Perspective Chapter: Ovarian Reproductive Aging and Rejuvenation Strategies

Abstract

The ovarian milieu, which includes increased vasculature, different growth factors, necessary hormone synthesis, and appropriate granulosa cell function, is essential for oocyte maturation. Keeping the microenvironment in a state of equilibrium is crucial for healthy ovarian function. However, as people age, their tissues rebuild less effectively, leading to an imbalance in the microenvironment’s homeostasis and ovarian fibrosis, which finally causes ovarian function to deteriorate. As a result, full restoration of ovarian microenvironment health is required to enhance ovarian function. The precise identification of the molecular pathways involved in ovarian aging can help to devise therapy techniques that can decrease ovarian decay and boost the amount and quality of oocytes available for IVF. Antioxidants, melatonin, growth hormones, and mitochondrial and cell therapy are among the available treatments. All of these treatments must be considered in light of every couple’s history and current biological parameters, and a personalized (patient-tailored) therapy program must be developed. In this chapter, we aim to give an overview on the identified mechanism involved in female reproductive aging and potential therapeutic approaches to amend reproductive efficiency.

1. Introduction

Aging in humans is an incredibly complex process characterized by time-dependent functional decline, resulting in a decrease in the quality of life [1]. There is a relationship between the longevity of a species and its reproductive capacity. The parameters that determine reproductive efficiency are (i) gestation length, (ii) litter size, (iii) time to reach adulthood, (iv) litter intervals, and (v) duration of fertility for the duration of the overall lifespan. We as humans are the longest-lived land mammal, at the top of the long-living mammals and far from other primates. Although our reproductive efficiency is low, our social structure and creative thinking create a formidable life-history combination that most likely played a significant role in the successful colonization of hunter-gatherers around the world.

Social behavior is evolving, and reproductive efficiency in humans is decreasing, the average age of motherhood has been increasing since 1980, reducing the reproductive time, litter size, and intervals. The risk of childlessness increases with age, with reproductive aging being the most obvious factor.

Reproductive aging is a process involving a variety of intrinsic and extrinsic factors that affect the entire organism. Specifically, the reproductive organs and germ cell quality [2]. Reproductive aging causes infertility, increased miscarriages, and birth defects because gamete quality declines more rapidly in women than in men [3]. Menopause is the final menstrual cycle in women and other primates, which can be ovulatory or not. Fertility usually ends before menopause [4].

For decades, researchers have studied reproductive aging in humans and laboratory animals at the physiological, hormonal, cellular, and molecular levels.

2. Reproductive system aging

Aging is characterized by the progressive decline of multiple organ functions and the onset of degenerative diseases [5]. One aspect of aging is the decline in reproductive function. The reproductive system is a network of organs that generate gametes together with sex hormones. It fosters the birth of healthy offspring and coordinates physiological functions by sustaining endocrine homeostasis. When a female partner is over the age of 35, the risk of infertility tends to increase [6]. Furthermore, aging-related menopause in women is accompanied by an endocrine disorder as well as an increased risk of several major health complications, which include osteoporosis, cardiovascular disease, recurrent depression, and others [7]. Male fertility declines with age as well, but it happens more gradually and is associated with endocrine equilibrium disorders like late-onset hypogonadism (LOH). LOH symptoms include libido loss, sexual dysfunction, and declines in bone and muscle mass density. Moreover, benign prostatic hypertrophy (BPH) as well as prostate cancer (PCa), are common age-related diseases that impact the male reproductive system and impair the elderly’s quality of life [8].

Reproductive aging leads to a variety of age-related disorders in both men and women since reproductive health is so tightly connected to overall health.

3. Female reproductive aging

Age-related infertility is a multifactorial process and understanding factors affecting follicle/oocyte aging requires the comparison of the theories on aging mechanisms based on studies on tissues and organs other than the ovary [9].

Female fecundity capacity peaks in their 20s, declines in their late 30s in defiance of regular menstrual cycles, and ends in menopause at the average age of 50–51 years [10]. Data from in vitro fertilization (IVF) shows that the mother’s age is an important factor that leads to impressive differences in clinical results [11]. While the fraction of first births in women over the age of 30 increased sixfold between 1970 and 2002. According to an American study of over 120.000 assisted reproduction technologies (ART), the successful delivery rate per embryo transfer reduced from 43.2% in women 35 years old to 15.1% in women 41–42 years old and 5.9% in women over 42 years old [12].

Changes in the ovarian follicle pool and a decrease in the ovarian reserve are the primary causes of the female reproductive ability decline in humans [13]. Despite uterine and neuroendocrine factors, it is well characterized that ovaries and ovarian follicles are outstanding regulators of reproductive aging. This statement is substantiated by oocyte donation from younger women in the treatment of age-related infertility [10, 14].

It is widely accepted that female mammals are born with a finite supply of primordial follicles (PMFs), which are gradually depleted over the course of their lives [15]. A follicle reserve is formed in the fetal or early postnatal ovaries. Composed of pregranulosa cells and primary oocytes arrested in the diplotene stage of meiotic prophase I. These latter are formed from primordial germ cells (PGCs), which originate outside the gonadal anlages and migrate into the forming ovaries [16]. After establishment, each PMF has three developmental options: I remain quiescent, (ii) die directly from the quiescent state, or (iii) be recruited into a growing follicle pool via a process known as follicle activation, which contributes to cyclic endocrine secretion.

The size of the ovarian PMF pool is estimated to be 7 million oogonia by the fifth month of prenatal development (Sadler, 2011). Except for a small number near the ovarian surface, many oogonia and primary oocytes become atretic at this time. During the female fertile age, a cohort of primordial follicles is recruited month by month in order to develop a single dominant follicle and ovulate its fertile oocyte. The dormant oocyte is arrested in prophase I in primordial follicles and is surrounded by a single layer of flattened granulose cells (GCs) [17]. Primordial follicle recruitment causes the oocyte to activate and mature, as well as the proliferation and differentiation of surrounding GCs, resulting in the formation of primary follicles, which are distinguished by a single layer of cuboidal GCs surrounding the oocyte. In secondary follicles, GC layers continue to proliferate. They differentiate into cumulus cells (CCs), which are cells contiguous with the maturing oocyte [18, 19]. CCs are also important in the recruitment of androgen-producing theca cells (later segregated into theca interna and externa layers) from the ovarian mesenchyme and mesonephros progenitor pools [20]. Folliculogenesis is a paracrine signaling gonadotropin-independent unit comprised of multiple locally active growth factors and small molecules secreted by early follicle GCs until the formation of the preantral follicle [21].

Maturing follicles eventually express gonadotropin receptors and become responsive to gonadotropins, first to follicle-stimulating hormone (FSH) and then to luteinizing hormone (LH).

Follicles become hormone-secreting units during this gonadotropin-dependent phase of folliculogenesis.

FSH promotes GC growth, estradiol production, and dormant follicle selection [22], whereas LH is in charge of androgen secretion from cholesterol, meiosis I completion in the oocyte, germinal vesicle breakdown [23, 24], and subsequent progression to metaphase II [25]. With the production of a mature oocyte capable of fertilization, ovulation marks the end of folliculogenesis [26].

Multiple follicles are arrested at different stages of the highly regulated and well-orchestrated folliculogenesis process and undergo irreversible atretic degeneration with increasing speed after the mid-30s [27, 28].

The development of an antral follicle from a dormant primordial follicle into a mature, healthy oocyte that is prepared for fertilization is a carefully orchestrated, complicated process that requires the precise synchronized timing of intraovarian molecules and cells. Aging causes progressive deterioration in follicle function and capacity to form an oocyte capable of fertilization. Age-related disruption in folliculogenesis is attributed to several changes that occur concurrently. Bioenergetics dysfunction, shortened telomere length, decreased DNA repair capacity with loss of chromosome cohesion and spindle aberration increase the risk of mutations and meiotic errors have been identified in aged oocytes [29, 30]. Additionally, the number of GCs surrounding the oocyte declines with aging as a result of decreased proliferation [31] and elevated GC apoptosis [32], which is accompanied by altered production of locally active growth factors [33], and disrupted steroidogenesis [34].

The decline in steroid hormone biosynthesis with age is primarily due to a decrease in the number of ovarian follicles, and thus a decrease in functional steroid-producing cells. Markers associated with reproductive aging and senescence in women are based on a decline in granulosa cell activity, such as a decrease in circulating levels of the anti-Müllerian hormone (AMH; produced by immature granulosa cells), decreased levels of estrogen (produced by mature granulosa cells), and elevated levels of FSH (due to a lack of inhibin production by granulosa cells).

AMH, also known as Mullerian Inhibitory Substance, is a peptide belonging to the TGF superfamily that functions by binding to the AMHR2 receptor. Beginning at roughly 36 weeks of gestation in female fetuses, AMH expression in the ovary reaches a peak in the middle of the 20s and then gradually declines until menopause [35, 36, 37]. The majority of AMH produced by antral follicles is released by CCs [38]. AMH and its receptor AMHR2 are largely expressed in the GCs of mature follicles in the ovary, with the levels of expression varying depending on the stage [38, 39]. Beginning in primary follicle GCs, AMH expression rises steadily until it reaches its peak in small antral follicles less than 6 mm in size [35].

Regarding AMH’s function in preventing the formation of primordial follicles, human studies on primordial reserve using ovarian cortex biopsies are still controversial [38]. AMH expression during fetal gonadal development has a detrimental effect on healthy ovarian and follicular pool development, AMH inhibits the developing follicle during the early gonadotropin-dependent and gonadotropin-independent phases of follicular development [36]. AMH is essential for choosing cyclic antral follicles as well [40]. AMH knockout mouse models recruit noticeably more developing follicles with ovarian stimulation than the control group [36]. AMH regulation and the production of its receptors are intricate processes that still need more research.

4. Vascular aging

In humans, vascular endothelial dysfunction appears to occur with aging even in the absence of clinical cardiovascular disease (CVD) and significant CVD risk factors, according to a number of lines of research. Older humans, rodents, and non-human primates have all shown signs of impaired endothelium-dependent dilation, reduced fibrinolytic activity, increased leukocyte adhesion, altered permeability, and/or other markers of endothelial dysfunction [41]. The function of the macro- and microvascular endothelium declines gradually with age [42] rarefaction, which affects the systemic microvasculature in all organs, being a crucial indicator of aging [43, 44, 45, 46, 47]. It is believed that decreased endothelium turnover and enhanced apoptosis cell death are two factors in age-related microvascular rarefaction. Age-related microvascular rarefaction causes blood flow to decrease, which worsens ischemia injury, especially in tissues with high metabolic activity like the brain and heart. This loss in blood flow also diminishes metabolic support and decreases microvascular flexibility and the circulatory system’s capacity to adapt to variations in metabolic demand [48].

Although more data are needed to link ovarian aging and vascular rarefaction, indirect evidence suggests a relationship between oocyte maturation, aging, and vasculature. As previously stated, follicular vasculature begins to develop inside the theca cell layer at the secondary follicle stage, with the GC layer remaining avascular and separated by the basement membrane.

The normal function of the ovaries and ovarian follicles is maintained by continuous angiogenesis, which is the development of new blood vessels from existing ones. Endothelial and mural cells become destabilized in response to an angiogenic stimulation such as hypoxia or injury. Following that, they migrate toward angiogenic stimuli and grow, resulting in the formation of a new vessel [49]. Follicular vasculature seems to be crucial in achieving dominance by the follicle. It is well known that dominant follicles ingest more serum gonadotrophins than other follicles, in addition to having a more vascular theca [50].

Microvasculature is critical for the follicle nutrients and oxygen, as well as ensuring an adequate supply of gonadotropins, steroid precursors, and other regulators. VEGF is thought to be crucial for follicular growth and for the formation of the antrum thecal angiogenesis and guarantee vascular permeability.

Microvasculature determines follicular dominance because the dominant follicle has more plentiful vasculature in its theca layer than other follicles in the same cohort. Oocytes derived from follicles with adequate vascularization and oxygen content (3%) had increased fertilization and developmental potential in prospective research based on pulsed Doppler ultrasonographic examination [51]. Studies of perifollicular vascularity also revealed a positive correlation between high-grade vascularity and better results during IVF cycles [52].

A diminished oxygen supply to the leading follicle, which is a state dependent on a defective perifollicular vascularization, was proposed as a possible representation of a key environmental component responsible for oocyte senescence [53, 54]. In fact, it has been noted that alterations in older MII oocytes, such as aberrant chromosome and spindle structure, are similar to those in young oocytes derived from Graafian follicles with lower perifollicular vascularization and oxygen concentration [54, 55].

Growing follicles definitely require a sufficient ingrowth of capillaries into the theca, as opposed to primordial and preantral follicles, which obtain their blood supply from the stromal arteries.

There is no question that VEGF and its receptors, VEGFR1 and VEGFR2 are regulatory factors in mammalian ovarian folliculogenesis controlling both follicular and luteal angiogenesis as well as new capillary creation within the ovulatory follicle. Its blockage causes a significant reduction in endothelial and granulosa cell proliferation in growing antral follicles, as well as inhibition of follicular growth and ovulation [56].

Small preantral follicles in the ovarian cortex lack their own blood supply and must rely on passive diffusion from stromal tissue for nourishment and oxygenation. Beginning with secondary follicles, outer stromal cells surrounding the oocyte develop into theca cells during follicular maturation [57]. For the follicle to expand, the inner theca layer, which is separated from the granulosa cell layer by a basement membrane, creates a capillary network [58, 59]. Direct injection of VEGF into the ovarian blood supply can improve angiogenesis, increase the number of primary and secondary follicles, and decrease follicular atresia [60, 61].

VEGF levels in follicular fluid rise with age in both natural and in vitro fertilization (IVF) cycles [33, 62, 63, 64, 65]. This aging-related rise in VEGF levels could be a result of selective gonadotropin elevation in older reproductive-age women, a compensatory response to follicular hypoxia, and a decrease in energy synthesis in the presence of impaired mitochondrial function [21].

Nitric oxide is an important angiogenesis mediator. NO is pro-angiogenic because it increases endothelial cell survival, proliferation, and migration. VEGF, FGF, and other growth factors increase endothelial NO synthesis, which is a significant mediator of their actions. Angiogenesis is hampered by NOS pathway abnormalities caused by pharmacological, metabolic, or genetic factors. Similarly, ADMA, an endogenous NOS inhibitor, functions as a natural anti-angiogenic agent [66].

The NOS pathway’s activity is thus critical in the response to endogenous or therapeutic angiogenic drugs. Manipulation of the NOS pathway could provide another strategy for therapeutically modify angiogenesis in folliculogenesis. Multiple isoenzymes of NO synthase (NOS) catalyze the oxidation of L-arginine to L-citrulline in a nicotinamide adenine dinucleotide phosphate reduced form (NADPH) and oxygen-dependent reaction in mammals. NOS1, NOS2, and NOS3 are the three NOS isoforms. The product of the NOS1 gene is known as neuronal NOS (nNOS), whereas the product of the NOS3 gene is known as endothelial NOS (eNOS). These isoforms are calcium-dependent and constitutive. The third isoform produced by the NOS2 gene is an inducible NOS (iNOS) that is calcium-independent. Only cytokines such as lipopolysaccharide, interleukin 1, and tumor necrosis factor-alpha (TNF) activate iNOS [67].

NO exerts remarkable functions within the ovary, including the control of steroidogenesis, folliculogenesis, and oocyte competence. NO can be produced in the ovary not only by ovarian cells but also by the ovarian vasculature and resident or invading macrophages [68].

In rats, eNOS is found in mural granulosa cells, theca layer, ovarian stroma, and ovarian blood vessels [69], but iNOS is found only in somatic cells of primary, secondary, and small antral follicles, as well as luteal cells.

In contrast, both eNOS and iNOS are expressed in theca and granulosa cells of the mouse ovary [69]. eNOS is expressed in theca and granulosa cells, as well as the surface epithelium and luteal cells, in the bovine ovary [70]. Additionally, eNOS is discovered more frequently than iNOS in granulosa cells in the porcine ovary [71]. Human granulosa and luteal cells have been shown to contain inducible NOS and eNOS [72].

NO represents a key regulator in ovarian steroidogenesis, NO exerts its inhibitory effect on aromatase activity, a key enzyme in the steroidogenic pathway. The direct inhibitory effect on the enzyme is mediated by the formation of a nitrosothiol group in the cysteine residue of the aromatase enzyme [73].

Age-related endothelium dysfunction is primarily caused by at least three NO-related events, including changed NOS enzyme expression and activity, decreased vascular antioxidant capacity, and NO consumption by excessive O₂.

NO has been linked favorably to delaying oocyte aging. As previously presented NO is a common molecule that plays a crucial role in the microenvironment of the oocyte from folliculogenesis to early embryo development.

When fresh oocytes are exposed to superoxide, the zona pellucida dissolution time of these oocytes increases significantly. Further, superoxide exposure of fresh oocytes exhibited increased ooplasm microtubule dynamics (OMD) and major CG loss. Both old and fresh oocytes exposed to NO have a considerable decrease in OMD and the zona pellucida dissolution time, as well as a reduction in spontaneous CG loss. Additionally, NO exposure lowers the frequency of aberrant spindles. Because of its capacity to neutralize peroxyl lipid radicals and cytotoxic ROS, NO may act as an unusual antioxidant. Through the activation of guanylate cyclase, which increases the generation of cyclic guanosine monophosphate and might greatly reduce zona pellucida dissolution time and OMD, NO may also contribute to the delay of oocyte aging. Overall, NO slows down oocyte aging and strengthens the microtubular spindle apparatus in older oocytes [74].

5. Mitochondria

A defining contributing factor in aging has been for a long time mitochondrial dysfunction [75, 76]. Deterioration of pleiotropic activities is the result of mitochondrial malfunction, which is linked to a number of characteristics of aging including dysregulation of cell signaling and inefficient energy production [77, 78]. The accumulation of somatic mtDNA mutations, decreased OXPHOS activity, increased oxidative damage, altered mitochondrial quality control, ineffective mitochondrial biogenesis or clearance, and dysregulation of mitochondrial dynamics are all aspects of mitochondrial dysfunction that have been linked to aging [79, 80, 81].

ROS formation occurs naturally as a byproduct of energy production in the mitochondria [40, 82]. The majority of endogenous ROS are produced by mitochondrial OXPHOS, which serves as the final step in the metabolism of substrates and the creation of ATP [83, 84].

The hypothesis for why mtDNA is more susceptible to oxidative damage than nuclear DNA is that it is close to the respiratory chain, lacks histones, and has ineffective repair mechanisms [47]. It was shown that the production of ROS, oxidative damage, and chronological age are all strongly correlated [48]. mtDNA mutations are expected to build up over time, causing cells to have less oxidative energy and, eventually, an aging phenotype. In reproductive cells, it has been hypothesized that age-related ROS buildup and oxidative damage in mtDNA cause a reduction in the rate of oocyte fertilization and developmental potential [49].

This hypothesis can not be confirmed because several studies have obtained contradictory results or even no differences in the frequency of mtDNA changes in the oocytes of older women [85, 86, 87, 88, 89, 90]. Furthermore, no increase in mtDNA mutations was observed in embryo samples from women above the age of 40 [91].

Because of inconsistencies in human data, accepting the free radical hypothesis of aging as a final mechanical explanation for ovarian aging is difficult (reviewed in [92]).

Mitochondrial dynamics are defined by fission and fusion. Fission produces smaller mitochondria, which could be better at driving cell proliferation and causing ROS, whereas fusion improves communication with the endoplasmic reticulum and dilutes accumulated mtDNA mutations and oxidized proteins [93]. Fission and fusion abnormalities have serious implications for ovarian aging. In C57BL/6 mice, oocyte-specific deletion of the mitochondrial fusion protein Mitofusin (Mfn1) results in rapid depletion of the ovarian follicular reserve. Based on immunofluorescence, it was shown that ceramide, a membrane sphingolipid involved in apoptosis and cell cycle arrest, was elevated in Mfn1−/− mice oocytes, suggesting that it is probably a factor in the mechanism of decreased ovarian reserve in this mouse model. Myriocin, a ceramide synthesis inhibitor, was administered to mice every day for 21 days in a row, which increased follicular growth and allowed the production of antral follicles, partially reversing the reproductive phenotype [94].

A vital and intricate homeostatic coordination mechanism of the nuclear and mitochondrial genomes is necessarily necessary to drive correct mitochondrial biogenesis.

It is reliant on nuclear genes encoding nuclear respiratory factor-1 and -2, estrogen-related receptor (ERR), peroxisome proliferator-activated receptor (PPAR) coactivator 1 (PGC-1), and PGC-1 (NRF-1, 2). Additionally, the mitochondrial-localized sirtuin (SIRT) family genes SIRT3, SIRT4, and SIRT5 are involved. The most thoroughly studied sirtuin, SIRT3, has been discovered to interact with PGC-1, a crucial regulator of mitochondrial biogenesis, suppress intracellular ROS, and perhaps even control longevity and aging phenotypes [95]. The primary regulator of mitochondrial homeostasis and a promoter of mitochondrial biogenesis is AMP-activated protein kinase (AMPK). In particular, AMPK interacts with PGC-1 in a variety of ways [96]. Mitophagy, the selective autophagic eradication of defective mitochondria, controls mitochondrial biogenesis. PTEN-induced kinase 1 (PINK1)-Parkin pathway as well as AMPK both have a role in controling mitophagy [97, 98].

Critical to ovarian function is mitochondrial biogenesis. From the immature germinal vesicle stage to the mature oocyte, there is a dramatic increase in mitochondrial biogenesis. A single egg contains hundreds of thousands of mitochondria at the time of fertilization, providing enough ATP to enable fertilization and support development until implantation when mitochondrial replication picks back up in the blastocyst.

During ovarian aging, decreasing mtDNA content, which indicates decreased mitochondrial biogenesis, is frequently seen. Premature ovarian aging patients had the lowest amounts of mtDNA, followed by IVF recipients who respond normally and recipients who do not respond well to IVF [99].

In humans undergoing in vitro fertilization (IVF), sirtuin 3 (SIRT3) active protein co-localized to mitochondria in follicular granulosa and cumulus cells. SIRT3 mRNA levels were also lower in advanced maternal-age women compared to control women [100]. Advanced maternal-age women showed lower PGC-1 expression in cumulus cells along with lower mtDNA concentration in cumulus and oocyte cells when compared to women with normal ovarian reserve women [101]. These correlations provide evidence in favor of the hypothesis that poor oocyte competence in IVF may be caused by insufficient mitochondrial biogenesis during oocyte maturation [102].

mtDNA copy number has lately been the subject of several research because it is one of the mitochondrial factors that may represent the reproductive capability of gametes and embryos.

Mammalian models have shown that mtDNA levels dramatically rise during oogenesis [103] remain constant during fertilization, and then resume replication at the blastocyst stage [104, 105].

As a result, each time a cell divides in the early preimplantation embryo, mtDNA is distributed among the different blastomeres [106] and the total amount of mtDNA in cleavage-stage embryos corresponds to the mtDNA content of the oocyte [107, 108].

In an effort to identify a reliable biomarker for the implantation potential of euploid embryos, several researchers looked at the mtDNA content of biopsy samples from the cleavage and blastocyst stages of euploid embryos. In the initial study, Fragouli et al. used a combination of array-comparative genomic hybridization, real-time quantitative polymerase chain reaction, and next-generation sequencing to examine day-3 and day-5 embryos. They found that older women’s embryos contained much more copies of mtDNA. Higher amounts of mtDNA, which were age-independent, were also seen in aneuploid embryos [91]. Importantly, they identified a threshold for mtDNA beyond which euploid embryos did not implant. Diez Juan et al. discovered poor implantation potential for day-3 and day-5 euploid embryos with higher quantities of mtDNA in a later investigation that also examined day-3 blastomere and day-5 trophectoderm biopsies. However, unlike Fragouli et al., Diez Juan et al. did not discover a rise in mtDNA copy number in embryos from older vs. younger reproductive-age women [109]. These results supported the quiet embryo hypothesis, which holds that under ideal circumstances, embryos would have little metabolic activity whereas under stress, embryos would boost mitochondrial replication as a coping mechanism [110] but no clear differences in embryos from women with advanced reproductive age.

6. Methods to prolong reproductive life and slow down ovarian aging

There are currently few therapy options available to help women with their ovarian reserve and oocyte quality. Infertility patients can get Coenzyme Q10 (CoQ10), Dehydroepiandrosterone (DHEA), vitamins including vitamins C and D, or dietary or supplement isoflavones as therapies for diminished ovarian reserve.

No study has categorically validated the routine administration of these bioactive substances, despite the possibility that they have some therapeutic effects on DOR. Therefore, more study is required to develop novel therapeutic approaches that will improve patients’ reproductive results.

6.1 Mitochondrial support therapy

It has been demonstrated that the mitochondrial electron transport chain component CoQ10 reduces mitochondrial dysfunction in infertile mice [111]. A 2014 human study evaluated the post-meiotic aneuploidy rates in embryos of IVF patients treated with 600 mg per day of CoQ10 versus placebo and found that the two groups had respective aneuploidy rates of 46.5% and 62.8%, respectively. CoQ10 is a soluble lipid transporter that is essential for complex III stability. Furthermore, it is a strong cellular antioxidant. Normal tissues produce their own supply, however, it has been found that tissue concentration decreases with aging [112]. In mice, research on the potential effects of CoQ10 supplementation on fertility found that it may boost mitochondrial activity, reduce ROS levels, and postpone the loss of ovarian reserve while also restoring the expression of the mitochondrial gene in oocytes [111, 113, 114]. Importantly, early results of a stopped (and solely existing) human research due to worries about the effects of polar body biopsy on embryos. The trial was halted, and statistical significance was not reached although suggested that CoQ10 supplementation might reduce aneuploidy rates [115, 116].

Several members of the sirtuin deacetylase family function as anti-aging agents in mammalian cells [117]. Sirtuin 3 (SIRT3) activation enhances mitochondrial activity [100]. Similar to this, ovarian reserve is improved both quantitatively and qualitatively when SIRT1 expression is activated by resveratrol [118, 119]. In oocytes, mtDNA concentrations, membrane potential, and ATP production were discovered to be elevated by the anti-aging chemical resveratrol [120, 121]. Additionally, resveratrol was discovered to increase the quantity and quality of oocytes in mice, protecting against the decline in fertility brought on by aging of the reproductive system [122]. Melatonin is thought to activate sirtuin [123, 124]. Melatonin and SIRT3 posttranslationally collaborate for the regulation of free radical equilibrium in mitochondria, increasing the size of the primordial follicle pool and delaying ovarian aging [125, 126]. Melatonin is a chemical that functions as both a direct antioxidant and a modulator of the mechanisms defending cells against oxidative stress. It was initially utilized in reproductive medicine to treat endometriosis and adenomyosis-related infertility [33, 34, 127, 128]. Since then, fresh research has shown how melatonin may delay the aging of the ovary [36]. Melatonin administration is clearly advised for women with age-related ovarian decay or POI even though the mechanism underlying the hormone’s anti-aging effect in the human ovary still needs to be fully understood [36]. It has no serious side effects and offers additional benefits to patients who receive it.

Multiple observations have revealed that aging-related pathologies are also significantly influenced by the mTOR signaling system [129]. Rapamycin, an inhibitor of mTOR, controls the sirtuin and mTOR pathways and prevents the first activation of follicles [130]. Rapamycin is being researched as a potential preventative agent against early ovarian failure and reproductive aging after it was discovered to partially reverse the infertile phenotype in Clpp knockout mice [92].

A potent antioxidant and mitochondrial metabolic facilitator, alpha-lipoic acid [131], has been shown to enhance in vitro follicular growth and oocyte maturation while reducing follicular ROS generation by improving mitochondrial metabolism [132, 133].

Overall, significant progress has been made in understanding the nutrients that could enhance mitochondrial activity, slow the aging of the ovaries, and benefit women with DOR or early ovarian insufficiency. A protective effect has not yet been demonstrated, though.

6.2 HGH administration

The first treatment proven to be effective in older women was GH injection during ovarian stimulation.

In a randomized controlled trial, 100 women over the age of 40 who were receiving treatment for assisted reproduction were randomly assigned to receive growth hormone treatment or a placebo, and the results showed that the GH arm had significantly higher delivery and live birth rates than the placebo arm [134]. These results were supported by subsequent research, which also expanded the use of GH therapy to include younger women with POI [135, 136].

The cell signaling pathways involved in cellular defense against oxidative stress are affected by GH [137], and adult GH deficiency results in insufficient cellular response to radical generation. This explains why ovarian degradation can be influenced by age-related or early GH insufficiency, even when it is largely brought on by other factors. GH might help a subset of patients who do not respond well to treatment—women with poor oocyte and/or embryonic development. Therefore, in women who have both age-related ovarian decline and POI, GH may be used as an adjuvant therapy during ovarian stimulation [136].

6.3 Dehydroepiandrosterone supplementation

The zona reticularis layer of the adrenal cortex and the theca cells of the ovary create DHEA, an important prohormone, when they synthesize testosterone and estradiol from cholesterol [138]. Its levels are seen to be high, particularly in the early stages of reproduction, and to decrease with age [116, 139]. Several phases of folliculogenesis have seen the identification of androgen receptors (AR) [140]. By stimulating primordial follicles in monkeys [141, 142] and mouse models, androgens were found to increase the number of primary follicles [143]. Additionally, they influence the development of preovulatory follicles, follicle maturation, and FSH-R mRNA synthesis in primate and mouse models [144, 145] as well as GC proliferation in vitro [146].

DHEA was shown to be crucial for folliculogenesis, just as androgens, and its administration may enhance the success of IVF, particularly in populations with DOR or weak ovarian response. One of the first to discuss the advantages of DHEA with DOR was Casson et al. They observed an increase in peak estradiol levels [147, 148] as well as an increase in ovarian responsiveness to gonadotropin stimulation. Following Casson’s original study, numerous studies in mice and humans with poor responders were carried out using different doses (10–80 mg per day) of DHEA administration for various lengths of time (pre-IVF treatment or concurrently with ovarian stimulation), and it was found that improved ovarian function was associated with an increase in ovarian response and a decrease in the number of atretic follicles [123, 139]. When DHEA was pretreated for at least 8 weeks prior to IVF treatment, Li et al. found that the CCs of women older than 38 years produced more energy and had higher-quality oocytes [124]. A premature ovarian insufficiency rat model with subfertility, decreased follicular number, and increased atresia was used in a different investigation by Sozen et al. Primal follicular recruitment and follicular development were both stimulated by DHEA [149].

One of the mechanisms by which DHEA can have positive effects, has been identified to be secondary to its involvement in raising IGF1, which may then improve responsiveness to gonadotropins and may have favorable effects on oocyte quality, particularly in those who respond poorly to these hormones [150]. Additionally, Zhang et al. [151] confirmed that 2-month DHEA supplementation enhanced BMP15 levels in cases of inadequate ovarian response. DHEA is also known to control AR expression and boost follicular development and recruitment [152]. Another mechanism for DHEA’s effects on aged follicles is that it enhances mitochondrial hemostasis, transports oxidative phosphorylation, increases cumulus cells’ mitochondrial oxygen consumption, and switches energy generation from anaerobic metabolism to aerobic metabolism [124] preventing mitochondrial malfunction with the alternating expression of mitochondrial dynamics genes. With DHEA administration in human CCs, it was observed that the expression of MFN1, a mitochondrial fusion gene, was elevated while PINK1 and PRKN, important proteins for mitophagy, were downregulated [153]. DHEA can also reduce the rate of CC apoptosis in aged follicles [154]. It is believed that DHEA will improve the ovarian microenvironment and reduce age-related embryonic aneuploidy [139].

Testosterone is another androgen that can be used as a potential modifier of folliculogenesis. Lower serum testosterone levels have been linked to reproductive aging [155]. The effects of testosterone supplementation in ovarian aging mammalian models or among older women undergoing IVF, however, are not currently known. As an aromatase inhibitor, letrozole prevents the conversion of testosterone to estrogen, which raises the levels of testosterone. It has been shown to increase the number of retrieved oocytes and the rate of implantation in poor responders [156] and lower IVF expenditures by reducing gonadotropin dosage [157, 158]. Data on its impact on clinical pregnancy rates or live birth rates, however, are limited [159].

6.4 Vitamin D

Vitamin D (VD) is a fat-soluble secosteroid. VD regulates the transcription of genes involved in a variety of cellular processes, including pro-differentiation, anti-proliferation, pro-apoptosis, immunosuppressive, and anti-inflammation activities, in target cells via binding to specific VD receptors (VDR). There is a growing understanding that VD is crucial for optimal folliculogenesis and maximizing women’s reproductive potential, in addition to its critical function in bone physiology and health [160, 161].

Ovarian follicles display VD production and signaling mechanisms [161]. VD administration enhanced follicle survival, size, and function as well as oocyte maturation and AMH production, according to in vitro experiments carried out on rhesus monkeys [162]. They also verified that VD production and signaling control follicular growth, raising the possibility that VD has endocrine, paracrine, and autocrine effects in the ovary. Reduced ovarian aromatase activity in VDR null mutant mice leads to compromised folliculogenesis [163]. Less oocytes were recovered from oviducts after gonadotropin stimulation in VD-deficient diets, which also delayed follicular development and lengthened estrous cycles [164]. Serum AMH levels have demonstrated a relationship between VD levels and ovarian reserve. The connection between circulating VD and AMH in premenopausal women with regular menstrual cycles suggests that VD deficit may be linked to a reduced ovarian reserve in late-reproductive-aged women [165]. Patients with uterine fibroids also showed an inverse correlation between blood VD levels and ovarian reserve [166]. Additionally, a VDR polymorphism was linked to lower antral follicle numbers in women receiving ovarian stimulation [167].

The capacity for and results of reproduction seem to be influenced by VD levels.

Higher FF levels of 25-hydroxyvitamin D were shown to be substantially related to greater clinical pregnancy and implantation rates in a study examining infertile women undergoing IVF [168]. The number of mature oocytes retrieved and the success rates of oocyte fertilization in patients undergoing IVF were favorably connected with blood VD levels in various prospective studies [65]. The same study’s multivariable logistic regression analysis found FF levels of VD as an independent predictor of an IVF cycle’s success after correcting for age, BMI, ethnicity, and the number of embryos transferred (Esencan et al., 2022).

The adjusted odds ratio for clinical pregnancy in women with vitamin D levels of 20 ng/mL was considerably higher as compared to women with serum levels of 20 ng/mL in another study [169] examining a cohort of women undergoing IVF. According to a subgroup study, women with the highest serum levels (> 30 ng/mL) had the best probability of getting pregnant [163]. However, there has been a lack of consistency in research evaluating the predictive usefulness of FF VD on IVF results. Despite the fact that a prospective cohort study by Kinuta et al., found that women with higher serum and FF vitamin D levels were significantly more likely to experience clinical pregnancy after IVF-embryo transfer, a different study found lower-quality embryos and significantly lower clinical pregnancy rates with higher levels of follicular VD [164].

7. Cell therapy

Stem cells’ therapeutic effects are carried out by differentiation, homing, and paracrine activation. The injured ovary attracts stem cells on their own, where they adhere and grow under a variety of conditions. Recent studies suggest that paracrine mechanisms may be in charge of the therapeutic effect of stem cell transplantation. Surrounding cells release a variety of physiologically active substances, such as cytokines, growth factors, regulatory factors, and signal peptides, in order to influence nearby cells. Injured ovaries’ health is improved by this technique through immunological modulation, angiogenesis, antiapoptosis, antifibrosis, and anti-inflammation, as presented in Figure 1.

Figure 1.

Mechanisms involved in stem cell-based therapies in POF. Adapted from [170].

Studies have thus far focused on providing women with weakened ovarian reserves with a suitable environment in an effort to restore the damaged ovarian niche. Autologous stem cells produced from various organs have drawn the attention of many researchers. As a result of the paracrine secretion of soluble factors [171] playing a role in the activation of primordial follicles in impaired ovaries, other researchers have concentrated their attention on various approaches, such as platelet-rich plasma (PRP) [172].

8. Platelet-rich plasma (PRP)

Platelet-rich plasma (PRP) is produced by centrifugation and separation of its various components from whole blood, which contains plasma (55%), red blood cells (41%), platelets, and white blood cells (4%). Red blood cells are removed during the centrifugation and separation process, and plasma is created with a 5–10 times higher concentration of growth factors [173]. Alpha granules, which are found in platelets in PRP, produce a variety of substances that promote angiogenesis, cell proliferation, and growth when triggered [174]. It has been demonstrated that the growth factors in PRP are crucial for promoting collagen synthesis, bone cell proliferation, fibroblast chemotaxis, macrophage activation, angiogenesis, immune cell chemotaxis, endothelial cell migration and mitosis, epithelial cell differentiation, and cytokine secretion by mesenchymal and epithelial cells. Table 1 summarizes the composition of PRP [175].

Function	Factors
Adhesive proteins	Von Willebrand factor, fibrinogen, trombospondin-1, trombospondin-2, laminin-8
Angiogenic factors	Vascular endothelium growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)
Chemokines	CCL5 (RANTES), CCL-3 (MIP-1a), CCL-2 (MCP-1), CCL-7 (MCP-3), CXCL8 (IL-8), CXCL2 (MIP-2), CXCL6 (LIX), CXCL-1 (GRO-a), CXCL5 (ENA-78), CXCL-12 (SDF-1a), CXCL4 (PF4)
Clotting factors and their inhibitors	Factor V, factor IX, antithrombin, factor S, protease nexin-1, protease nexin-2, tissue factor pathway inhibitor,
Growth factors	Epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), transforming growth factor ß (TGF-ß)
Immune mediators	Complement C3 precursor, complement C4 precursor, factor D, factor H, C1 inhibitor, IgG
Integral membrane proteins	Integrin aIIb3, GPIba-IX-V, GPVI, TLT-1, p-selectin

Table 1.

Platelet granule content [175].

After a venous blood sample, autologous PRP therapy administers injections of the patient’s own concentrated platelets and plasma. The natural healing process is the body’s initial response to tissue injury by sending activated platelets and releasing growth factors, according to the theory underlying the use of this method for treatment. Over the past ten years, the clinical application of PRP has grown significantly, and it is currently used to treat conditions like alopecia, musculoskeletal injuries, arthritis, periorbital rejuvenation, regenerative dentistry, and wound healing [176]. For women who have a poor ovarian reserve (POR), premature ovarian insufficiency (POI), or even menopause, PRP treatment has recently been employed as an adjuvant in assisted reproductive technology, in particular, as an intraovarian injection in conjunction with in vitro fertilization (IVF). Reviewed in [172].

The impact of PRP on the development and viability of isolated early human follicles was examined in one experimental research [177]. After brain death was determined in three postmortem women under the age of 35, ovarian tissue samples were taken. After removing the ovarian cortex tissue, half of the sample was vitrified for future research while the other half was used right away. Following that, ovarian follicle isolation was carried out under a stereomicroscope. Fresh samples’ follicles were grown in fetal bovine serum (FBS), platelet-rich plasma (PRP), or PRP + FBS. Additionally, FBS, PRP, PRP + FBS, or human serum albumin were cultured with the follicles from vitrified-thawed samples. A 5 ml of blood was taken into tubes containing 0.5 ml of acid citrate solution to prepare PRP (anticoagulant). The top and middle layers of the blood were transferred to fresh tubes and centrifuged at 3000 g for 15 minutes after the blood had been centrifuged at 200 g for 20 min at 20°C. The remaining 0.5 mL of plasma with precipitated platelets was mixed uniformly and utilized for PRP after the supernatant plasma was discarded. To release the growth factor from the platelets, 20 IU/mL thrombin was added to the PRP, causing it to clot. The platelet fragments were finally removed from the clot by centrifuging it at 4000 g for 5 minutes. The samples were compared after 10 days of cultivation. Sixty primordial follicles in all were extracted and cultivated from the fresh samples. The PRP alone group demonstrated the greatest changes in size (p < 0.001) and viability (p < 0.05) after 10 days, with 91.4% 1.86 vs. 61% 0.89 in the fresh group with FBS and 82% 1.70 vs. 59% 1.00 in the vitrified group with FBS. The follicles’ sizes also significantly increased (p < 0.001) after 10 days in all groups. A total of 240 primordial follicles were extracted and cultivated from the vitrified-thaw group. Once more, the follicles that received PRP supplementation changed the most in size and viability. Fresh samples displayed a greater growth rate and more viable follicles when compared to vitrified-thawed ones. These findings suggest that PRP more effectively supports the vitality and proliferation of human primordial and primary follicles that have been separated and/or enclosed.

The results of this well-conducted study may not be extended to infertile patients beyond the age of 35 because the study was only able to include three participants and relatively younger patients without an infertility diagnosis. There is a need for additional research to ascertain whether the use of thrombin had an impact on the outcomes compared to the use of pure PRP without thrombin in that study’s modification of PRP to release growth factors.

Non-autologous PRP was investigated by Ahmadian et al. in rats given gonadotoxic IP chemical agents [178]. Female rats were used in the non-autologous production of PRP. The relative expression of the angiogenic-related genes ANGPT2 and KDR was used to study how dramatically PRP reduced follicular atresia and inflammatory response. After PRP, the birth rate in POI rats also increased. Due to the use of intraovarian PRP (rather than IP) and the reporting of birth rate, this study has greater clinical value (which is the end goal of this therapy). In order to treat mice models of gonadotoxin-induced POI, Vural et al. combined non-autologous PRP with rat mesenchymal stem cells (MSCs) from adipose tissue [179]. AMH and estradiol (E2) levels considerably increased when MSC was introduced to PRP. The expressions of CXCL12, BMP-4, TGF-, and IGF-1 (insulin-like growth factor-1) were likewise elevated in that group. CXCL12 stands for C-X-C motif chemokine ligand 12. The study came to the conclusion that PRP alone did not increase follicular regeneration, whereas MSC with or without PRP did.

The first controlled trial incorporating ovarian PRP injection was released by Stojkovska et al. [180]. This prospective, controlled, non-randomized pilot trial included 40 patients who met the POR requirements (at least two of the Bologna criteria) and had normal partner semen tests. The population under study was 35–42 years old.

PRP was administered intravenously in the intervention group, and IVF was carried out in the control and PRP groups two months later. There was no statistically significant difference between the groups in terms of clinical pregnancies and live birth rate. Clinical pregnancy and live birth rates were, respectively, 33.33 ± 44.99, 40.00 ± 50.71, and 10.71 ± 28.95, 14.29 ± 36.31, in the PRP group and 10.71 ± 28.95, 14.29 ± 36.31, and in the control group. Hormone levels also did not considerably improve. Only individuals whose IVF procedures finally led to an embryo transfer were included in the study, which is a significant limitation.

Another prospective, controlled, non-randomized pilot trial using intraovarian PRP injection and 120 patients who were monitored for three months was published by Sfakianoudis et al. [181]. POR (matching Bologna criteria), POI (age 40, amenorrhea at least 4 months, and increased FSH > 25 IU/L), perimenopause (age 40, irregular menstrual cycles), or menopause (age 45–55, with amenorrhea at least 1 year without HRT, and FSH > 30 IU/L) were the criteria for inclusion. In each of these four distinct study groups, bilateral ovaries received an injection of 4 mL of activated PRP with 1X10⁹ platelets/mL. In 60% of POI patients, menstruation returned, and levels of AMH, FSH, and AFC showed statistically significant improvement. In the menopausal group, 43% of the women had lowered FSH or started menstruating again. For 80% of the perimenopausal women, normal menstruation, increased hormone levels, and AFC were noted. Within the study groups, conceptions through IVF and natural means were both successful. The results showed a considerable improvement in the POR group’s hormonal profile, ovarian reserve indicators, and ICSI cycle performance.

The actual understanding of PRP administration therapy seems to have a non-negligible encouraging effect on women who had previously displayed decreased ovarian function, and it should not be disregarded as a potential therapeutic option that may increase the chance for both natural conception and IVF conception, as well as even improvement of perimenopausal symptoms. PRP injection is thought to activate some of the ovaries’ latent oocytes, hence enhancing hormonal profiles and any symptoms of estrogen deficiency. Finally, to ascertain whether autologous intraovarian PRP injection is advantageous in female reproduction, particularly for women with POR, POI, and early menopause, it needs to be researched on a larger scale in a clinical trial setting with standardized preparation, injection, and follow-up techniques [176].

9. Adult stem cells

Mesenchymal stem cells (MSCs) can be detected in a variety of adult tissues and exhibit strong replication capability as well as in vitro differentiation potential into chondrocytes, osteocytes, and adipocytes [182].

Liu et al. [183] used human amniotic fluid MSCs in a POF mice model based on cyclophosphamide to conduct the first study on the ability of human MSCs to survive, engraft, and proliferate into the ovaries. By lowering atresia, preserving the growth of surviving follicles, and reestablishing estrous cyclicity, direct ovarian infusion of mouse amniotic fluid MSCs enhances ovarian function and permits the production of offspring and short-term fertility recovery [184]. After delivery, amniotic membranes can also be easily separated from amniotic epithelial cells (AEC) and amniotic mesenchymal cells (AMSCs), allowing the recovery of clinically important cell values. In mice with various degrees of chemotherapy-induced ovarian damage, ranging from DOR to established POF, human AECs and AMSCs have both been successfully evaluated [185]. After the infusion of hAECs, it has been reported that hormone synthesis, differentiation into granulosa cells, and restoration of folliculogenesis have all returned [185], though hAMSCs have even more positive effects. MSCs have been successfully extracted from umbilical cord blood, which has shown promise in treating a number of non-reproductive degenerative illnesses. Umbilical cord blood MSCs injected into ovaries shield follicular cells from death [186], boosting follicle development and estradiol release. These findings, which have been confirmed in perimenopausal rat ovaries treated with chemotherapy and aged naturally, appear to be mediated by an indirect effect on the ovarian epithelium and niche via expression of key regulators for apoptosis and folliculogenesis, such as cytokeratin 8/18, transforming growth factor (TGF-b), and proliferating cell nuclear antigen [171].

The lack of an autologous source for these MSCs, however, should be viewed as a con to their use for cell treatment in already old and POI patients without previously cryopreserved umbilical cord blood or amniotic membranes, as well as in the absence of menses, such as women with POI. Other autologous cell sources, including adipose tissue and bone marrow, have been investigated as a result of these problems. Adipose MSCs actually induce the expression of POF-related genes and the production of paracrine cytokines, which reduce ovarian apoptosis and restore ovulation in a chemoablated mouse model [187, 188]. However, ovarian effects appear to be smaller than those seen for MSCs generated from amniotic tissue [189].

Recent clinical findings show that stem cell therapy improves ovarian function as seen by resumed menstruation, controlled hormone levels, and, in very rare circumstances, the capacity to become pregnant. It is crucial to choose the right individuals while conducting an analysis of stem cells’ therapeutic effects. The inclusion and exclusion criteria were mostly comparable among the clinical studies included in Table 2. The majority of studies comprised patients with FSH levels above 20 or 25, who were younger than 40, had a normal karyotype, and were diagnosed with POI.

Stem cell type	Sponsor	Clinical Trial Number
OCT4 marker measured	Al-Azhar University	NCT02151890
hUCMSC and hCBMNC transplantation	Shenzhen Beike Bio-Technology Co., Ltd	NCT01742533
autologous MSCs injection	El-Rayadh Fertility Centre	NCT02043743
autologous MSCs treatment + OCT4 marker measured	Sayed Bakry	NCT02062931
BMSC treatment directly to ovary	University of Illinois at Chicago	NCT02696889
embryonic stem cell-derived MSC-like cell transplantation directly into bilateral ovaries	Chinese Academy of Sciences	NCT03877471
VSELs from the patient’s peripheral blood injected in bilateral oviducts + hormone and menstrual conditions measured	Fuda Cancer Hospital, Guangzhou	NCT03985462
Derivation of hESC lines	Hadassah Medical Organization	NCT00353197

Table 2.

Clinical study of stem cell therapy on POF (192).

hUCMSC human umbilical cord mesenchymal stem cell, hCBMNC human cord blood-mononuclear cells, BMSC bone marrow derived mesenchymal stem cells, VSEL very small embryonic-like stem cell, hESC human embryonic stem cell.

Studies using animal models and clinical trials have previously demonstrated the therapeutic benefits of stem cells. It is clear that stem cells can support and restore ovarian function, which in turn has a favorable impact on folliculogenesis, guard against GC apoptosis, and manages ovarian hormones (Figure 1). The use of stem cells does present significant ethical and technical challenges, and stem cell therapies are still illegal in several nations. Although employing MSCs instead of ESCs can address ethical issues about their use, there are still some unanswered safety questions with the extraction and transplantation of stem cells for therapeutic purposes. Minimally invasive techniques that do not injure the donor can be used to extract MSCs from adipose cells, the placenta, or the umbilical cord. Direct transplantation can be invasive and may result in adverse reactions such as immunological responses. Additional in vivo research and clinical trials should be conducted to assess these problems.