Kidney-tonifying Chinese Medicine in Alleviating Oxidative Stress in Oocyte in Older Infertile Women: A Ｒeview

Abstract Assisted reproductive technology( AＲT) is a big hope for older infertile women． However, the clinical outcomes are unsatisfactory because of the poor ovarian reserve, low antral follicle count, low quality of oocytes, and small number of high-quality embryos． Oocyte quality is the key to the pregnancy． Oxidative stress is the main reason for granulosa cell apoptosis and poor oocyte quality． Kidney-tonifying Chinese medicine plays an essential part by alleviating oxidative stress damage, inhibiting granulosa cell apoptosis, improving egg quality, and improving pregnancy rate． Kidney-tonifying Chinese medicine has been widely used in different stages of in vitro fertilization-embryo transfer( IVF-ET)． It is a new way for the treatment of older infertile women．

 Keywords：Kidney-tonifying Chinese medicine; Older; IVF-ET; Oocyte; Oxidative stress; Apoptosis; Ｒeview

HERBAL  FORMULATION FOR LOW QUALITY OF OOCYTES

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In recent years, with the development of society and the increase in work and life pressure, the childbearing age of women has gradually increased. The proportion of women giving birth to their first child after age 35 increased from 1/100 in 1970 to 1/12 in 2006 [1-2]. Age is closely related to ovarian reserve function and can be an independent factor in predicting female fertility. Female fertility declines significantly after age 35 [3-4]. With the relaxation of the two-child policy, there are 90 million couples in China who meet the childbearing conditions [], of which 60% are women over 35 years old [5-7]. These women have decreased ovarian reserve function and low clinical pregnancy rate. How to improve the fertility of elderly infertile women is an important issue related to the national economy and people's livelihood.

At present, the number of elderly infertile women over 35 years old who choose assisted reproductive technology (Assisted Reproductive Technology, ART) for treatment is rapidly increasing worldwide [8-9]. However, the results of assisted pregnancy are often not ideal. Foreign studies have shown that the embryo implantation rate of women over 35 years old in the IVF-ET cycle begins to decline. For every year of age, the embryo implantation rate decreases by 2.77%. The embryo implantation rate of women over 44 years old is only 2.3% [10]. At the same time, the clinical pregnancy rate drops sharply and the spontaneous abortion rate increases [11]. The number of antral follicles in elderly infertile women decreases, and the quality of oocytes decreases. The use of a large amount of exogenous ovulation-inducing drugs in the IVF-ET cycle and repeated ovarian stimulation can induce oxidative stress and reduce oocyte quality [12]. Oocyte quality is a key factor affecting pregnancy success and decreases with age. To solve this problem, reproductive physicians use a variety of strategies in clinical practice to improve ovarian response, such as adding coenzyme Q10, dehydroepiandrosterone (DHEA), growth hormone (GH), etc., but the research results are still controversial. As an important part of traditional Chinese medicine, kidney-tonifying Chinese medicine has been shown in a large number of studies to improve oocyte quality, reduce the dosage of gonadotropins (Gn), increase the normal fertilization rate and high-quality embryo rate, and improve the clinical pregnancy outcomes of elderly infertile women during IVF-ET cycles [11-36]. It is speculated that this may be related to kidney-tonifying Chinese medicine improving oxidative stress damage in oocytes of elderly women and inhibiting apoptosis of granulosa cells, but its exact mechanism is still unclear.

Research progress on the relationship between advanced-age infertility and oxidative stress The rapid decline in fertility in advanced-age women is closely related to the decline in the number and quality of oocytes. Female babies have 1 million to 2 million oocytes in their ovaries at birth, and no new oocytes are produced thereafter. Instead, they are continuously lost. Studies have shown that during puberty, the number of oocytes has decreased from 100,000 to 300,000. In the late 30s, especially after the age of 35, the number of oocytes decreases more significantly [17]. Although the reasons for the decline in the number and quality of oocytes are relatively complex, the impact of oxidative stress has attracted increasing attention. It is generally believed that oxidative stress caused by reactive oxygen species (ROS) in the body can lead to mitochondrial dysfunction in the oocytes of advanced-age women. It has been reported that the decline in antioxidant function caused by aging is related to the decline in oocyte quality [18-19]. Oxidative stress refers to an imbalance between oxidation and antioxidant activity caused by excessive production of ROS, which exceeds the body's antioxidant defense level, thus causing tissue and cell damage [20-21]. Under physiological conditions, 90% of ROS originate from the mitochondrial oxidative phosphorylation process [22]. ROS plays an important role in the reproductive process, affecting the growth, development, and maturation of oocytes, as well as ovulation, fertilization, embryo implantation, and embryonic development [23]. During ovulation, due to the increase in luteinizing hormone (LH) and the formation of new blood vessels in the follicles, vascular endothelial cells produce a large amount of ROS, which provides the necessary conditions for oocyte maturation and follicle rupture. However, excessive ROS can cause cell damage. As a defense mechanism against ROS, antioxidant enzymes, and antioxidants exist in follicles and oocytes to protect oocytes from damage by oxidative stress. Enzyme antioxidants include superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), glutathione peroxidase (GSH-Px), etc.; non-enzyme antioxidants include vitamin C, vitamin E, GSH, carotenoids, melatonin, etc. [2-25]. If the ROS level is unbalanced with the antioxidant system, oocytes, and granulosa cells are easily damaged by oxidative stress, affecting the quality of oocytes. Oxidative stress plays an important role in the physiological and pathological processes of the female reproductive system and is an important factor causing the decline of female reproductive function.

1. 1 Oxidative stress and decrease in oocyte quality

Oxidative stress is the main reason for the decline in oocyte quality in elderly women [26]. Oxidative stress may be the "initiator" of oocyte aging and reproductive diseases and is the main cause of abnormal follicle atresia, abnormal meiosis, reduced fertilization rate, and delayed embryonic development [27]. Mitochondria are closely related to the decline in oocyte quality. Oocyte mitochondria contain a unique genetic system called mitochondrial DNA (mtDNA). Some studies have pointed out that aging can cause oxidative damage to mtDNA, and insufficient mtDNA copy numbers can cause oocyte damage.

Cell development potential is reduced [28]. The copy number of mtDNA is closely related to age and ovarian reserve. The copy number of mtDNA is significantly decreased in elderly women with poor ovarian reserve [29-31]. According to the theory of reproductive aging, the decline in oocyte quality in older women is caused by the shortening of mitochondrial telomeres. Excessive ROS levels can cause different types of telomere DNA damage, triggering (local telomere) single-strand breakage (Single-strand Breakage). , SSB), affecting the transcription of telomere repeating RNA (TERRA) or the binding of protective proteins on telomeres, leading to the shortening of telomeres and a decrease in oocyte quality [32-34].

Mitochondrial dysfunction is the focus of oocyte aging [35]. Jönsson et al. [36] found that when a redox reaction occurs, electrons in mitochondria are transferred to oxygen through complexes I and III of the respiratory chain, which enhances oxidative phosphorylation activity and increases ROS production. Due to the particularity of the tDNA structure, when ROS is too high, it is easy to combine with mtDNA, causing mutations, causing mitochondrial dysfunction, affecting the electron transport chain, causing electron leakage, and combining with oxygen molecules to generate a large amount of ROS, causing a vicious cycle and causing egg damage. Mother cell damage and mitophagy cannot produce enough Adenosine Triphosphate (ATP), affecting egg and embryo development.

Oxidative stress can lead to poor embryonic development and reduced clinical pregnancy rates in older women [37-38]. High levels of ROS are the main cause of IVF-ET failure [39]. Patients with high granulosa cell ROS in follicular fluid have a lower probability of obtaining eggs and a lower embryo implantation rate [40]. Carbone et al [41] found that the levels of CAT and SOD in the follicular fluid of elderly women were significantly lower than those of women of childbearing age. Wen Canxin et al. [42] found that ROS in follicular fluid of elderly women increased, SOD activity decreased, and CAT and GSH levels in granulosa cells significantly decreased. Moreover, SOD activity in follicular fluid affected oocyte quality and fertilization rate, ultimately affecting embryo development potential and Blastocyst formation. Al-Saleh et al. [43] found that the higher the content of malondialdehyde (MDA) in follicular fluid, the more serious the damage to the DNA of granulosa cells; CAT can remove ROS in follicular fluid and has antioxidant effects. The increase in the level of CAT in the fluid improves the fertilization rate and the live birth rate, which has a positive impact on the clinical outcomes of IVF-ET.

1.2 Oxidative stress and apoptosis

Increased levels of ROS may cause oxidation of DNA, RNA, carbohydrates, proteins, and lipids, resulting in irreparable cell damage and leading to apoptosis [44]. Apoptosis plays an important role in the maintenance of the body's internal environment and the development of multiple systems. It is strictly regulated by multiple genes such as the B-cell Lymphoma-2 (Bcl-2) family and the caspase family. Bcl-2 family proteins are a class of key proteins that maintain mitochondrial integrity by regulating the balance between pro-apoptosis and anti-apoptosis [45].

Bcl-2-associated X protein (BAX) can enhance mitochondrial membrane permeability and change the transmembrane potential, thereby promoting the release of mitochondrial cytochrome C into the cytoplasm, causing a cascade reaction of caspase, and ultimately leading to the activation of caspase-3, inducing apoptosis [46-48]. Bcl-2 protein can exert anti-apoptotic effects by inhibiting a series of signal activations induced by apoptotic proteins [49-50]. Oxidative stress can cause granulosa cell apoptosis, leading to decreased 17β-estradiol (Estradiol, E2) levels, decreased ovulation rate, and affected oocyte quality [51]. Oxidative stress-mediated granulosa cell apoptosis reduces the connection between granulosa cells and oocytes, thereby reducing the nutrient supply of oocytes, further affecting the quality of oocytes [52]. Lee et al. [53] reported that granulosa cell apoptosis is related to age, and the apoptosis rate of granulosa cells in elderly IVF-ET patients is high. Some research results show that the ROS level and apoptosis rate of granulosa cells hurt the pregnancy outcome of IVF-ET, and the expression levels of both in the non-pregnant group are significantly higher than those in the pregnant group [54]. There are three main pathways of cell apoptosis: the mitochondria-mediated intrinsic apoptosis pathway, the death receptor-mediated extrinsic apoptosis pathway, and the endoplasmic reticulum-mediated apoptosis pathway. Among them, the mitochondria-mediated intrinsic apoptosis pathway is an important pathway for oxidative stress to cause oocyte apoptosis [55-56]. Studies have shown that oxidative stress is a key factor leading to apoptosis of oocytes and granulosa cells. Excessive ROS activates the mitochondria-mediated apoptosis signaling pathway, induces apoptosis of granulosa cells, and leads to oocyte atresia [57-59]. The essence of oocyte apoptosis is the result of the disruption of the dynamic balance between the intracellular oxidative system and the antioxidant system.