Exploring the Role of Hyaluronic Acid in Reproductive Biology and Beyond: Applications in Assisted Reproduction and Tissue Engineering

Hyaluronic acid (HA) plays a prominent role in various aspects of reproductive biology and assisted reproductive technologies (ART). This review describes the multifaceted influence of HA, ranging from primordial germ cell migration, ovarian follicle development, and ovulation in females to sperm structure, physiology, motility, and capacitation in males. In addition, HA also plays an important role in fertilization and promotes embryo implantation by mediating cellular adhesion and communication within the uterus. Against this physiological background, the review examines the current applications of HA in the context of ART. In addition, the article addresses the emerging field of reproductive tissue engineering, where HA‐based hydrogels offer promising perspectives as they can support the development of mature oocytes and spermatogenesis in vitro. Overall, this review highlights the integral role of HA in the intricate mechanisms of reproductive biology and its growing importance for improving ART outcomes and the field of tissue engineering of the reproductive system.


Introduction
Hyaluronic acid (HA), also known as hyaluronan, is a linear polysaccharide composed of repeating disaccharide units DOI: 10.1002/adbi.202300621consisting of D-glucuronic acid and N-acetyl-D-glucosamine.This glucosaminoglycan (GAG) has been conserved throughout evolution and is expressed in high quantities intracellularly, within the extracellular matrix (ECM), and on the surface of mammalian cells.The human body synthesizes significant amounts of HA, which mainly plays a role in various cellular interactions.These include processes such as cell differentiation, proliferation, development, and molecular recognition.In addition, HA contributes to several important physiological functions that include lubrication, maintenance of hydration balance, structural support within the ECM, and steric interactions. [1]hile HA is known for its multiple roles in cellular interactions and physiological functions, it is intriguing to consider how this versatile macromolecule relates to the complicated landscape of the male and female reproductive systems, where HA has a significant role starting from the initial stage of gonad formation, continuing the migration of primordial germ cells, [2] and the development of gametes [3] up until the process of fertilization. [4]In the context of human reproductive health, its importance becomes even more apparent when considering the spectrum of disorders that can affect the male and female reproductive systems and lead to infertility.According to the World Health Organization (WHO), infertility ranks as the fifth most significant global disability among the younger generation, affecting ≈10% of the global population. [5]Common causes of male infertility include poor semen quality, difficulties in sperm delivery, and excessive exposure to certain environmental variables.In females, infertility can be attributed to disorders related to ovulation, abnormalities in the uterus or cervix, damage or obstruction in the fallopian tubes, endometriosis, and primary ovarian insufficiency. [6]Furthermore, many structural anomalies in the male and female reproductive systems can be influenced by diseases, trauma, and some medicines, including cancer treatments, resulting in the loss of reproductive stem cells and ultimately leading to infertility. [7]arious techniques are used to preserve and promote human fertility, including assisted reproductive technology (ART), egg and sperm freezing, embryo freezing, ovarian tissue freezing, and methods to preserve fertility in cancer patients, each of which addresses different aspects of reproductive health.Cryopreservation of sperm and oocytes is a current method employed to preserve fertility in individuals of both genders.Nevertheless, this approach is ineffective in cancer cases, particularly in prepubertal children.This is because boys only have spermatogonia stem cells (SSC), which are the precursor cells for mature sperm. [8]n the other hand, retrieving oocytes from pre-pubescent girls is not feasible due to the immaturity of their ovaries, which are incapable of producing functional oocytes. [9]n response to these challenges, alternatives such as testicular and ovarian tissue freezing have emerged to facilitate the development of new options for germ cell transplantation, tissue transplantation, and in vitro maturation of gametes to restore fertility. [10]In addition, tissue engineering has emerged as a viable platform to treat infertility by culturing germ cells or tissue fragments in a controlled in vitro environment.Biomaterials are the main components of these tissue engineering approaches, including both synthetic and natural variants.Among natural biomaterials such as collagen, gelatin, and fibrin, HA has gained prominence in the biomedical field, where it is used either alone or in various combinations to develop coatings, nanoparticles, and hydrogels. [11]In particular, HA hydrogels are currently used in various applications, such as space fillers, mucoadhesive agents, wound healing, cancer therapy, and cosmetic products. [12,13]In the field of regenerative medicine, the ability of HA hydrogels to modulate the 3D architecture of the extracellular matrix (ECM) in a manner that promotes the release of growth factors, cytokines, and therapeutic molecules is a critical element. [14]HA has found wide application in tissue repair and regeneration strategies in this direction, particularly in cartilage, bone, and smooth muscle therapies. [15]he intricate interplay of HA in regenerative medicine and tissue engineering becomes even more important when considering the role HA plays in the reproductive system.In recent years, HA's key physiological role in the reproductive system has been increasingly recognized, leading to an increasing focus on its importance in the treatment of infertility and in the in vitro culture of reproductive cells and tissues.This article provides an overview of the recent applications of HA in ART and reproductive tissue engineering, highlighting significant advances that may lead to higher fertility rates and birth rates.

HA Characteristics and Formation In Vivo
HA was initially discovered in 1934 when Meyer and Palmer isolated it from the bovine vitreous humor. [16]As a non-sulfated GAG, HA is structurally made up of D-glucuronic acid (GlcUA) and N-acetyl glucosamine (GlcNAc) groups via exchanging ß-1,4, and ß-1,3 glycoside linkages (Figure 1).These disaccharides bond to each other, creating a coiled chain structure with lengths varying up to 10 nm and relative molecular weights ranging from 10 to 1000 kDa. [17]Although the presence of HA in the cytoplasm and nucleus was suggested as early as the 1970s, it was clearly demonstrated and confirmed in the 1990s.The ECM in all mammalian tissues is composed of GAGs, with HA being structurally the simplest of these GAGs, [18] and synthesized by the three distinct and highly conserved HA synthases (HAS) 1, 2, and 3 isoenzymes. [19]HAS1 exhibits less catalytic activity than HAS2, which is less active than HAS3.Moreover, HAS1 and HAS2 are responsible for the synthesis of long HA polymer chains (≈2000 kDa), whereas HAS3 generates shorter chains (200 kDa). [20]he pathway of GAG biosynthesis begins with glucose and glucosamine (GlcN) entering the cell via GLUT transporters.First, glucose-1-phosphate is used by UDP-glucose pyrophosphorylase (UGPP) and dehydrogenase (UGDH) to produce UDP-glucose (UDP-G) and UDP-glucuronic acid (UDP-GlcUA), respectively.UDP-GlcUA and uridine diphosphate-Nacetylglucosamine (UDP-GlcNAc) can be used directly to produce HA by plasma membrane HAS enzymes or transported into the ER/Golgi to synthesize various glycoconjugates, including proteoglycans. [21]HA is found in varying concentrations in body fluids (from 0.01-0.1 g g −1 in blood serum to 1400-3600 g g −1 in synovial fluid) and connective tissues (from 8.5-18 g g −1 in thoracic lymph nodes to 140-338 g g −1 in the vitreous body). [22]he molecular weight of HA can vary in certain disease states.For example, rheumatoid fluid has a molecular weight of 3000-5000 kDa compared to 6000-7000 kDa in normal human synovial fluid. [23]he biological function of HA is influenced by variable molecular weight.Low molecular weight HA (LMW-HA) (< 35 kDa) is proinflammatory and functions as an intracellular signaling molecule during inflammation, whereas high molecular weight HA (HMW-HA) (> 103 kDa) has antioxidant, antibacterial, antiinflammatory, and lubricating properties. [24]In wound healing, partially degraded fragments of HMW-HA and hyaluronan oligosaccharides (10 disaccharide units) stimulate angiogenesis and endothelial cell migration. [25]Due to its remarkable waterbinding capacity, HA significantly affects tissue water transport and hydration.HA effectively binds water molecules in its structure and maintains osmotic pressure by utilizing the negative charge originating from its carboxyl groups. [26]A is generally depolymerized by endoglycosidases known as hyaluronidases (HYALs) or destroyed nonspecifically by oxidative damage caused by reactive oxygen species (ROS).The ß-1,4 glycosidic link between the GlcNAc and GlcUA of HA is hydrolyzed by HYALs in mammals.In humans, six members of the HYAL family have been discovered, with ≈40% identity overlap. [27]The HYAL1, HYAL2, and HYAL3 genes are located on human chromosome 3p2 and produce specific HYALs that cleave HMW-HA into fragments at the plasma membrane level (HYAL2) and are then internalized into the cell via endocytosis and further degraded in the lysosome (HYAL1). [28]A second mechanism involves the natural degradation by ROS, which is produced in large quantities during inflammatory responses, tissue injury, and tumor development.
Due to its diverse molecular sizes and chemical configurations, HA plays an important role in many biological processes, including cell migration, inflammation, angiogenesis, remodeling of ECM, and scar formation. [29]Several HA functions depend on its interaction with cell surface receptors, including cluster determinant 44 (CD44), receptor of hyaluronan-mediated motility (RHAMM), lymphatic vessel endothelial hyaluronan receptor (LYVE-1), hyaluronan receptor for endocytosis (HARE), liver endothelial cell clearance receptor (LEC receptor), and toll-like receptor 4. [30] CD44 and RHAMM are both known to be associated with the cytoskeleton and play a role in regulating cell functions.While CD44 mediates the migration of cancer cells, fibroblasts, and endothelial cells, [31] RHAMM is known as a cell surface receptor that facilitates hyaluronan-induced motility and also as a microtubule-associated protein that interacts with dynein and stabilizes the spindle poles. [32,33]RHAMM is a G2/M cell cycle protein.This was confirmed by comparing its expression to cyclin B2, another G2/M protein.However, in contrast to the subcellular location of cyclin B2, RHAMM maintains mitotic spindles in both anaphase and metaphase. [34]Intracellular HA accumulates at the mitotic spindle and is colocalized with tubulin and RHAMM in the smooth muscle cells.At this time, forming the pericellular matrix facilitates cell detachment and rounding. [35]n addition to cell mitosis, HA mediates cell differentiation and proliferation. [36]Biologically and at the cellular level, HA polymers are most abundant in highly hydrated connective tissues, skeletal structures, and supporting tissues.

HA Synthesis and Modification In Vitro
In industry and research laboratories, HA is usually derived from human umbilical cords or animal tissues such as rooster comb, shark skin, or bovine vitreous humor. [37]HA from animal origin often has a very high molecular weight (up to 20 000 kDa).For example, the MW of HA obtained from rooster combs is 1200 kDa, and it is 770-1700 kDa from bovine vitreous humor.HA from human umbilical cords has a MW of 3400 kDa.Bacteria also produce HA with a molecular weight between 1000 and 4000 kDa.However, using an enzymatic process, it is possible to produce polysaccharides with molecular weights between 550 and 2500 kDa. [23]tudies have shown that in tissue regeneration and the extracellular matrix (ECM) of fetal tissue, the increased presence of HA plays an important role in preventing scar formation. [38]HA is often modified to fabricate hydrogels for tissue engineering applications, and crosslinking is a common method to improve mechanical properties, viscosity, solubility, degradation, and biological properties. [39]Crosslinking can be achieved by physical methods that include variations in pH, temperature, ionic strength, and physicochemical interactions. [40]In the chemical crosslinking, three of HA's most common covalent modification sites (carboxylic groups, hydroxyl groups, and NHCOCH3 groups) are the target of acidic, neutral, and alkaline environments crosslinking.Numerous techniques for functionalizing hyaluronan with side groups have also been developed to provide novel medications for targeted and regulated release. [41]HA's carboxylic acid and hydroxyl sites are two commonly utilized covalent modification sites for chemical cross-linking.The carboxyl groups on HA may be crosslinked via an ester linkage, whereas the hydroxyl groups may be crosslinked via an ether linkage. [39]The carboxylate group is easily targeted by ester or amide bond-forming reagents, resulting in a reactive product, allowing a second reaction to target and link it to a polymer, like gelatin.The polysaccharide derivatives alginate and HA are routinely employed in biopolymers to make injectable hydrogels using the Schiff-base reaction.Schiff's base creation is one of the chemical crosslinking techniques actively researched due to its quick crosslinking and great biocompatibility. [42,43]Other techniques for cross-linking HA include thiol modification, an enzyme-driven approach triggered by ROS, a substitution reaction with bromoacetic anhydride, and cross-linking with methacrylate.Thiolate HA is an optimal choice for injectable hydrogels due to its inherent biocompatibility and ability to form a hydrogel through a thiol-disulfide exchange reaction. [44]However, the most widely employed technique is the covalent crosslinking of hyaluronic acid to polymers, generating hydrogels. [36]Hydrogels made of HA are similar to human tissues regarding water content, oxygen exchange, nutrient absorption, and metabolic waste removal. [45]Given the hydrophilic nature of HA, approaches to encapsulate hydrophobic substances involve conjugation with hydrophobic components, which also alters its properties such as degradation, mechanical stability, and tendency to aggregation. [46]he chemical modulation of HA, specifically through the alteration of HA carboxyl sites individually with thiol (HA-SH) [47] and N-hydroxysuccinimide (NHS), [48] has gained attention in the field of follicular culture and embryo implantation in reproductive research.

The Physiological Role of HA in the Reproductive System
HA exerts a multifaceted influence on the entire reproductive system, affecting various critical phases of reproductive biology.With its involvement in forming primordial germ cells and their migration to the genital ridges, HA plays a central role in facilitating the fundamental step of gametogenesis.HA remains essential in gonadal development and supports the intricate cellular processes leading to gamete formation, fertilization, and embryo implantation.In the female reproductive system, the involvement of HA extends to the development of ovarian follicles and the formation of cumulus-oocyte complexes, which are critical for successful ovulation.Notably, disruption of HA has been associated with atypical follicular maturation, which may contribute to ovarian dysfunction.In the male reproductive system, HA is expressed in key regions and secretions, affecting sperm structure and physiology.HA modification of sperm membranes improves their binding ability, influences their motility, and plays an essential role in capacitation and fertilization.In addition, HA helps promote embryo implantation by mediating cellular adhesion and communication in the uterus, ultimately supporting successful early pregnancy.The following subsections address these critical aspects of HA's physiological role in the reproductive system and examine each stage in more detail.

Gonad Formation
Primordial germ cells (PGC) are the fundamental undifferentiated stem cell type from which gametes like spermatozoa or oocytes originate.In mice, PGCs first emerge on embryonic day 7.25 (E7.25) during gastrulation, positioned posterior to the primordial streak at the base of the allantois within the extraembryonic mesoderm.Over four days, PGCs travel from this location to their final destination, the genital ridges. [49]This migration is favored by the chemical structure of HA, as shown by the positive Alcian blue staining confirming HA's presence on the left side of the mesentery. [50]Following the migration of PGCs to the genital ridge and the subsequent formation of gonads, the HA plays significant roles in various aspects of embryonic development, such as gametogenesis, fertilization, and embryo implantation.During fetal growth, neural crest cells move along a hyaluronan-rich path.This is due to HA's capacity to confer strength and flexibility to various tissues, enabling hyaluronanrich regions to separate physical structures under internal pressure, creating "highways" for cellular movement. [51]

Ovarian Follicles
The HA concentration in ovaries is comparatively lower than other urogenital organs, including the urinary bladder, uterus, vagina, and penis.In ovarian follicles, the extracellular matrices of the theca interna, follicular fluids, and zona pellucida of large antral follicles are the primary sites of HA accumulation, whereas the ECMs of small preantral follicles do not exhibit significant HA accumulation.
The cumulus-oocyte complex (COC) in mammals comprises multiple layers of compacted cumulus cells surrounding the maturing oocyte.Upon exposure to gonadotropin stimulus, COCs initiate the phenomenon of cumulus expansion. [58]Cumulus expansion involves the synthesis of HA in the cumulus cells regulated by the expression of HAS2, which is, in turn, controlled by FSH. [53]In contrast to the outermost layers of granulosa cells, which are called mural granulosa cells, HA synthesis is expanded to the granulosa cells near the COCs, and the innermost layers of granulosa cells adjacent to the follicular antrum, called antral granulosa cells.The hyaluronan secreted from the cumulus cells binds to hyaluronan-associated proteins (ITI, TSG6, and PTX3), forming an enlarged hyaluronan-rich matrix in the COCs. [52]arious proteins, including serum-derived inter-alpha-trypsin inhibitor, tumor necrosis factor-alpha protein 6, pentraxin-3, and versican, contribute to the stabilization of HA within the extracellular matrix during cumulus expansion. [58]The enlarged extracellular matrix interconnects the oocyte and cumulus cells to protect against proteolytic and mechanical factors throughout ovulation, increasing the chance of successful and efficient reproductive outcomes. [52]Research on mice, [55] porcine, [56] and cattle [57] has confirmed the higher expression of HAS2 mRNA in cumulus cells when activated by FSH/CG and its crucial role in regulating cumulus enlargement.
HA is synthesized by the enzymatic activity of hyaluronan synthase genes HAS1 and HAS2, and the mRNA molecules of these genes are predominantly located in the theca and granulosa cell layers during follicle development. [54]he binding of hyaluronan and CD44 during cumulus expansion is essential for maturation-promoting factor (MPF) activity and oocyte meiosis.MPF, a serine/threonine protein kinase that interacts with cyclin B and p34cdc2, plays a crucial role in initiating meiosis, and cAMP regulates the activation of MPF in the oocyte.cAMP is produced in the cumulus cells and transferred to oocytes through COC gap junctions. [52]Connexin 43 (Cx43), the most common gap junction protein, has been identified in ovarian follicles of many species.After preovulatory gonadotropin surges, Cx43 is phosphorylated and leads to the closure of the gap junction channel. [59]Gap junction channels subsequently support the free flow and exchange of small molecules, such as cAMP, between follicular cells and interact with the regulatory subunits of cAMP-dependent protein kinase (PKA), which activates PKA activity. [60]The interaction between HA and its main surface receptor CD44 affects the tyrosine phosphorylation of Cx43 in cumulus cells, leading to gap junction closure and activation of meiosis resumption. [61]hroughout the expansion of the COCs, notable alterations occur in the morphology of cumulus cells.These changes involve a substantial reorganization of the cytoskeleton, characterized by the assembly of actin microfilaments.Additionally, the synthesis of HA is induced, transforming densely packed cumulus cells into a considerably larger aggregation of adhesive cells. [62]On the contrary, the synthesis of HA is facilitated by many growth factors, including transforming growth factor- (TGF-1), which enhances the expression of HAS2.The primary intracellular signaling pathway responsible for the upregulation of HA synthase expression and subsequent hyaluronan synthesis in human granulosa-lutein (hGL) cells is the canonical SMAD2/SMAD3-SMAD4 signaling pathway. [63]he crucial function that HA plays in oocyte maturation is evident in polycystic ovary syndrome (PCOS), one of the most common endocrine disorders.Previous studies have demonstrated a significant association between oocyte morphology disruption and HA's presence.Typically, during the formation of COCs before successful ovulation, follicles exhibit the presence of HA and hyaluronan-binding protein 1 (HABP1), TNF-a-stimulated gene/protein 6 (TSG-6), and pentraxin-3 (PTX-3) in a coordinated manner.Disruption of HABP1 in the ovaries, together with concomitant changes in expression and excessive activation of associated matrix metalloproteinase (MMP), could lead to atypical follicle maturation, ultimately resulting in ovarian dysfunction in rats with PCOS. [64]

Sperm Parameters
HA is expressed in various regions and secretions of the male reproductive tract (e.g., testis and seminal plasma). [65]Spermiogenesis entails a series of transformations within sperm that are pivotal for establishing binding sites for the zona pellucida and HA.Consequently, the lack of sperm membrane modification greatly impacts the formation of the zona pellucida and HA binding sites.Additional attributes associated with robust sperm development, including the absence of cytoplasmic retention, the absence of persistent histones, and elevated levels of DNA chain integrity, are also evident in HA-bound sperm. [66]This modification is correlated with better morphological characteristics, sperm maturation, and increased progressive motility.The location of the RHAMM protein along the tail, midpiece, and head of the sperm has been found to effectively regulate sperm motility during contact with HA.Additionally, it has been observed that the plasma membrane of spermatozoa from human, boar, bull, and ram species exhibit the expression of CD44, which may facilitate their ability to bind to the hyaluronan in the tubal fluid. [67]ntricate modifications in the structure and physiology of sperm, including the depletion of cholesterol in the membrane, translocation of glycoconjugates, phosphorylation of protein residues, and hyperactivation, characterize sperm capacitation.Research has shown that sperm capacitation necessitates the elevation of the tyrosine phosphorylation (TP) of membrane proteins.
TP has been observed in multiple regions of the sperm cell, such as the acrosomal cap, equatorial head region, neck, and principal piece.Tyrosine-phosphorylated proteins are primarily located in the spermatozoa's flagellum, where they perform a vital support function for the sperm as it prepares to connect to the zona pellucida and fuse with gametes.The phosphorylation pattern observed in the head and neck regions of sperm remains consistent upon attachment to either the zona pellucida or HA.Furthermore, it has been observed that TP levels increase over time during the capacitation process. [68]n contrast, to achieve effective fertilization, the sperm must release HYALs situated within the acrosome.This enzymatic activity allows the sperm to enter the zona pellucida and merge with the egg membrane. [69]The expressions of HYAL1, HYAL2, and HYAL3 occur in somatic cells.In contrast, the genes HYAL4, HYAL6, and sperm hyaluronidase 1 (SPAM1) are expressed in germ cells and are found on human chromosomes 3p21.3 and 7q31.3. [70]SPAM1, also known as PH-20, is a GPI-anchored hyaluronidase located in the head of the sperm.Its significant hyaluronidase activity is required to bind the zona pellucida and break down the extracellular matrix HA. [71] Recent research has determined that the distribution of SPAM1 inside the sperm head is essential for effective interaction with the cumulus-oocyte matrix. [71]nother sperm surface protein implicated in sperm-oocyte adhesion is HABP1.The maturation and functional alterations of sperm in the epididymis may be related to the expression of HABP1 on their surface.The study results show that hyaluronan may enhance in vivo phosphorylation of HABP1 and cellular phosphorylation.The fact that non-motile spermatozoa exhibit a blockage of this phosphorylation step raises the possibility that hyaluronan aids in sperm motility promotion. [72]esearch has shown that HA regulates sperm motility in mammals by interacting with HA-binding proteins, which could be phosphorylated in active cells, and that HA enhances the progesterone-induced acrosome response in human spermatozoa [73] as well as the zona-induced acrosome reaction in macaque spermatozoa. [74]HA has been shown to increase the intracellular calcium concentrations (Ca 2+ ) in sperm to promote motility. [75]The GPI-anchored plasma membrane protein PH-20 is responsible for this Ca2+ rise, as evidenced by treatment with Fab fragments of anti-PH-20 IgG preventing the increase. [76]mportant elements of the acrosome response signaling system are inositol 1,4,5-trisphosphate-gated calcium channels.Acrosome responses were triggered by the translocation of 1,4,5-trisphosphate into spermatozoa after increased calcium flux. [77]Other channel proteins, in addition to the inositol channel, are primarily found in the principal part of spermatozoa.The primary mechanism governing Ca2+ influx in spermatozoa is the opening of CatSper channels, which allow Ca2+ entry into the cells. [78]

Embryo Implantation
HA has been detected in uterine fluid and observed to promote adhesion between cells and the extracellular matrix.HA potentially plays a crucial function in enhancing the adhesion of embryos to the endometrial surface of the uterus, therefore facilitating the invasion of the epithelium and maternal circulation for the formation of the placenta.CD44 is a prominent receptor that directly binds to HA, which is crucial in facilitating certain cellcell and cell-matrix interactions. [79]A single 20-exon gene found on the short arm of chromosome 11 (11p13) codes for the integral transmembrane protein CD44, which has a molecular mass of 85 to 90 kDa.Ten of the 20 exons are transcribed in the common form, CD44s.There are several different isoforms of CD44 (CD44v1-v10), with sizes varying from 80 to 250 kDa, which result from alternative mRNA splicing of the remaining ten exons.Individual cells may simultaneously express one or several isoforms or even "switch" between isoforms in response to environmental cues. [80]efore embryo implantation, a substantial alteration of the uterine environment is required during the menstrual cycle to adequately prepare for the process of embryo implantation.The process encompasses cellular proliferation and subsequent differentiation, transforming endometrial fibroblasts into a distinct cellular phenotype referred to as decidual cells.Three stages make up the menstrual cycle: proliferative, secretory, and menstrual. [81]Studies investigating the existence of HA and CD44 throughout the menstrual cycle indicated that both the endometrial glands and stroma exhibited a lack of CD44s and CD44v6 expression during the proliferative phase.During the secretory phase, the endometrial glands lacked CD44 expression, while CD44v6 was expressed.
In contrast, the stromal cells exhibited the expression of CD44s while lacking CD44v6 expression.Hematoxylin and eosin (H&E) staining demonstrated the presence of HA in the endometrial stroma throughout the menstrual cycle.However, the staining was found to be most prominent and diffusely distributed during the midsecretory phase.Perivascular staining for HA was observed consistently throughout the menstrual cycle.The staining intensity was highest near the spiral arterioles during the secretory phase.Accordingly, the endometrium seemed most receptive to embryo implantation during the peak staining of HA and CD44s in the stroma and overexpression of CD44v6 in secretory glands. [82]Moreover, even though CD44 is present in developing embryos and pre-hatched blastocysts, CD44-HA interactions seemed to be linked to embryo attachment during the early stages of implantation.The endometrial epithelium and the embryo are thought to be linked by HA through CD44 (and possibly other receptors). [83]n addition to CD44, RHAMM regulates various cellular and dynamic processes, such as cell-to-cell adhesion between the uterus and embryo.The expression RHAMM is observed to vary across all stages of preimplantation of human embryos and human embryonic stem cells (hESC).It has been found that suppressing RHAMM leads to the downregulation of multiple markers associated with pluripotency in hESCs. [84]The spatiotemporal expression of RHAMM in the uterus during the estrous cycle and peri-implantation period is a way through which the uterus becomes receptive to developing an embryo.The expression of RHAMM in the rat uterus during the estrous cycle and implantation period has demonstrated the role of RHAMM in cell proliferation, differentiation, and angiogenesis. [85]levated levels of HA are thought to act as a binding agent that facilitates the initial attachment between blastocyst-stage embryo trophectoderm (TE) and endometrial epithelial cells by linking receptors on both surfaces [86] However, due to ethical restrictions on studying human embryos, the mechanism of action of HA after implantation in humans is still unknown, and the only data available are from animal models.

HA and Assisted Reproductive Technologies (ART)
In this section, the critical role that HA plays in ART will be addressed, including its involvement in sperm selection and its influence on embryo transfer and implantation.

Sperm Selection
The physiological importance of HA for sperm maturation and integrity has paved the way for its application in diagnostic tools based on the marked selectivity of sperm for binding to hyaluronan.(Figure 2). [87]Sperm attaching to the zona pellucida (ZP) surrounding the oocyte is the first stage of fertilization.Unlike the ECM covering somatic cells, the ZP is a group of glycosylated proteins termed ZP1-4 in mammals.In addition to its vital role in promoting the physiological development of oocytes, ZP also acts as a species-specific receptor, enabling motile sperm to attach to eggs during fertilization. [88]Moreover, the female reproductive system has a significant amount of HA, which plays a role in the interactions between sperm and oocytes.COC also contains a high concentration of HA, found in the zona pellucida and the perivitelline space of mammalian oocytes.During fertilization, HA-receptors CD44 and RHAMM help the mature sperm bind to COC through hyaladherins.This interaction enables the release of PH20/SPAM1, which has no link to HA and enhances sperm penetration in the COC.Hence, the absence or inadequate expression of these receptors in immature spermatozoa impairs or reduces the ability of sperm to bind to the HA. [89]his binding can be intentionally created in vitro using easily accessible processes, such as depositing of HA droplets in the lower portion of a plastic culture dish in the PICSI approach, in gel-like material such as in Sperm Slow (Medicult Origio), and chambers covered in hyaluronan in the HBA assay (Cooper Surgical) to distinguish mature spermatozoa with plasma membrane hyaluronan receptors from immature ones. [90]In scientific literature, the HA-binding score (HBS) is commonly used to describe the proportion of sperm that attaches to an immobilized solid-state HA-coated surface, as determined by the HBA assay.In this technique, a drop of the sperm was applied to a slide coated with HA, and 100-200 sperm were counted to determine the percentage of unbound sperm that moved freely versus those bound to HA. [91] It is reported that the attached sperm HA shows better motility, morphology, nuclear DNA maturity, [92] a lower percentage of aneuploidies, and sperm DNA fragmentation. [93]he analysis of 1247 semen samples, which were studied for HBS and DNA integrity, revealed a significant negative relationship between HBA and three DNA fragmentation assays (Acridine orange, Comet, and TUNEL assay).Furthermore, it is worth noting that the older women who participated in this study exhibited comparable live birth rates to their younger counterparts following the evaluation of sperm DNA fragmentation and sperm selection by HBA. [94]A-binding can also be used to select fully developed sperm for the insemination process.Highly viscous media containing HA, such as SpermSlow, offer potential substitutes for polyvinylpyrrolidone (PVP) in the immobilization of sperm during the intracytoplasmic sperm injection (ICSI) process. [95,96]On the other hand, a study on 45 IVF cycles, including 257 oocytes fertilized with PICSI and 294 with traditional ICSI, suggested that sperm selected by HA can effectively increase fertilization rate and ART outcome.Most patients in the study had experienced past fertility problems, poor fetal development, implantation failure, or miscarriage, and at least one failed IVF cycle (except for two individuals).The outcomes showed higher fertilization and transfer-eligible embryo rates in the PICSI-fertilized group. [97]According to current research, the widespread use of HAB in clinics suggests that HA has found its place in ART, while some uncertainty remains over the efficiency of HBA in enhancing ART success and achieving greater live birth rates after employing HBA for sperm selection.

Embryo Transfer and Implantation
HA has been incorporated into embryo culture and transfer media to enhance the prospects of success in ART (Figure 2).Ongoing research and practical applications are focused on the investigation of using synthetic albumin with HA or replacing albumin with HA in culture media, with results indicating that supplementing albumin with HA can serve as an effective replacement for blood-derived albumin in enhancing reproductive outcomes. [98]ccording to a Cochrane review (2020) which examined 26 studies and 6704 participants, the addition of functional HA concentrations (0.5 mg mL −1 ) to the embryo transfer media could increase live birth rates (Risks Ratio (RR): 1.21, 95% confidence intervals:1.1 to 1.31; 10 RCTs, N = 4066 participants; I 2 = 33%; moderate-quality evidence).This suggests that incorporating HA into the culture media may increase the probability of live birth from the baseline of 33% to a range between 37% and 44%. [99]dditionally, a cohort study by Adeniyi et al. (2021) confirmed that using an HA-rich medium for embryo transfer was positively and significantly associated with improved clinical pregnancy rate and a singleton or twin live birth, regardless of the duration of exposure evaluated in this study.However, using an HA-rich medium for ET did not affect the gestation length or birthweight. [100]he introduction of commercial hyaluronan-rich culture media like EmbryoGlue (Vitrolife, Sweden) and UTM (Origio, Denmark), created specifically for embryo transfer, was prompted by the efficacy of HA-rich culture medium in improving ART results following embryo transfer.Studies on EmbryoGlue indicated that it may enhance the chances of achieving a clinical pregnancy in individuals who have encountered multiple IVF embryo transfer (ii) The HA coating interacts with endometrial epithelial cells via native ligand-receptor interactions, while the remaining NHS groups of the HA coating allow for additional binding to endometrium surface via formation of covalent amide bonds with surface proteins.(iii) Over time, HA coating undergoes enzymatic digestion into fragments which may act as useful signaling factors.(iv) At the time of hatching, the HA-conjugated ZP detaches from embryo and implantation progresses with native ligand receptor interaction.Adapted and reproduced with permission. [48]2022, Elsevier.
failures. [101]Valojerdi et al. investigated the effectiveness of Em-bryoGlue in 417 patients as a human embryo transfer medium, reporting an increase in the clinical pregnancy and implantation rate in the tubal factors and repeated implantation failures.Furthermore, compared to 398 patients in the control group, these patients had considerably higher rates of live births and triplet deliveries. [102]hile several studies have reported that HA-rich medium improved embryo growth and implantation, there is also conflicting research that questions the extent of the positive effects of hyaluronan enrichment on IVF success and pregnancy rates. [103,104]Ganji et al. ( 2022) made small alterations to the HA's chemical structure using N-hydroxysuccinimide (NHS) to generate new artificial binding sites, allowing the HA to form strong covalent connections with the surfaces of the embryo and endometrium.This change increased the implantation rate in mice to 80%, compared to 53% with HA-thiol and 56% with Embryo-Glue (Figure 3). [48]Based on the findings mentioned above, ongoing research efforts are currently concentrated on augmenting the activity that supports HA by employing techniques to introduce novel binding sites onto the HA structure.

Tissue Engineering of Ovary and Testis
The increasing number of cancer diagnoses in children, adolescents, and young adults, as well as improved survival rates due to effective chemotherapy and radiotherapy, [105] have raised concerns about the possible negative effects of these treatments on female fertility.Ovarian tissue cryopreservation is currently the primary method for cancer patients at risk of premature ovarian failure following cancer therapy. [106]This technique offers the possibility of ovarian tissue transplantation as a proactive approach to fertility preservation.However, the risk of reintroducing malignant cells during ovarian tissue transplanta-tion has led to a search for non-surgical methods to generate mature oocytes in vitro from ovarian tissue, including in vitro follicle culture and oocyte maturation.
Conventional 2D culturing techniques fail to maintain the 3D spatial arrangement essential for human follicles, which lose their 3D structure and cellular connections, resulting in immature oocytes. [107][110] A significant portion of the ECM of ovarian follicles consists of HA, reflecting its physiological importance in the female reproductive system.HA plays a structural role in ovarian follicles and is involved in signaling pathways critical for oocyte development and ovulation (Figure 4). [111]Research has therefore investigated the use of HA in the treastment of ovarian disease using animal models.In a rat model of endometriosis, HA-danazole hydrogel reduced the size of endometrial cysts without affecting the estrous cycle. [112]n addition, administering decidual endometrial stromal cells to mice encapsulated in an HA-fibrin composite has restored the damaged endometrium, leading to successful embryo implantation and development (Figure 5). [113]3D culture of mouse preantral follicles, either vitrified or freshly maintained in an HA and ECM-HA hydrogel, have provided an optimal microenvironment and reduced follicle flattening. [108]Incorporation of HA into the ECM resulted in a significantly higher level of estradiol secretion per follicle as compared to the HA or control groups.In another study, transplantation of 402 preantral follicles in HA hydrogel resulted in the formation of 314 COCs.Of the 314 COCs, 73% of the in vitro matured oocytes underwent meiosis and reached metaphase II (MII).After insemination, 83% of these oocytes were successfully fertilized, resulting in a basculation rate of 46%.In a pilot experiment with three recipient mice, one pup was delivered. [114]Another study compared the encapsulation of Model of the regulation of HA biosynthesis, degradation, and function in the reproductive system.The authors hypothesize that at least in ungulates, steroid hormones orchestrate a sequential expression pattern for HA of different sizes in the reproductive system, with estradiol (E2) inducing expression of HAS-2 resulting in the production of large-molecular-weight HA to support ovulation and fertilization, followed by the progesterone (P4)dominated phase, which upregulates CD44 expression and stimulates small-size HA production by HAS3 and HA fragments Hyal-2.Hyal-2 and HA fragments support early embryo development and induce the expression of adhesion molecules and signaling cascades required for the attachment of the blastocyst to the uterine luminal epithelium (LE) and establishment of pregnancy.FSH, follicle-stimulating hormone; GE, glandular epithelium; LH, luteinizing hormone; IFNt, interferon tau; MUC1, mucin 1; OPN, osteopontin; OTr, oxytocin receptor; PG″2a, prostaglandin F2 alpha; PGE, prostaglandin E; St, uterine stroma cells.Reproduced (Adapted) with permission. [111]© 2017, reproduced by permission from Society for Reproduction and Fertility.
follicles in alginate and alginate-fibrin with that of alginate and HA-alginate, showing that a greater proportion of follicles encapsulated in alginate and HA-alginate reached the antral stage.In contrast to oocytes developed from alginate, a greater proportion of oocytes formed from HA-alginate continued meiosis to the germinal vesicle breakdown (GVBD) phase/MII.In addition, compared to follicles encapsulated in alginate and alginate-fibrin, follicles in HA-alginate exhibited significant upregulation of most growth and differentiation genes and released greater amounts of estradiol. [115]n addition, stem cell therapy has received considerable attention in recent years in assisted reproductive research.Research has shown that HA hydrogels, in combination with umbilical cord-mesenchymal stem cells (UC-MSCs), resulted in prolonged survival of UC-MSCs in the ovary, supporting their paracrine activity.UC-MSCs support follicular survival by activating the PI3K-AKT pathway through a paracrine mechanism in vitro and in vivo.These findings highlight the potential of HA scaffolds for enhancing the effectiveness of UC-MSC in treating ovarian aging. [116]n ovarian tissue transplantation, HA may be useful to prevent the loss of primordial follicles in grafts due to ischemia, hypoxia, and tissue damage after transplantation.Up to 90% of primordial follicles may perish by apoptosis in the first few days after transplantation due to ischemia-induced tissue damage.HA is also known to promote angiogenesis and regulate blood vessel development.In conjunction with growth factors such as VEGF and bFGF, HA hydrogel-encapsulated ovaries appear to maintain the number of primordial and primary follicles, reduce apoptosis, and restore ovarian function after autotransplantation. [117] Together with growth factors, HA hydrogels can help maintain encapsulated ovarian tissue during transplantation by reducing ischemia-induced follicular loss, maintaining the follicular pool, promoting follicular survival, supporting angiogenesis, and restoring hormone levels. [118]Although HA hydrogel successfully preserved the viability of intact follicles in fresh ovarian tissue, it was not as successful in vitrified tissue.Nevertheless, it has been suggested that growth factors should be incorporated into HA-hydrogel to achieve results equivalent to those of fresh tissue during autotransplantation. [119]egarding tissue engineering in the male reproductive system, although HA is commonly used for sperm selection in ART (Figure 2), it has been used only to a limited extent for testicular tissue engineering and in vitro spermatogenesis.In general, other natural biomaterials such as collagen, [120] alginate, [121] Matrigel, [122] and agarose [121] have been widely used for culturing testicular tubules and spermatogonial stem cells (SSCs), but recent studies have shown promise in using HA-based hydrogels.Notably, a study using a composite of HA, chitosan, and decellularized mouse testicular matrix (DTM) resulted in the expression of key markers such as promyelocytic leukemia zinc finger (PLZF), transition protein 1 (TP1), and tectin 1 (TEKT1) by SSCs, indicating stem cell maintenance and post-meiotic differentiation. [123]These results indicate that HA-containing scaffolds have the potential to promote proliferation and differentiation of SSCs in vitro. [123]In another study the authors cultured SSCs in a hydrogel comprising HA and DTM and demonstrated proliferation and differentiation of SSCs in the hydrogel and later in the testicular tubule of a mouse model of azoospermia.In contrast to the in vitro seeded scaffolds, the scaffolds used in ex vivo Figure 5. Histological and functional assessment of endometrium treated with injectable HA/fibrin hydrogels in a murine model of uterine damage.The function of the regenerative endometrium was evaluated by analyzing the implantation of transferred embryos using hematoxylin and eosin staining and immunohistochemistry. Implantation and normal early gastrulation were confirmed, and the birth of offspring was also evaluated.A) Expression of three germ layer markers, Sox2/Brachyury/HNF4a, at E7.5 and B) Nestin/Brachyury/aFP at E8.5, in developing embryos.C) E13.5 embryos and neonates from the hydrogel-treated group showed normal development comparable to nondamaged control animals.*E: embryonic day, F: fibrinogen, HA: hyaluronic acid, T: thrombin.Adapted and reproduced with permission. [113]2019, Elsevier.
studies showed a significant and remarkable increase in the expressions of PLZF, TP1, and TEKT1. [124]

In Vitro Production of Embryos in Livestock
The production of animal embryos through in vitro fertilization and embryo culture has increased considerably in recent years.In 2019, almost one million transferable embryos were produced worldwide for cattle, thirty thousand for sheep, and three thousand for goats. [125]Ovum pickup and in vitro production (IVP) are two extremely important tools that lead to the availability of sufficient embryos for livestock production. [126]As in humans and animal models, HA has beneficial effects on livestock IVP at different levels, ranging from oocyte maturation to embryo implantation.

Oocyte Maturation
Producing good quality and quantity of mature oocytes is an important criterion for fertilization and the blastocyst rate.Cumulus cell expansion, which occurs during the maturation of oocytes following LH surge in vivo or after FSH or EGF treatment in vitro, requires the synthesis and organization of an ECM in which HA is a predominant component. [127]Genes and proteins responsible for HA production and its receptor CD44 are present in COCs across various animal species.To examine the impact of HA's molecular size and concentration on oocyte maturation and subsequent embryonic development, bovine oocytes were cultured in vitro with or without HA, Hyal-2, or 4-methylumbelliferone (4-MU), which is an inhibitor of HA formation.The results showed that the Hyal-2 prevented cumulus cell growth without affecting oocyte maturation or subsequent embryonic development.
In contrast, 4-MU decreased the growth of cumulus cells, the rate of oocyte maturation, and the progression of embryo development; the addition of exogenous HA somewhat mitigated these effects. [3]Moreover, supplementation of culture medium with different concentrations of HA has been reported to be an effective substrate for IVM.Remarkably, the culture of buffalo's COCs in TCM-199 medium for in vitro production of bovine embryos supplemented with 2.5 μg mL −1 HA resulted in a higher COC maturation rate. [128]Like bovine, CD44 was detected in porcine oocytes.Porcine cumulus cells express HAS2 and CD44 mRNA, but HAS3 was only found in oocytes.CD44 likely plays a role in the connection between cumulus cells and oocytes [56] and is strongly linked to gap junction communication in the complexes during meiotic resumption during cumulus expansion. [129]The 85-kDa hyaluronic acid-binding protein (HABP) is another related HA protein located on the membrane of cumulus cells.Through IVM, the increased expression level of HABP has been reported as cumulus expansion progressed. [130]As in the modification of IVM medium with HA to support the maturation of bovine oocytes, the addition of HA to IVM has led to similar results in pigs.When the HA components such as 0.05 mM glucuronic acid and 0.05 mm N-acetyl-Dglucosamine were added to the IVM medium, they were able to improve the cortical granule exocytosis, cytoplasmic maturation, cumulus expansion, and decrease the ROS levels. [131]Thus, it ap-pears that HA can efficiently assist in retrieving mature oocytes from the ovary of prepubescent farm animals after IVM.

Embryo Development
Due to the increased embryo production after ovum retrieval, IVP is a desirable option compared to multiple ovulation and embryo transfer.Oviductal culture of IVP embryos promotes better growth until the blastocyst stage. [132]However, only 30% of all embryo transfers occur from IVP embryos.Several factors influence the success of IVP embryos, such as maternal/paternal fertility, embryonic characteristics such as aneuploidies, and in vitro culture conditions such as light exposure, culture medium composition, and temperature variations. [126]Researchers have studied modifying culture conditions in different species by adding growth factors and ECM components like HA to mimic the natural microenvironment.HA is present in the oviductal and uterine fluids of bovines, while its receptors are in the preimplantation bovine embryo.Throughout embryonic development, RHAMM/IHABP mRNA levels decrease from the 2-cell to the 16cell stage, increase at the morula stage, and peak at the expanded blastocyst stage.
In contrast, HA production increases from the 2-cell to the 8-cell stage and decreases at the 16-cell stage.Finally, the secretion of HA increases during the enlarged and hatched blastocyst phases. [133]Accordingly, the strategy of adding HA to the culture medium also confirmed the increase in blastocyst production, better survival after vitrification, and improved survival rates after transfer of bovine embryos when cultured in modified synthetic oviductal fluid (SOF) with different concentrations of HA (0, 0.1, 0.5, or 1 mg mL −1 ). [134]The probable mechanism involves a reduction in the levels of ROS and an increase in the hatching rate after the addition of HA from the zygote to the blastocyst stage.HA can decrease the number of apoptotic nuclei and improve the development of blastocysts.This efficiency may be linked to the age of the embryo because when blastocysts are cultured with epidermal growth factor (EGF) + HA until the 16-cell stage, an increase in the expression of genes associated with the focal adhesion pathway was observed as compared to supplementation after the 16-cell stage. [135]A similar method has proven successful in improving IVP in pigs.The study found that adding 1 mg mL −1 , HA increased the percentage of cleaved embryos to ≈95%, morulae to ≈87%, and blastocysts to ≈77%.This effect is due to a significant reduction in ROS levels caused by decreased inner mitochondrial membrane potential in response to HA. [136] Although several studies support the positive effect of HA on oocyte maturation and embryo development, the specific advantages of HA for IVP embryos are still unclear and require further research.

Prospects for HA in ART and Reproductive Tissue Engineering
The clinical importance of HA has witnessed a recent upsurge, as it has assumed a more significant role in the treatment of various diseases and regenerative medical therapies.Furthermore, incorporating hyaluronic acid plays a key part in the real-world application of assisted reproductive techniques and associated research www.advanced-bio.comactivities.The utilization of hyaluronic acid as a sperm selection method and its application as an appropriate medium for in vitro oocyte maturation and embryo transfer has proven to be an effective approach to enhance the success of assisted reproductive technology.
HA has proven to be efficacious in ART procedures, particularly when patients possess viable oocytes and sperm.In this context, this biomaterial is not suitable for infertile couples who encounter challenges such as azoospermia or the presence of lowquality oocytes.Although the available research is limited in the present scenario, there is a growing interest in exploring the use of hyaluronic acid and its prospective uses in ovarian and testicular tissue engineering.
Using HA in ovarian tissue engineering exhibits encouraging prospects for developing artificial ovaries and the potential achievement of fully developed oocytes.Likewise, applying HA in the context of testis tissue engineering may pave the way for new directions in in-vitro spermatogenesis.Additionally, HA proposes a promising application as a bioactive hydrogel that encourages stem cell migration toward the formation of organoids, considering that testis organoids represent the most recent advancements in the field of testicular tissue engineering.
While there have been reports on the employing of HA in combination with other biomaterials, such as alginate, for the creation of hydrogels, as well as the use of modified HA to enhance embryo transfer and implantation, the combination of HA with decellularized extracellular matrix represents a potentially important application in the field of ovary and testis tissue engineering.A blend might be more successful in laboratory settings for gamete differentiation.
However, the processes and activities of HA in this situation are still not well comprehended.Therefore, conducting further research to assess the effectiveness of HA and elucidate its underlying mechanism in experiments could offer novel opportunities for exploring its impact on the development of SSCs and follicles, as well as its potential therapeutic uses for treating infertility in both males and females.

Figure 1 .
Figure 1.Chemical structure of hyaluronic acid.Hyaluronic acid is a non-sulfated glycosaminoglycan consisting of repeating disaccharide units of Dglucuronic acid and N-acetyl-D-glucosamine linked by alternating -1,4and -1,3-glycosidic bonds (shown in red).The green dotted lines indicate the hydrophilic functional groups, the blue color indicates the hydrophobic moieties.

Figure 2 .
Figure 2. Hyaluronic acid applications in assisted reproductive techniques and reproductive tissue engineering.

Figure 3 .
Figure 3. Schematic illustration of possible mechanisms underlying supportive action of the hyaluronan derivative (HA-NHS) during embryo implantation.(i) Upon incubation of blastocyst in HA-NHS solution, a uniform coating of HA is quickly attained via amide bond formation between amine-reactive groups (NHS) of HA and amine-bearing glycoproteins of ZP.The free-floating HA-surrounded blastocyst is transferred in vivo by routine procedures.(ii)The HA coating interacts with endometrial epithelial cells via native ligand-receptor interactions, while the remaining NHS groups of the HA coating allow for additional binding to endometrium surface via formation of covalent amide bonds with surface proteins.(iii) Over time, HA coating undergoes enzymatic digestion into fragments which may act as useful signaling factors.(iv) At the time of hatching, the HA-conjugated ZP detaches from embryo and implantation progresses with native ligand receptor interaction.Adapted and reproduced with permission.[48]2022, Elsevier.

Figure 4 .
Figure 4. Model of the regulation of HA biosynthesis, degradation, and function in the reproductive system.The authors hypothesize that at least in ungulates, steroid hormones orchestrate a sequential expression pattern for HA of different sizes in the reproductive system, with estradiol (E2) inducing expression of HAS-2 resulting in the production of large-molecular-weight HA to support ovulation and fertilization, followed by the progesterone (P4)dominated phase, which upregulates CD44 expression and stimulates small-size HA production by HAS3 and HA fragments Hyal-2.Hyal-2 and HA fragments support early embryo development and induce the expression of adhesion molecules and signaling cascades required for the attachment of the blastocyst to the uterine luminal epithelium (LE) and establishment of pregnancy.FSH, follicle-stimulating hormone; GE, glandular epithelium; LH, luteinizing hormone; IFNt, interferon tau; MUC1, mucin 1; OPN, osteopontin; OTr, oxytocin receptor; PG″2a, prostaglandin F2 alpha; PGE, prostaglandin E; St, uterine stroma cells.Reproduced (Adapted) with permission.[111]© 2017, reproduced by permission from Society for Reproduction and Fertility.
Pablo Pennisi received his Ph.D. in Biomedical Engineering from Aalborg University (Denmark) in 2008 and is currently an Associate Professor at the Department of Health Science and Technology.He conducts research in cell and tissue engineering, focusing on biomaterials for the development of 3D cell microenvironments.He is an active contributor to the academic community with over 60 peer-reviewed publications and eight book chapters in engineering and biology.He serves on editorial boards of international journals and is an active member of the IEEE-EMBS, the Scandinavian Society of Biomaterials, and the Danish Stem Cell Society.Abodolhossein Shahverdi obtained his Ph.D. in Anatomy in 2006 and currently leads the Royan Institute's esteemed Sperm Biology Department.With a focused expertise in sperm cryopreservation, he has pioneered innovative approaches to sperm processing in Assisted Reproductive Technology (ART).His prolific research contributions include over 180 articles and the authorship of 5 seminal books on sperm biology.Fereshteh Dardmeh earned her Ph.D. in Clinical Biomedicine with a focus on reproductive medicine from Aalborg University, Denmark, in 2017.Currently, an Associate Professor at the Department of Health Science and Technology, Aalborg University, Dr. Dardmeh has led "The Probiotic Research Initiative™" since 2019, exploring the role of the microbiome and probiotic interventions in a wide range of domains, including male fertility.Holding editorial responsibilities in several international journals, she has actively contributed to the academic community with more than 20 peer-reviewed publications and over 25 presentations in scientific meetings.Hiva Alipour earned his Ph.D. in Clinical Science and Biomedicine from Aalborg University, Department of Health Science and Technology, in 2017.Currently an Associate Professor within the Regenerative Medicine Group at the same institution in Aalborg, Denmark, his research primarily focuses on Reproductive and Regenerative Medicine, particularly Andrology.With a distinguished academic career, he has authored over 30 publications in national and international journals, including two book chapters, and has delivered more than 45 invited lectures, workshops, or presentations.Additionally, he holds editorial board positions in multiple international journals and scientific meetings, actively contributing to several scientific associations.