48 hours.

[0033] FIG. 4 is a graphical depiction of a MEGA™ Assay evaluating nine experimental conditions at 96 hours.

[0034] FIG. 5 is a graphical depiction of a mouse embryo assay (MEA) evaluating eight experimental conditions at 96 hours.

[0035] FIG. 6 is a graphical depiction of a bovine fertilization assay evaluating eighteen experimental conditions.

[0036] FIG. 7 is a graphical depiction of a bovine embryo assay evaluating nine experimental conditions.

DETAILED DESCRIPTION

[0037] After reading this description it will become apparent to one skilled in the art how to implement the invention in various alternative embodiments and alternative applications. However, all the various embodiments of the present invention will not be described herein. It will be understood that the embodiments presented here are presented by way of an example only, and not limitation. As such, this detailed description of various alternative embodiments should not be construed to limit the scope or breadth of the present invention as set forth below.

[0038] Before the present invention is disclosed and described, it is to be understood that the aspects described below are not limited to specific compositions, methods of preparing such compositions, or uses thereof as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [0040] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term“about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0041] It must be noted that as used herein and in the appended claims, the singular forms“a”,“an”, and“the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“an embryo” includes a plurality of embryos.

[0042] The term“about” when used before a numerical designation, e.g.,

temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by ( + ) or ( - ) 10 %, 5 %, 3 %, 2 %, 1 %, 0.1%, or 0.01 %.

[0043] As used herein, the term“biological equivalent thereof’ is intended to be synonymous with“equivalent thereof’ when referring to a reference protein, antibody, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. For example, an equivalent intends at least about 70% homology or identity, or at least 80 % homology or identity and alternatively, or at least about 85 %, or alternatively at least about 90 %, or alternatively at least about 95 %, or alternatively 98 % percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid.

[0044] As used herein, the term“CD44 ligand” refers to any molecule capable of binding to CD44, a CD44 variant, a CD44 glycoform, or an equivalent of each thereof.

CD44 is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. In humans, the CD44 antigen is encoded by the CD44 gene on Chromosome 11 (EntrezGene: 690; UniProt: P16070). CD44 variants are proteins produced through alternative splicing of one or more CD44 exons (e.g., CD44vRA). In some embodiments, the CD44 ligand binds to CD44, a CD44 variant, a CD44 glycoform, or equivalent thereof with a KD of greater than or equal to 10^-6 M, 10^-7 M, 10^-8 M, 10^-9 M, or 10^-10 M. In some embodiments, the CD44 ligand binds to CD44, a CD44 variant, a CD44 glycoform, or an equivalent thereof with high affinity: a KD ranging from nanomolar range (10^'9)

to picomolar (10 ¹²) range or greater. Non-limiting examples of CD44 ligands include CD44 matrix metalloproteinases, hyaluronic acid, osteopontin, serglycin, chondroitin, fibrin, P- selectin, basic Fibroblast Growth Factor (b-FGF), heparin binding epidermal growth factor (HB-EGF), collagen, fibronectin, galectin-8 (gal-8), and laminins. In some embodiments, the CD44 ligand is recombinant.

[0045] Collagen is a structural protein that is generally found in connective tissue and the extracellular space of animals. Collagen is classified into several types including but not limited to type I ( e.g . COL1 A1 (Entrez gene: 1277, UniProt: P02452); COL1 A2 (Entrez gene: 1278, UniProt: P08123)), type II (e.g. COL2A1 (Entrez gene: 1280, UniProt: P02458)), type III (e.g. COL3A1 (Entrez gene: 1281, UniProt: P02461)), type IV (basement membrane collagen, e.g. COL4A1 (Entrez gene: 1282, UniProt: P02462), COL4A2 (Entrez gene: 1284, UniProt: P08572), COL4A3 (Entrez gene: 1285, UniProt: Q01955), COL4A4 (Entrez gene: 1286, UniProt: P53420), COL4A5 (Entrez gene: 1287, UniProt: P29400), COL4A6 (Entrez gene: 1288, UniProt: Q14031)), type V (e.g. COL5A1 (Entrez gene: 1289, UniProt: P20908), COL5A2 (Entrez gene: 1290, UniProt: P05997), COL5A3 (Entrez gene: 5059, UniProt: P25940)), type VI (e.g. COL6A1 (Entrez gene: 1291, UniProt: P12109), COL6A2 (Entrez gene: 1292, UniProt: P12110), COL6A3 (Entrez gene: 1293, UniProt: P12111), COL6A5 (Entrez gene: 256076, UniProt: PA8TX70, H0Y935)), type VII (e.g. COL7A1 (Entrez gene: 1294, UniProt: Q02388)), type VIII (e.g. COL8A1 (Entrez gene: 1295, UniProt: P27658), COL8A2 (Entrez gene: 1296, UniProt: P25067, Q4VAQ0)), type IX (e.g. COL9A1 (Entrez gene: 1297, UniProt: P20908), COL9A2 (Entrez gene: 1290, UniProt: P05997), COL9A3 (Entrez gene: 5059, UniProt: P25940)), type X (e.g. COL 10A1 (Entrez gene: 1300, UniProt: A03692)), type XI (e.g. COL11A1 (Entrez gene: 1301, UniProt: P12107), COL11A2 (Entrez gene: 1302, UniProt: P13942)), type XII (e.g. COL 12 A1 (Entrez gene: 1303, UniProt: Q99715)), or type XIII (e.g. COL 10A1 (Entrez gene: 1300, UniProt: A03692). In particular embodiments, the collagen is type IV collagen. Collagens are available from, for example, Sigma Aldrich, St. Louis, Missouri, U.S.A. (e.g. CAS# 9007-34-5, cat.#: C6745).

[0046] The term“comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. For example, a composition consisting essentially of the elements as defined herein would not exclude other elements that do not materially affect the basic and novel characteristic(s) of the claimed invention.“Consisting of’ shall mean excluding more than trace amount of other ingredients and substantial method steps recited. Embodiments defined by each of these transition terms are within the scope of this invention.

[0047] The terms“culture” or“culturing” refer to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of cell(s) grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.

[0048] Extracellular vesicles (also referred to as cell derived vesicles), are membrane surrounded structures that are released by cells in vitro and in vivo. Extracellular vesicles can contain proteins, lipids, and nucleic acids and can mediate intercellular communication between different cells, including different cell types, in the body. Two types of extracellular vesicles are exosomes and microvesicles. Exosomes are small lipid-bound, cellularly secreted vesicles that mediate intercellular communication via cell-to-cell transport of proteins and RNA (El Andaloussi, S. et al. (2013) Nature Reviews: Drug Discovery

12(5):347-357). Exosomes range in size from approximately 30 nm to about 200 nm.

Exosomes are released from a cell by fusion of multivesicular endosomes (MVE) with the plasma membrane. Microvesicles, on the other hand, are released from a cell upon direct budding from the plasma membrane (PM). Microvesicles are typically larger than exosomes and range from approximately 100 nm to 1 pm. Cell-derived vesicles (e.g., exosomes and/or microvesicles) can be isolated from eukaryotic cells. Non-limiting examples of cells that cell-derived vesicles can be isolated from include stem cells. Non-limiting examples of such stem cells include adult stem cells, embryonic stem cells, embryonic-like stem cells, neural stem cells, or induced pluripotent stem cells. In some embodiments, the stem cell is an adult stem cell that is optionally a mesenchymal stem cell. In one aspect the stem cell, e.g., the mesenchymal stem cells, has been cultured under conditions of hypoxia and low serum or serum-free conditions. [0049] Fibronectin is an extracellular matrix glycoprotein that is capable of binding integrins. Fibronectin is encoded by the FN1 gene (Entrez gene: 2335, UniProt: P02751) and is available from, for example, Sigma Aldrich ( e.g . CAS# 86088-83-7; cat.#: F2006).

[0050] Recombinant fibronectin is a multifunctional glycoprotein that can be expressed in various cells and organisms, including, but not limited to, insect cells.lt is available from, for example, Sigma Aldrich.

[0051] Laminins are heterotrimeric proteins formed by combinations of an alpha chain

(e.g. LAMA1 (Entrez gene: 284217, UniProt: P25391), LAMA2 (Entrez gene: 3908, UniProt: P24043), LAMA3 (Entrez gene: 3909, UniProt: Q16787), LAMA4 (Entrez gene: 3910, UniProt: Q16363), or LAMA5 (Entrez gene: 3911, UniProt: 015230)), beta chain (e.g. LAMB1 (Entrez gene: 3912, UniProt: P07942), LAMB2 (Entrez gene: 3913, UniProt: P55268), LAMB3 (Entrez gene: 3914, UniProt: Q13751), or LAMB4 (Entrez gene: 22798)), and gamma chain (e.g. LAMC1 (Entrez gene: 22798, UniProt: P25391), LAMC2 (Entrez gene: 284217, UniProt: P25391), or LAMC3 (Entrez gene: 284217, UniProt: P25391)) of laminin. Laminins include but are not limited to the following trimers: laminin 111, laminin 211, laminin 121, laminin 221, laminin 332, laminin 3A32, laminin 3B32, laminin 311, laminin 3 Al l, laminin 312, laminin 3A21, laminin 411, laminin 421, laminin 511, laminin 521, laminin 213, laminin 423, laminin 522, and laminin 523. Laminin is available from, for example, Sigma Aldrich (e.g. Laminin from Engelbreth-Holm- Swarm murine sarcoma basement membrane, CAS #: 114956-81-9, cat.#: L2020).

[0052] Hyaluronic acid is an unsulfated, anionic glycosaminoglycan abundant in extracellular matrices and having the chemical formula (Ci4H2iNOn)n (e.g. Sigma cat.# N9164).

[0053] In vitro fertilization (IVF) is the process of fertilization of an egg (oocyte) with sperm outside of the body of the mother in a laboratory dish. The fertilized egg, or embryo, is usually cultured in vitro for some period of time or until a desired developmental stage is reached. For example, some embryos are cultured until the embryo reaches 6-8 cells, or until reaching the blastocyst stage. Other embryos may be cultured for a period of time such as 5 or 6 days, for example. After the desired culturing period has lapsed , embryo selection and embryo transfer occur by which an embryo is selected and transferred into the uterus of a female patient, and/or in some cases preserved (e.g., cryopreservation) for later implantation.

[0054] The term“isolated” as used herein refers to molecules or biologicals or cellular materials being substantially free from other materials.

[0055] A“marrow stromal cell” also referred to as“mesenchymal stem cells,” or MSC, is a multipotent stem cell that can differentiate into a variety of cell types. Under some conditions, MSCs secrete extracellular vesicles such as exosomes. Cell types that MSCs have been shown to differentiate into in vitro or in vivo include osteoblasts, chondrocytes, myocytes, and adipocytes. Methods to isolate such cells, propagate, culture, differentiate such cells, and harvest MSC exosomes are known in the technical and patent literature, e.g., U.S. Patent Application Publications 2007/0224171, 2007/0054399, 2009/0010895, and International Patent Publication WO2017/117585, each of which are each incorporated by reference in their entirety.

[0056] The“mouse embryo assay” (MEA) and the MEGA™ are assays used to examine the detection of embryotoxicity of culture media components, products, methods, and/or environment without involving human materials. The basic techniques and protocols employed for performing MEA are set forth in In Vitro Fertilization and Embryo Transfer: A Manual of Basic Techniques (Don P. Wolf, Editor), 1988, pages 57-75; the contents of which are hereby incorporated by reference in their entirety. Briefly, the assay involves superovulation of female mice with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). The mice are placed with males at the time of hCG injection and killed 24 hours following hCG to obtain one-cell embryos or 36 hours after injection to obtain two-cell embryos. One-cell embryos are selected for use if they have two polar bodies visible; two cell embryos are selected for use if they look morphologically normal. The basic protocol for performing MEGA is disclosed in US Patent Application No. 2014/0302513, incorporated by reference herein in its entirety. In addition to assessing morphology of the mouse embryo, the MEGA assay further comprises assessing expression of one or more reporter genes in the developing mouse embryo. See Gilbert et al., Reprod Biol Endocrinol. 2016; 14: 13, incorporated by reference herein in its entirety.

[0057] “Optional” or“optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0058] As used herein, the term“subject” is used interchangeably with“individual” and“patient,” and indicates a mammal, in particular a human, equine, bovine, porcine, feline, canine, murine, rat, or non-human primate. In preferred embodiments, the subject is a human. In some embodiments, the subject is undergoing infertility treatment. In some embodiments, the subject is suffering from or at risk of suffering from infertility.

Media and Supplemental Compositions

[0059] In some aspects provided herein are supplemental compositions for cell culture comprising, consisting of, or consisting essentially of: (a) Human Serum Albumin (HSA); (b) dextran; and (c) one or more CD44 ligands. In some embodiments, the supplemental compositions further comprise, consist of, or consist essentially of insulin, epidermal growth factor (EGF), and/or platelet-derived growth factor (PDGF).

[0060] In some embodiments of the supplemental compositions, the HSA is recombinant HSA (rHSA). In some embodiments, the rHSA is expressed in bacteria, yeast, insects, or plants. In some embodiments, the rHSA is expressed in yeast.

[0061] In some embodiments of the supplemental compositions, the CD44 ligand is selected from the group of matrix metalloproteinase, hyaluronic acid, osteopontin, serglycin, chondroitin, fibrin, P-selectin, basic Fibroblast Growth Factor (b-FGF), heparin binding epidermal growth factor (HB-EGF), collagen, fibronectin, and laminin. In some

embodiments, the collagen is selected from collagen I, collagen II, collagen III, collagen IV, and collagen V. In some embodiments of the supplemental compositions, the collagen is collagen IV. In some embodiments of the supplemental compositions, the CD44 ligand is fibronectin. In some embodiments of the supplemental compositions, the fibronectin is recombinant fibronectin. In some embodiments of the supplemental compositions, the laminin is selected from laminin-111, laminin-211, laminin-121, laminin-221, laminin-332, laminin-3 A32, laminin-3B32, laminin-311, laminin-3al 1, laminin-321, laminin-3 A21, laminin-411, laminin-421, laminin-511, laminin-521, laminin-213, laminin-423, laminin-522, and laminin-523.

[0062] In some embodiments of the supplemental compositions, the compositions further comprise, consist of, or consist essentially of one or more embryotrophic factors. In some embodiments of the supplemental compositions, the embryotrophic factor is an anti oxidant. In some embodiments of the supplemental compositions, the embryotrophic factor is media conditioned by stem cells and/or extracellular vesicles secreted by stem cells. In some embodiments, the stem cells are mesenchymal stem cells. In some embodiments, the extracellular vesicles are exosomes.

[0063] The medium can be any suitable medium for embryo culture, sperm culture, egg culture, fertilization, and/or cryopreservation, including, for example, a sequential medium or a single medium that is replenished at least once during the culture process, or a non- sequential medium or a medium that is not replenished (e.g., uninterrupted or continuous culture) during the culturing of the embryo. Existing media that are known to those of skill in the art or that are commercially available can be modified with the supplemental composition described herein. Some non-limiting examples of existing media that can be utilized in the methods or that can be modified for use include, without limitation,

Continuous Single Culture™ media (Irvine Scientific), any Global® media (LifeGlobal), G1 medium (Vitrolife), G2 medium (Vitrolife), a Human Tubal Fluid (HTF) medium, HTF medium with or without glucose or phosphate, Whitten’s Medium, Ham’s F-10 Medium, Sage Media, and the like. In some embodiments, the medium is a novel medium not previously published or commercialized.

[0064] Components of published exemplary, non-limiting media that may be used with the supplement composition disclosed herein are given in Table 1. Components of exemplary, non-limiting media based on currently available Irvine Scientific culture media that may be modified for use in the present invention are given in Tables 2-6.

Table 1: Embryo Culture Media

Table 2: Irvine Scientific HTF media

HTF

Component . mM

Sodium Chloride . 97.8

Potassium Chloride . 4.69

Magnesium Sulfate, Anhydrous . 0.20

Potassium Phosphate, Monobasic . 0.37

Calcium Chloride, Anhydrous . 2.04

Sodium Bicarbonate . 25.0

Glucose . 2.7

Sodium Pyruvate . 0.33

Sodium Lactate . 21.4

Gentamicin . 10 gg/mL

Phenol Red . 5 mg/L

Protein Supplementation . None (User Option)

Complete HTF with SSS

Component . mM

Sodium Chloride . 91.44

Potassium Chloride . 4.22

Magnesium Sulfate, Anhydrous . 0.18

Potassium Phosphate . 0.33

Calcium Chloride . 1.84

Sodium Bicarbonate . 22.50

Glucose . 2.50

Sodium Pyruvate . 0.30

Sodium Lactate . 19.26

Phenol Red . 4.5 mg/L

Gentamicin . 9 pg/mL

Human Serum Albumin . 5 mg/mL

Globulins . 1 mg/mL

Modified HTF

Component . mM

Sodium Chloride . 97.8

Potassium Chloride . 4.69 Magnesium Sulfate, Anhydrous . 0 . 2 0

Potassium Phosphate, Monobasic . 0.37

Calcium Chloride, Anhydrous .. 2.04

Sodium Bicarbonate .. 4.0

HEPES 2 1 . 0

Glucose .. 2.78

Sodium Pyruvate .. 0.33

Sodium Lactate . 21.4

Gentamicin . 10 pg/mL Phenol Red . 5 mg/L

Protein Supplementation .None (User Option)

Table 3: Irvine Scientific Single Step Medium™

Component . mM

Sodium Chloride . 101.5

Potassium Chloride . 2.5

Potassium Phosphate . 0.35

Calcium Chloride, Anhydrous . 1.7

Magnesium Sulfate, Anhydrous . 0.2

Sodium Bicarbonate . 25.0

Sodium Pyruvate . 0.2

Glucose . 0.5

Sodium Citrate . 1.0

Sodium Lactate (D/L) . 20

EDTA, Disodium, Dihydrate . 10 mM

Alanyl-glutamine . 1.0

Alanine . 0.05

Arginine . 0.3

Asparagine . 0.05

Aspartic Acid . 0.05

Cysteine . 0.05

Glutamic Acid . 0.05

Glycine . 0.05

Histidine . 0.1

Isoleucine . 0.2

Leucine . 0.2

Lysine . 0.2

Methionine . 0.05

Phenylalanine . 0.1

Proline . 0.05

Serine . 0.05

Taurine . 0.05

Threonine . 0.2

Tryptophan . 0.02 Tyrosine . . 0.1

Valine 0.2

Phenol Red 4.8 mg/L

Gentamicin 10 pg/mL

Table 4: Irvine Scientific P-1^® (Preimplantation Stage One) Media™

P-1® Medium*

Component . mM

Sodium Chloride . 101.6

Potassium Chloride . 4.69

Magnesium Sulfate, Anhydrous . 0.20

Calcium Chloride, Anhydrous . 2.04

Sodium Bicarbonate . 25

Sodium Pyruvate . 0.33

S odium Lactate . 21.4

Taurine . 0.05

Sodium Citrate . 0.15 mg/L

Phenol Red . 5 mg/L

Gentamicin . 10 pg/mL

Complete P-1® Medium with SSS™

Component mM

Sodium Chloride 91.44 Potassium Chloride 4.22 Magnesium Sulfate, Anhydrous 0.18 Calcium Chloride, Anhydrous 1.84 Sodium Bicarbonate 22.50 Sodium Pyruvate 0.30 Sodium Lactate 19.26 Taurine 0.05

Sodium Citrate 0.14 mg/L Phenol Red 4.50 mg/L Gentamicin 9 pg/mL Human Serum Albumin 5 mg/mL Globulins 1 mg/mL

Complete P-1® Medium with DSS

Component . mM

Sodium Chloride . 91.44

Potassium Chloride . 4.22

Magnesium Sulfate, Anhydrous . 0.18 Calcium Chloride, Anhydrous . 1.84

Sodium Bicarbonate . 22.50

Sodium Pyruvate . 0.30

Sodium Lactate . 19.26

Taurine . 0.05

Sodium Citrate . 0.14 mg/L

Phenol Red . 4.50 mg/L

Gentamicin . 9 pg/mL

Human Serum Albumin . 5 mg/mL

Dextran . 2 mg/mL

Table 5: Irvine Scientific Early Cleavage Media™

Component . mM

Glucose . 0.50

Sodium Chloride . 102.7

Potassium Chloride . 2.50

Magnesium Sulfate, Anhydrous . 0.20

Calcium Chloride, Anhydrous . 1.70

Sodium Bicarbonate . 25.0

Sodium Pyruvate . 0.33

Sodium Lactate (D/L) . 20.77

Alanyl-glutamine . 0.50

Taurine . 0.05

Sodium Citrate . 0.15 mg/L

EDTA, disodium, dehydrate . 10 mM

Phenol Red . 4.8 mg/L

Gentamicin . 10 pg/mL

Table 6: Irvine Scientific MultiBlast Media TM

Component . mM

Sodium Chloride . 101.5

Potassium Chloride . 2.5

Potassium Phosphate . 0.35

Calcium Chloride, Anhydrous . 1.7

Magnesium Sulfate, Anhydrous . 0.2

Sodium Bicarbonate . 25.0

Sodium Pyruvate . 0.2

Glucose . 3.0

Sodium Citrate . 1.0

Sodium Lactate (D/L) . 20

Alanyl-glutamine . 1.0

Alanine . 0.05 Arginine . 0.3

Asparagine . 0.05

Aspartic Acid . 0.05

Cysteine 0.05

Glutamic Acid 0.05

Glycine 0.05

Histidine 0.1

Isoleucine 0.2

Leucine 0.2

Lysine 0.2

Methionine 0.05

Phenylalanine 0.1

Proline 0.05

Serine 0.05

Taurine 0.05

Threonine 0.2

Tryptophan 0.02

Tyrosine 0.1

Valine 0.2

Phenol Red 4.8 mg/L

Gentamicin 10 gg/mL

Table 7: Exemplary Concentration Ranges of Select Supplemental Composition Components

[0065] In some embodiments, the supplemental composition described herein is provided in a 2X, 4X, 5X, 10X, 15X, 20X, 25X, 50X, 100X, 250X, 500X, or 1000X concentration, In some embodiments, the supplemental composition is dilutable with culture medium and/or a carrier such as saline or phosphate-buffered saline.

[0066] In some embodiments, the supplemental composition may contain at least the following components: HSA or rHSA, dextran, CD44 ligand, matrix metalloproteinase, hyaluronic acid, osteopontin, serglycin, chondroitin, fibrin, P-selectin, Basic Fibroblast Growth Factor (b-FGF), heparin binding epidermal growth factor (HB-EGF), collagen (e.g., collagen IV), fibronectin, Laminin, insulin, EGF, and PDGF.

[0067] In some aspects, provided herein is a culture medium comprising, consisting of, or consisting essentially of: (a) a culture medium suitable for use in culturing an embryo, fertilizing an egg, or culturing sperm; (b) rHSA; (c) dextran; and (d) one or more CD44 ligands. In some embodiments of the medium, the HSA is recombinant HSA (rHSA). In some embodiments of the medium, the rHSA is expressed in bacteria, yeast, or plants. In some embodiments, the rHSA is expressed in yeast.

[0068] In general, embryo culture media contains at least one energy substrate, at least one salt/ion, and preferably a buffer. Optionally, embryo culture media may contain at least one essential amino acid, at least one non-essential amino acid, at least one antioxidant, a pH indicator, and/or an antibiotic. In some embodiments, the components of the media set forth in Tables 1-6 can be modified up or down by up to about 40%, for example about 1%, about 2%, about 5%, about 10%, about 20%, or about 40%. In some embodiments, the supplemental composition described herein further comprises at least one energy substrate, salt/ion, and/or buffer.

[0069] In some embodiments, the medium and/or supplemental composition described herein comprises at least one energy substrate. In some embodiments, the energy substrate(s) are selected from the group consisting of glucose, pyruvate, and lactate. [0070] In some embodiments, the at least one energy substrate comprises, consists of, or consists essentially of lactate. In some embodiments, the lactate is present between about 0 mM and about 10 mM, or any value or subrange there between. In some embodiments, the lactate is present between about 0.1 mM and about 3 mM, or any value or subrange there between (e.g., about 0.1 mM, about 0 2 mM, about 0.3 mM, about 0.32 mM, about 0.33 mM, about 0.37 mM, about 0.5 mM, about 2.0 mM, about 2.27 mM). In some embodiments, lactate is present in the medium at one of the concentrations indicated in Tables 1-6 above.

[0071] In some embodiments, the at least one energy substrate comprises, consists of, or consists essentially of pyruvate (e.g., sodium pyruvate). In some embodiments, the pyruvate is present between about 0 mM and about 3 mM, or any value or subrange there between. In some embodiments, the pyruvate is present between about 0.1 mM and about 2.3 mM, or any value or subrange there between (e.g., about 0.1 mM, about 0 2 mM, about 0.3 mM, about 0.32 mM, about 0.33 mM, about 0.37 mM, about 0.5 mM, about 2.0 mM, about 2.27 mM). In some embodiments, pyruvate is present in the medium at one of the

concentrations indicated in Tables 1-6 above.

[0072] In some embodiments, the at least one additional energy substrate comprises, consists of, or consists essentially of glucose. In some embodiments, glucose is present between about 0 mM and about 10 mM, or any value or subrange there between. In some embodiments, glucose is present between about 0 mM and about 6.5 mM, or any value or subrange there between. In an especially preferred embodiment, glucose is present at less than about 3 mM, or any value or subrange there between (e.g., about 0.3 mM, about 0.5 mM, about 0.6 mM, about 0.8 mM, about 1.0 mM, about 1.5 mM, about 2.0 mM, about 2.5 mM, or about 3.0 mM). In some embodiments, no glucose is present. In some embodiments, glucose is present in the medium at one of the concentrations indicated in Tables 1-6 above.

[0073] In some embodiments, the medium and/or supplement composition described herein comprises at least one salt. Salts that can be used in the medium and/or supplement composition described herein include, but are not limited to, sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, potassium phosphate, sodium phosphate, cupric sulfate, ferric sulfate, and zinc sulfate. [0074] In some embodiments, the medium and/or supplement composition further comprises sodium chloride. In some embodiments, the sodium chloride is present in the medium at a concentration between about 65 mM and about 150 mM, or any value or subrange there between. In some embodiments, sodium chloride is present in the medium at a concentration between about 85 mM and about 120 mM. In some embodiments, sodium chloride is present in the medium at a concentration between about 85 mM and about 110 mM. In some embodiments, sodium chloride is present in the medium at one of the concentrations indicated in Tables 1-6 above.

[0075] In some embodiments, the medium and/or supplement composition further comprises potassium chloride. In some embodiments, potassium chloride is present at a concentration of between about 2 mM and about 10 mM, or any value or subrange there between. In some embodiments, potassium chloride is present at a concentration of between about 2.5 mM and about 6 mM. In some embodiments, potassium chloride is present in the medium at one of the concentrations indicated in Tables 1-6 above.

[0076] In some embodiments, the medium and/or supplement composition further comprises magnesium sulfate. In some embodiments, magnesium sulfate is present at a concentration of between about 0 mM and about 2 mM, or any value or subrange there between. In some embodiments, magnesium sulfate is present at a concentration of between about 0.1 mM and about 1 mM. In some embodiments, magnesium sulfate is present in the medium at one of the concentrations indicated in Tables 1-6 above.

[0077] In some embodiments, the medium and/or supplement composition further comprises calcium chloride. In some embodiments, calcium chloride is present at a concentration of between about 0 mM and about 5 mM, or any value or subrange there between. In some embodiments, calcium chloride is present at a concentration of between about 1 mM and about 2.5 mM. In some embodiments, calcium chloride is present in the medium at one of the concentrations indicated in Tables 1-6 above.

[0078] In some embodiments, the medium and/or supplement composition further comprises potassium phosphate. In some embodiments, potassium phosphate is present at a concentration of between about 0 mM and about 2 mM, or any value or subrange there between. In some embodiments, potassium phosphate is present at a concentration of between about 0 mM and about 0.5 mM. In some embodiments, potassium phosphate is present in the medium at one of the concentrations indicated in Tables 1-6 above.

[0079] In some embodiments, the medium and/or supplement composition further comprises sodium phosphate. In some embodiments, sodium phosphate is present at a concentration of between about 0 mM and about 2 mM, or any value or subrange there between. In some embodiments, sodium phosphate is present at a concentration of between about 0 mM and about 0.8 mM. In some embodiments, sodium phosphate is present in the medium at one of the concentrations indicated in Tables 1-6 above.

[0080] In some embodiments, the medium and/or supplement comprises a buffer.

Buffers and concentrations thereof for media solutions are well-known in the art. In some embodiments, the buffer is sodium bicarbonate. In some embodiments, sodium bicarbonate is present at a concentration of between about 0 mM and about 30 mM, or any value or subrange there between. In some embodiments, sodium bicarbonate is present at a concentration of between about 20 mM and about 25 mM. In some embodiments, sodium phosphate is present in the medium at one of the concentrations indicated in Tables 1-6 above.

[0081] In some embodiments, the medium and/or supplement composition further comprises at least one buffer. Buffers and concentrations thereof for media solutions are well-known in the art. In some embodiments, the buffer is HEPES. In some embodiments, HEPES is present at a concentration of between about 0 mM and about 30 mM, or any value or subrange there between. In some embodiments, the buffer is sodium bicarbonate. In some embodiments, sodium bicarbonate is present at a concentration of between about 0 mM and about 30 mM, or any value or subrange there between. In some embodiments, the buffer is present at a concentration of between about 20 mM and about 25 mM. In some embodiments, the buffer is present in the medium at one of the concentrations indicated in Tables 1-6 above.

[0082] In some embodiments, the medium and/or supplement composition further comprises at least one amino acid. In some embodiments, the medium comprises at least one essential amino acid. The essential amino acids include arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, taurine, tryptophan, tyrosine, and valine. In some embodiments, the medium comprises at least one non-essential amino acid. The non-essential amino acids include alanine, asparagine, aspartate, glutamate, glycine, proline, and serine. In some embodiments, glutamine is present as alanyl-glutamine. In some embodiments, the medium comprises at least one synthetic or unnatural amino acid. In some embodiments, the at least one amino acid is present at a concentration of between about 0 mM and about 1.5 mM. In some embodiments, the at least amino acid is present at a concentration of between about 0 mM and about 0.6 mM. In some embodiments, amino acid is present in the medium at one of the concentrations indicated in Tables 1-6 above.

[0083] In some embodiments, the medium and/or supplement composition further comprises at least one antioxidant. Antioxidants and concentrations thereof for media solutions are well-known in the art. In some embodiments, the antioxidant is sodium citrate. In some embodiments, sodium citrate is present at a concentration of between about 0 mg/L and about 5 mg/L, or any value or subrange there between (e.g., about 0.1 mg/L, about 0.5 mg/L, about 1 mg/L, about 2 mg/L). In some embodiments, the antioxidant is EDTA. In some embodiments, EDTA is present at a concentration of between about 0 mM and about 50 mM, or any value or subrange there between (e.g., about 1 pM, about 5 pM, about 10 pM, about 25 pM, about 50 pM). In some embodiments, the antioxidant is present in the medium at one of the concentrations indicated in Tables 1-6 above.

[0084] In some embodiments, the medium and/or supplement composition further comprises a pH indicator. In some embodiments, the pH indicator is phenol red. In some embodiments, phenol red is present at a concentration of between about 0 mg/L and about 10 mg/L, or any value or subrange there between (e.g., between about 0 mg/L and about 5 mg/L, about 2 mg/L, about 3 mg/L, about 4 mg/L). In some embodiments, the pH indicator is present in the medium at one of the concentrations indicated in Tables 1-6 above.

[0085] In some embodiments, the medium and/or supplement composition further comprises at least one antibiotic. Antibiotics and concentrations thereof for media solutions are well-known in the art. In some embodiments, the antibiotic is gentamicin sulfate. In some embodiments, the antibiotic is penicillin. In some embodiments, the antibiotic is

streptomycin. In some embodiments, the antibiotic is present at a concentration between about 0 pg/mL and about 200 pg/mL, or any value or subrange there between (e.g., about 1 pg/mL, about 5 pg/mL, about 10 pg/mL, about 20 pg/mL, about 50 pg/mL, about 100 pg/mL). In some embodiments, the antibiotic is present at a concentration between about 0 IU/mL and about 500 IU/mL, or any value or subrange there between (e.g., about 1 IU/mL, about 5 IU/mL, about 10 IU/mL, about 20 IU/mL, about 50 IU/mL, about 100 IU/mL, about 250 IU/mL, about 500 IU/mL). IU = international units.

[0086] In some embodiments of the methods, the concentration of recombinant HSA in the supplemented medium is about 1 mg/mL. In some embodiments of the methods, the concentration of dextran in the supplemented medium is about 2 mg/mL. In some embodiments of the methods, the concentration of insulin in the supplemented medium is about 0.1. mg/L to about 3 mg/L. Insulin further may be included at a concentration of about 0.2 mg/L, about 0.3 mg/L, about 0.4 mg/L, about 0.5 mg/L, about 0.6 mg/L, about 0.7 mg/L, about 0.8 mg/L, about 0.9 mg/L, about 1.0 mg/L, about 1.1 mg/L, about 1.2 mg/L, about 1.3 mg/L, about 1.4 mg/L, about 1.5 mg/L, about 1.6 mg/L, about 1.7 mg/L, about 1.8 mg/L, about 1.9 mg/L, about 2.0 mg/L, about 2.1 mg/L, about 2.2 mg/L, about 2.3 mg/L, about 2.4 mg/L, about 2.5 mg/L, about 2.6 mg/L, about 2.7 mg/L, about 2.8 mg/L, about 2.9 mg/L, or about 3.0 mg/L. In certain embodiments, the product concentration of insulin is about 2 mg/L. In certain embodiments, the working concentration is about 0.2 mg/L. In certain embodiments, the product concentration is about 2 mg/L.

[0087] In some embodiments of the methods, the concentration of EGF in the supplemented medium is about 0 pg/L to about 12 pg/L. For product concentrations, EGF further may be included at a concentration of about 8.5 pg/L, about 8.6 pg/L, 8.7 pg/L, 8.8 pg/L, 8.9 pg/L, 9.0 pg/L, 9.1 pg/L, 9.2 pg/L, 9.3 pg/L, 9.4 pg/L, 9.5 pg/L, about 9.6 pg/L, about 9.7 pg/L, about 9.8 pg/L, about 9.9 pg/L, about 10.0 pg/L, about 10.1 pg/L, about 10.2 pg/L, about 10.3 pg/L, about 10.4 pg/L, about 10.5 pg/L, about 10.6 pg/L, about 10.7 pg/L, 10.8 pg/L, about 10.9 pg/L, about 11 pg/L, about 11.1 pg/L, about 11.2 pg/L, about 11.3 pg/L, about 11.4 pg/L, about 11.5 pg/L, about 11.6 pg/L, about 11.7 pg/L, about 11.8 pg/L, about 11.9 pg/L, or about 12.0 pg/L. For working concentrations, EGF further may be included at a concentration of about 0.1 pg/L, 0.2 pg/L, 0.3 pg/L, 0.4 pg/L, 0.5 pg/L, 0.6 pg/L, 0.7 pg/L, 0.8 pg/L, 0.9 pg/L, 1.0 pg/L, 1.1 pg/L, 1.2 pg/L, 1.3 pg/L, 1.4 pg/L. In certain embodiments, the product concentration of EGF may be about 10 pg/L. In certain embodiments, the working concentration of EGF may be about 1 pg/L.

[0088] In some embodiments of the methods, the concentration of PDGF in the supplemented medium is about 0.01 to about 0.4. For product concentrations, PDGF further may be included at a concentration of about 0.017 pg/L, about 0.018 pg/L, about 0.019 pg/L, about 0.020 pg/L, about 0.021 pg/L, about 0.022 pg/L, about 0.023 pg/L, about 0.024 pg/L, about 0.025 pg/L, about 0.026 pg/L, about 0.027 pg/L, about 0.028 pg/L, about 0.029 pg/L, about 0.030 pg/L, about 0.031 pg/L. For working concentrations, PDGF further may be included at a concentration of about 0.17 pg/L, about 0.18 pg/L, about 0.19 pg/L, about 0.20 pg/L, about 0.21 pg/L, about 0.22 pg/L, about 0.23 pg/L, about 0.24 pg/L, about 0.25 pg/L, about 0.26 pg/L, about 0.27 pg/L, about 0.28 pg/L, about 0.29 pg/L, about 0.30 pg/L, about 0.31 pg/L. In certain embodiments, the product concentration of PDGF may be about 0.24 pg/L. In certain embodiments, the working concentration of PDGF may be about 0.024 Pg/L.

Kits

[0089] In some aspects, provided herein are kits with a composition comprising, consisting of, or consisting essentially of: (a) Human Serum Albumin (HSA); (b) dextran; and (c) one or more CD44 ligands and instructions for use. In some embodiments, the compositions further comprise, consist of, or consist essentially of insulin, epidermal growth factor (EGF), and/or platelet-derived growth factor (PDGF). In some embodiments, the kits further comprise, consist of, or consist essentially of a culture medium suitable for use in culturing an embryo, fertilizing an egg, or culturing sperm.

[0090] In some embodiments of the kits, the HSA is recombinant HSA (rHSA). In some embodiments of the supplemental compositions, the rHSA is expressed in bacteria, yeast, insects, or plants. In some embodiments, the rHSA is expressed in yeast. Methods

[0091] Some embodiments herein are generally related to methods of culturing an embryo in vitro in a medium for embryo culture that has been supplemented with the supplemental composition described herein. Examples of embryos that can be cultured utilizing the methods described herein include any animal cell, but preferably include mammalian embryos. Mammalian embryos that can be used include, without limitation, bovine, equine, porcine, canine, feline, ovine, simian, lupine, murine, leporine, and more preferably include human or homo sapien embryos. In some embodiments, the embryo is from an endangered species. In some embodiments, the embryo is from a domesticated species (e.g., cattle, horses, sheep, pigs, dogs, cats, and the like, without limitation). In another embodiment, the embryo is from a non-human primate. It should be understood that one or more of the species listed above can be specifically excluded from one or more of the methods described herein.

[0092] In some embodiments, the embryo is transferred into a patient suffering from infertility. In some embodiments, the embryo is transferred into a patient undergoing fertility treatments. In some embodiments, the embryo is transferred into a surrogate. That is, the embryo need not be transferred back to the patient who provided the oocyte. In some embodiments, the embryo is cryopreserved. In some embodiments, a cryopreserved embryo is later transferred to a female patient.

[0093] In some aspects, provided herein are methods of culturing an embryo in vitro , comprising, consisting of, or consisting essentially of culturing the embryo in a medium supplemented with a composition comprising, consisting of, or consisting essentially of: (a) Human Serum Albumin (HSA); (b) dextran; and (c) one or more CD44 ligands. In some embodiments, the supplemental compositions further comprise, consist of, or consist essentially of insulin, epidermal growth factor (EGF), and/or platelet-derived growth factor (PDGF).

[0094] In some aspects, provided herein are methods of assessing a product for use in human Assisted Reproductive Technologies, comprising, consisting of, or consisting essentially of: (a) culturing an embryo in a medium supplemented with a composition comprising, consisting of, or consisting essentially of: (a) Human Serum Albumin (HSA); (b) dextran; and (c) one or more CD44 ligands in the presence of the product; (b) evaluating the embryo during at least one stage of development; and (c) determining the acceptability of the product based on the evaluation. In some embodiments, the medium does not contain serum. In some embodiments, the supplemental compositions further comprise, consist of, or consist essentially of insulin, epidermal growth factor (EGF), and/or platelet-derived growth factor (PDGF). In some embodiments, the method of assessing a product is an MEA or MEGA assay. In some embodiments, the supplemental composition described herein is for use in an MEA or MEGA assay.

[0095] In some aspects, provided herein are methods of fertilizing an egg, comprising, consisting of, or consisting essentially of fertilizing the egg in a medium supplemented with a composition comprising, consisting of, or consisting essentially of: (a) Human Serum Albumin (HSA); (b) dextran; and (c) one or more CD44 ligands. In some embodiments, the egg is from a subject suffering from infertility or undergoing infertility treatment. In some embodiments, the medium does not contain serum. In some embodiments, the supplemental compositions further comprise, consist of, or consist essentially of insulin, epidermal growth factor (EGF), and/or platelet-derived growth factor (PDGF).

[0096] In some aspects, provided herein are methods of enhancing sperm survival, comprising culturing the sperm in a medium supplemented with a composition comprising, consisting of, or consisting essentially of: (a) Human Serum Albumin (HSA); (b) dextran; and (c) one or more CD44 ligands. In some embodiments, the sperm is from a subject suffering from infertility or undergoing infertility treatment. In some embodiments, the medium does not contain serum. In some embodiments, the supplemental compositions further comprise, consist of, or consist essentially of insulin, epidermal growth factor (EGF), and/or platelet-derived growth factor (PDGF).

[0097] In some embodiments of the methods, the medium is a sequential medium or a medium that is replenished at least once during the culturing of the embryo. In some embodiments of the methods, the medium is a medium suitable for culturing a human embryo. In some embodiments of the methods, the medium is Continuous Single Culture™ medium, P-1 medium, G1 medium, G2 medium, Human Tubal Fluid (HTF) medium, modified HTF medium, Whitten’s medium, Ham’s F-10 medium, KSOM medium, Global Medium, or Single Step Medium™. In some embodiments, the medium does not contain serum.

[0098] In some embodiments of the methods, at least one biological parent of the embryo is suffering from infertility or undergoing infertility treatment. In some embodiments of the methods, the methods further comprise, consist of, or consist essentially of implanting the embryo into a female patient.

[0099] In some embodiments, the supplemental compositions further comprise, consist of, or consist essentially of insulin, epidermal growth factor (EGF), and/or platelet- derived growth factor (PDGF).

[00100] In some embodiments of the methods, the HSA is recombinant HSA (rHSA). In some embodiments of the supplemental compositions, the rHSA is expressed in bacteria, yeast, or plants. In some embodiments, the rHSA is expressed in yeast.

[00101] In some embodiments of the methods, the embryo is a mouse embryo. In some embodiments of the methods, the embryo expresses a reporter gene. In some embodiments of the methods, the evaluation comprises evaluating the morphology of the embryo. In some embodiments of the methods, the evaluation comprises evaluating the expression of the reporter gene.

[00102] The embryo can be cultured in the medium for any desired period of time.

For example, the embryo can be cultured for 24 hours up to 7 days or any time period following fertilization. Preferably, the embryo is cultured for 3-6 days before transfer. The embryo can be cultured until developmental milestones are met such as achieving a certain number of cells (e.g., 6-8 cells) or reaching a stage (e.g., the blastocyst stage). In some embodiments, a cryopreserved embryo is thawed and cultured using the methods described herein before transfer. In some embodiments, the embryo was cultured using methods as described herein prior to cryopreservation. In some embodiments, the embryo was not cultured using methods as described herein prior to cryopreservation. [00103] The culturing can be performed utilizing any suitable incubation or culturing system or device with about 5 % to about 6.5 % CO2 and maintaining at about 35 °C to about 40 °C. In some aspects, a dedicated incubator is utilized. Non-limiting examples of incubators include the MINCTM incubator (Cook Medical), Labotect C02 Mini Incubator (Origio MidAtlantic Devices), Origio Planer Benchtop Incubator BT37 (Origio), G185 IVF Tri-Gas Incubator (Lab IVF), and the like. The methods further can include imaging systems that can obtain images of the embryos at one or more stages of development. In some embodiments, the incubator itself can include integrated imaging systems or devices. One example of such a device is the EmbryoScope incubator (Fertilitech).

[00104] FIG. 1 shows a graphical depiction of dextran evaluation in the upper graph, and collagen I and collagen IV evaluation in the lower graph. Percent of development is compared across different compositions, including i) 2 mg/ml dextran + 1 mg/ml rHSA, ii) 0.4 mg/ml HPC + 1 mg/ml rHSA, iii) 0.25 mg/ml HA + 1 mg/mL rHSA and iv) 1 mg/ml rHSA alone. The control groups were H.S.A. and SSS. The results demonstrate that at 96 hours, 2 mg/mL dextran and 1 mg/ml rHSA exhibited less 2.0% early blastocyst

development, 2.6 % blastocyst development, 15.8% expand blast, and 78.9% hatching Blastocyst. In the lower graph, CSCM2 + rHSA served as a control study. The two experimental groups comprised i) CSCM2 + rHSA + collagen I (mg/ml) at three separation concentrations, and ii) CSCM2 +rHSA + collagen IV (mg/ml) at three separate

concentrations. The results demonstrate that at concentration #1 the CSCM2 + rHSA + Collagen IV (mg/mL) achieved 88% Grade A (> Blastocyst) and 12% Grade B (<Blastocyst). At concentration #2, the CSCM2 + rHSA + Collagen IV (mg/mL) led to 84% Grade A (> Blastocyst) and 16% Grade B (<Blastocyst). At concentration # 3, the CSCM2 + rHSA + Collagen IV (mg/mL) achieved 64% Grade A (> Blastocyst) and 36% Grade B (<Blastocyst).

[00105] FIG. 2 shows the results of an Enzyme Linked ImmunoSorbent Assay

(ELISA), with different subject growth factors and cytokines in measured in different cell types, including oviduct, endometrium, AD MSC, BM MSC, UCMSC/WJMSC

mesenchymal cells. The growth factors of interest were insulin, EGF, PDGF, GM-CSF, and LIF. [00106] FIG. 3 illustrates a graphical representation of embryo grading of a MEGA™ Assay at 48 hours under nine test conditions. Condition 1 (10% SSS + NX) exhibited 50%

FI 0-1 and 50% FI 2-3. Condition 5 (NX +rHSA + Dextran + Insulin) exhibited 26% FI 0-1 and 74% FI 2-3. Condition 9 (BEA #6 NX + rHSA + Dextran + Coll. IV + Insulin + EGF + PDGF) exhibited 13% FI 0-1 and 88% FI 2-3.

[00107] FIG. 4 shows a graphical depiction of embryo grading of a MEGA™ Assay at 96 hours under 9 test conditions. Condition 1 (10% SSS + NX) exhibited 21% <Blastocyst, 25% Blastocyst, and 54% hatching. Condition 5 (NX +rHSA + Dextran + Insulin) exhibited 4% <Blastocyst, 83% Blastocyst, and 13% hatching. Condition 9 (BEA #6 NX + rHSA + Dextran + Coll. IV + Insulin + EGF + PDGF) exhibited 4% < Blastocyst, 33% Blastocyst, and 63% hatching.

[00108] FIG. 5 shows a graphical depiction of embryo grading of a mouse embryo assay (MEA) at 96 hours under 8 test conditions. Condition 2 (10% SSS + CSCM NX) exhibited 8% < Blastocyst, 36% Blastocyst, and 56% hatching. Condition 3 (CSCM NX + rHSA + Dextran + Insulin) exhibited 16% < Blastocyst, 56% Blastocyst, and 28% hatching. Condition 8 (CSCM NX + rHSA + Dextran + Insulin + Collagen IV + PDGF + EGF) exhibited 16% < Blastocyst, 60% Blastocyst, and 24 % hatching.

[00109] FIG. 6 shows a graphical depiction of the results of Bovine Fertilization Assays (BFA) with two Batches of test conditions. In Batch One, Condition 1 (10% SSS + NX) is highlighted along with Condition 5 (NX + rHSA + Dextran + Insulin). Condition 1 resulted in 5 total cleaved and over 80% non-cleaved. In Batch Two, Condition 1 (CSCM- NX + 10% SSS) is highlighted along with Condition 6 (NX1 + Collagen IV+Insulin + PDGF + EGF). Condition 1 resulted in 5 total cleaved and over 80% non-cleaved. Condition 6 resulted in 24 total cleaved and under 20% non-cleaved.

[00110] FIG. 7 illustrates a graphical depiction of the results of a Bovine Embryo Assay with one set of nine experimental conditions. Attendant blastocyst rates are shown. Condition 1 (NX + 10% SSS) resulted in a 41% blastocyst rate, Condition 5 resulted in a 34% blastocyst rate, and Condition 9 resulted in a 42% blastocyst rate. Condition 1 led to 32% stage 4 and 5 (compact morula and early to mid blast, respectively), and 9% Stages 6, 7, and 8 (expanding blast, expanded blast, and hatching blast, respectively). Condition 5 led to 13% stage 4 and 5, and 22% stage 6, 7, and 8. Condition 9 led to 35% Stages 4 and 5, and 35% Stages 6, 7, and 8.

EXAMPLES

[00111] The bovine fertilization assay (BFA) comprised a total of four experiments. Each assay comprised 30 eggs per condition. Test conditions included 5 drops per plate at 40 pL and 6 - 7 oocytes per drop. At 48 hours post-fertilization, the samples were checked for cleavage. If there was no cleavage at or around 48 hours, then the samples were mounted, stained, and eggs were squashed to check for sperm penetration and pronuclei formation.

The results of the BFA are shown in FIG. 6 and discussed above.

[00112] The bovine embryo assay (BEA) comprised a total of six experiments. There were 3 drops per condition at 20 pL per drop, and 10-12 embryos per drop. Embryos were checked and graded at Day 7. The results of the BEA are shown in FIG. 7 and discussed above.

[00113] All experiments were outsourced and blinded.

EQUIVALENTS

[00114] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[00115] The present technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed. [00116] Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology.

[00117] The present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[00118] In addition, where features or aspects of the present technology are described in terms of Markush groups, those skilled in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[00119] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[00120] Other aspects are set forth within the following claims.

Claims

WHAT IS CLAIMED IS:

1. A supplemental composition for cell culture comprising:

(a) recombinant Human Serum Albumin (rHSA);

(b) dextran; and

(c) one or more CD44 ligands.

2. The supplemental composition of claim 1, further comprising insulin.

3. The supplemental composition of claim 1 or 2, further comprising epidermal growth factor (EGF).

4. The supplemental composition of any one of the previous claims, further comprising platelet-derived growth factor (PDGF).

5. The supplemental composition of any one of the previous claims, wherein the CD44 ligand is selected from the group of matrix metalloproteinase, hyaluronic acid, osteopontin, serglycin, chondroitin, fibrin, P-selectin, basic Fibroblast Growth Factor (b-FGF), heparin binding epidermal growth factor (HB-EGF), collagen, fibronectin, and laminin.

6. The supplemental composition of claim 5, wherein the collagen is selected from collagen I, collagen II, collagen III, collagen IV, and collagen V.

7. The supplemental composition of claim 6, wherein the collagen is collagen IV.

8. The supplemental composition of claim 5, wherein the CD44 ligand is fibronectin.

9. The supplemental composition of claim 8, wherein the fibronectin is recombinant fibronectin.

10. The supplemental composition of claim 5, wherein the laminin is selected from laminin-111, laminin-211, laminin-121, laminin-221, laminin-332, laminin-3 A32, laminin- 3B32, laminin-311, laminin-3al 1, laminin-321, laminin-3 A21, laminin-411, laminin-421, laminin-511, laminin-521, laminin-213, laminin-423, laminin-522, and laminin-523.

11. The supplemental composition of any one of the previous claims, wherein the rHSA is expressed in bacteria, yeast, or plants.

12. The supplemental composition of claim 11, wherein the rHSA is expressed in yeast.

13. The supplemental composition of any one of the previous claims, further comprising one or more embryotrophic factors.

14. The supplemental composition of claim 13, wherein the embryotrophic factor is an anti-oxidant.

15. The supplemental composition of claim 13, wherein the embryotrophic factor is conditioned media derived exosomes from mesenchymal stem cells.

16. A method of culturing an embryo in vitro , comprising culturing the embryo in a medium supplemented with the supplemental composition of any one of claims 1-15.

17. The method of claim 16, wherein the medium is a sequential medium or a medium that is replenished at least once during the culturing of the embryo.

18. The method of claim 16 or 17, wherein the medium is a medium suitable for culturing a human embryo.

19. The method of any one of claims 16-18, wherein the medium is Continuous Single Culture™ medium, P-1 medium, G1 medium, G2 medium, Human Tubal Fluid (HTF) medium, modified HTF medium, Whitten’s medium, Ham’s F-10 medium, KSOM medium, Global Medium, or Single Step Medium™.

20. The method of any one of claims 16-19, wherein at least one biological parent of the embryo is suffering from infertility or undergoing infertility treatment.

21. The method of any one of claims 16-20, wherein the embryo is cultured for about 1 day to about 7 days.

22. The method of claim 21, wherein the embryo is cultured for about 3 days to about 6 days.

23. The method of any one of claims 16-22, wherein the embryo is cultured until it reaches an 8-cell stage or a blastocyst stage.

24. The method of any one of claims 16-23, further comprising implanting the embryo into a female patient.

25. A method of assessing a product for use in human Assisted Reproductive

Technologies, comprising:

(a) culturing an embryo in a medium supplemented with the supplemental composition of any one of claims 1-15 in the presence of the product;

(b) evaluating the embryo during at least one stage of development; and

(c) determining the acceptability of the product based on the evaluation.

26. The method of claim 25, wherein the embryo is a mouse embryo.

27. The method of claim 25 or 26, wherein the embryo expresses a reporter gene.

28. The method of any one of claims 25-27, wherein the evaluation comprises evaluating the morphology of the embryo.

29. The method of claim 27, wherein the evaluation comprises evaluating the expression of the reporter gene.

30. A method of fertilizing an egg, comprising fertilizing the egg in a medium

supplemented with the supplemental composition of any one of claims 1-15.

31. The method of claim 30, wherein the egg is from a subject suffering from infertility or undergoing infertility treatment.

32. A method of enhancing sperm survival, comprising culturing the sperm in a medium supplemented with the supplemental composition of any one of claims 1-15.

33. The method of claim 32, wherein the sperm is from a subject suffering from infertility or undergoing infertility treatment.

34. The method of any one of claims 16-33, wherein the medium does not contain serum.

35. The method of any one of claims 16-34, wherein the concentration of recombinant HSA in the supplemented medium is about 0.4 g/L to about 2.4 g/L.

36. The method of any one of claims 16-35, wherein the concentration of dextran in the supplemented medium is about 1.6 g/L to about 2.4 g/L.

37. The method of any one of claims 16-36, wherein the concentration of insulin in the supplemented medium is about 0.16 mg/L to about 0.24 mg/L.

38. The method of any one of claims 16-37, wherein the concentration of EGF in the supplemented medium is about 0.8 pg/L to about 1.2 pg/L.

39. The method of any one of claims 16-38, wherein the concentration of PDGF in the supplemented medium is about 0.0192 pg/L to about 0.0288 pg/L.

40. A kit comprising the supplemental composition of any one of claims 1-15 and instructions for use.

41. The kit of claim 40, further comprising a culture medium suitable for use in culturing an embryo, fertilizing an egg, or culturing sperm.

42. A culture medium comprising:

(a) a culture medium backbone suitable for use in culturing an embryo, fertilizing an egg, or culturing sperm;

(b) rHSA;

(c) dextran; and

(d) one or more CD44 ligands.