Within the female reproductive tract, the ovary is the organ most commonly treated with human umbilical cord (hUC) cells, especially MSCs. The main reports of the use of human umbilical cord (hUC) mesenchymal stem cells (hUC-MSCs) and UC blood derivatives were included in Table 1. In 2013, Wang et al. ^[1][35] pioneered the use of hUC-MSCs to treat POI in mice, restoring ovarian function, serum estrogen levels, adequate follicle development, and notably reducing cell apoptosis.

Similar to how other cell therapies have been infused in rodents ^[2][179], hUC-MSCs have been administered via tail vein injection, to foster their natural migration towards the injured site ^{[3][4][5][6][7][8][9][10][11]}[84,180,181,182,183,184,185,186,187]. This strategy not only improved ovarian morphology, primordial follicle development, and hormone production, but also restored estrous cyclicity ^{[3][5][7][10]}[84,181,183,186], implying ovarian performance was recovered. More importantly, systemic treatment with hUC-MSCs restored fertility in all cases ^[5][9][11][181,185,187]. Furthermore, hUC-MSC therapy provoked ovarian secretion of cytokines such as HGF, VEGF, and insulin growth factor 1 (IGF1), suggesting ovarian regeneration was mediated by paracrine mechanisms, and these factors could improve the ovarian function and delay ovarian senescence ^[4][180]. Using metabolomics, Zhao et al. ^[5][181] showed that hUC-MSCs activated the phosphoinositide 3-kinase (PI3K) pathway by stimulating synthesis of free amino acids, improved lipid metabolism, and decreased concentration of monosaccharides ^[5][181]. Meanwhile, Lu’s group ^[6][182] also focused on the protective effect of the hUC-MSCs on the theca-interstitial cells. In vitro, they observed an interesting reduction in autophagy levels in the theca-interstitial cells, induced by the decrease in oxidative stress and regulation of the AMP-activated protein kinase (AMPK)/Mammalian target of rapamycin (mTOR) signaling pathway. Augmented tropomyosin receptor kinase A (TrkA) and nerve growth factor (NGF) and NGF following hUC-MSC treatment was also reported by Zheng et al. ^[3][84], corroborating the involvement of the NGF/TrkA signaling pathway in ovarian regeneration. Moreover, the efficacy of multiple intravenous injections in a chemotherapy-induced POI mouse model was analyzed by Lv et al. ^[11][187]. After introducing cells, the ovarian morphology, follicle count, and fertility improved, regardless of whether mice received a single or triple administration. However, higher levels of serum anti-Müllerian hormone (AMH) and local Ki67 expression suggested that multiple doses of hUC-MSCs have a superior therapeutic effect than a single hUC-MSC bolus.

Alternatively, local intraovarian injections were also reported ^[12][13][14][37,188,189], and were capable of restoring follicle development and fertility in rodents. Interestingly, Shi et al. ^[13][188] analyzed the toxicity associated with injecting increasing doses of hUC-MSCs. The maximum tolerated dose was 10⁶ cells/ovary, with severe toxicity and lethality observed after exceeding this limit. Meanwhile, Pan et al. ^[14][189] found that hUC-MSC and amniotic MSC treatment, comparably restored damaged ovarian morphology, functionality, elasticity, and toughness. Finally, intraperitoneal treatment with hUC-MSCs showed similar results in terms of follicle populations, hormone regulation, and fertility restoration ^[15][190].

Despite these promising findings, administration routes for hUC-MSCs remain controversial. Three independent groups compared the efficacy of intravenous or local administration for POI models ^[16][17][18][31,36,191]. In these studies, both routes reduced cell apoptosis, augmented the proportion of growing follicles, and restored the ovarian function (as determined by regulated hormonal levels and recuperated estrous cyclicity). On the other hand, Song’s group ^[17][36] did not find any differences, Zhang et al. ^[18][191] reasoned there was a better restoration of the ovarian function after intravenous treatment, and Zhu et al. ^[16][31] found that local injection was more efficient. In terms of ovarian aging models, Zhang et al. ^[19][192] observed the positive effects of hUC-MSCs regardless of the injection route (intravenous or intraovarian), with increased follicle development, restored fertility, and reduced apoptosis of granulosa cells and reactive oxygen species (ROS) production. Finally, in humans, ovarian injection of hUC-MSCs rescued ovarian function, increased follicle development, and ultimately, led to live births ^[20][193].

Recently, Lu et al. ^[21][194] elegantly studied the impact of hUC-MSCs throughout the ovary and the endometrium. The SCs were introduced via tail injection, two weeks after inducing a POI condition. They reported improved ovarian morphology and folliculogenesis, as well as restored serum hormone levels. Regarding the endometrium, an increased number of glands and enhanced neoangiogenesis were observed, in addition to a noticeable overexpression of Homeobox A10 (HOXA10, an essential regulator of endometrial decidualization ^[22][195]), altogether revealing improved morphology and functionality. Furthermore, the type 1/2 T helper (Th1/Th2) cell ratio, and expression of NK cells, significantly decreased in the endometrium, suggesting treatment significantly regulated the ovarian function and endometrial receptivity. Likewise, Aygün et al. ^[23][196] loaded hUC-MSCs in hyaluronic acid scaffolds, and transplanted intraperitoneally in an abdominal adhesion rat model, and demonstrated the synergistic effect of the SCs on restoring hormone levels, improving follicle development and endometrial angiogenesis (after reducing the adhesions macroscopically).

While hyaluronic acid is the most popular scaffold used to develop alternative treatments in murine models of ovarian pathologies ^[24][25][197,198], other synthetic scaffolds have been mixed with biological products to enhance their pharmacodynamics ^[26][199]. Notably, only Sun’s group ^[27][200] has translated the use of collagen scaffolds from mice ^[28][201] to humans. Nevertheless, in mice, both hyaluronic acid and collagen scaffolds sustained the effects of the hUC-MSCs, improving ovarian morphology, cell proliferation, angiogenesis, and ultimately, restoring fertility ^[24][25][28][197,198,201]. Likewise, women treated with the hUC-MSC-collagen complex achieved a complete restoration of fertility, with primordial follicle activation (determined by the expression of phosphorylation of Forkhead box O3a (FOXO3a) and Forkhead box O1 (FOXO 1)), increased proportion of growing follicles, and consequent high serum estradiol concentration ^[27][200].

When hUC-MSCs are isolated and applied for regenerative treatments, the bioactive executors of hUC-MSCs effects (including EVs) remain present in the cell secretome ^[29][202]. Based on this premise, independent groups have employed the medium collected from hUC-MSC culture as a reparative treatment for POI models ^[24][30][82,197], and found it increased AMH levels and folliculogenesis.

Specifically, several studies have described that hUC-MSCs secrete EVs, such as exosomes and microvesicles, to deliver biomolecules (i.e., lipids, carbohydrates, nucleic acids, and proteins) that facilitate cellular and host cell reprogramming ^[31][32][62,203]. Consistent with the findings of two other independent groups ^[33][34][49,107], Liu et al. ^[35][204] reported that administering EVs, derived from the hUC, through the tail vein efficiently improved fertility of mice with POI, by improving ovarian and follicle morphology, balancing hormone levels, returning estrous cyclicity, and reducing apoptosis.

The intraperitoneal transplantation of exosomes also proved beneficial in mice, by returning folliculogenesis and hormones to nearly normal levels ^[36][205]. This improvement in reproductive outcomes was associated with the regulation of the Hippo pathway, which is critical for regulating follicle activation and survival, and thus, ovarian function ^[37][206]. Furthermore, Ding et al. ^[38][48] demonstrated, in a murine model, that exosomal miRNA-17-5p was strongly associated with improvements in ovarian function. Indeed, the inhibition of this miRNA via a knockdown approach in hUC-MSCs diminished the regenerative effects of the exosomes.

The hUC, and its cells, actively secrete GFs that can potentially be used for regenerative applications, since they stimulate cell proliferation and differentiation ^[39][207]. Specifically, the hUC-MSCs are reservoirs that, in response to specific regenerative signals in their microenvironment, release molecules to promote tissue repair. In fact, Wang et al. ^[40][208] reported that intraperitoneal treatment with GM-CSFs (which are present in the hUC-MSC secretome and immunomodulate hematopoietic cells ^[40][208]) promoted follicle development (as validated through the expression of folliculogenesis-related biomarkers) ^[41][158]. Similarly, other GFs, such as HGF (enriched in the hUC-MSC secretome) ^[25][198], or EGF (loaded into collagen-based scaffolds as Matrigel^®) ^[42][209] have also been associated with positive fertility outcomes.

In terms of ovarian regeneration, Buigues et al. ^[43][61] compared the effect of hUC plasma (rich in many GFs) to that of mobilizing G-CSF treatment and demonstrated that both therapies increased the populations of growing follicles, proliferation, and angiogenesis, in addition to restoring fertility of mice with gonadotoxic damage. Furthermore, Wang et al. ^[44][210] observed the ample benefits of hUC-PRP treatment in a murine model of POI, including preserved ovarian morphology, regulated hormonal levels and estrous cyclicity, increased angiogenesis, and reduced apoptosis, with respect to the untreated groups.