Reactive oxygen species

All living aerobic multicellular organisms require molecular oxygen to survive. Oxygen has two unpaired electrons in separate orbits in its outer shell. This electron structure makes it susceptible to forming radicals. The sequential reduction of oxygen through the addition of electrons leads to the formation of ROS (Valko et al., Reference Valko, Leibfritz, Moncol, Cronin, Mazur and Telser2007; Liu et al., Reference Liu, Ren, Zhang, Chuang, Kandaswamy, Zhou and Zuo2018). The term ‘ROS’ is a phrase used to describe a number of reactive molecules and free radicals derived from molecular oxygen. ROS are formed as by-products of normal oxygen metabolism during mitochondrial electronic transport and play an important role in cellular signalling, homeostasis, physiological processes, cell proliferation, hypoxia adaptation and cell fate determination. ROS concentration is important and determines their physiological effects (Van Blerkom, Reference Van Blerkom2009; Scialo et al., Reference Scialo, Fernandez-Ayala and Sanz2017; Zhao et al., Reference Zhao, Jiang, Zhang and Yu2019). ROS generated by mitochondria, such as superoxides, are involved in multiple cell signalling pathways that control the rates of cell proliferation and other cellular activities such as molecular responses to hypoxia (Bell et al., Reference Bell, Emerling and Chandel2005; Bell and Chandel, Reference Bell and Chandel2007; Van Blerkom, Reference Van Blerkom2008). Regarding the role of hypoxia-inducible factor-1 (HIF-1) in cell survival under hypoxic conditions, ROS can regulate HIF-1 during low oxygen conditions. ROS regulates HIF-1 directly or indirectly through the ERK and PI3K/AKT signalling pathways. ROS increase the signalling activity of ERK and PI3K/AKT, and cause HIF-1 transcription and translation. These processes lead to cell proliferation (Movafagh et al., Reference Movafagh, Crook and Vo2015; Zhao et al., Reference Zhao, Jiang, Zhang and Yu2019). ROS produced by electron leak during electron transfer chain in the mitochondria play an important role in cellular signal transduction and the physiology of cells (Zhao et al., Reference Zhao, Jiang, Zhang and Yu2019). Moreover, ROS are important second messengers that mediate different intracellular pathways. ROS act through the oxidative modification of many types of proteins, receptors, phosphatases, caspases, kinases, ion channels, and transcription factors (De Giusti et al., Reference De Giusti, Caldiz, Ennis, Perez, Cingolani and Aiello2013; Zhao et al., Reference Zhao, Jiang, Zhang and Yu2019), therefore small amounts of ROS are needed for the natural function of cells (Scialo et al., Reference Scialo, Fernandez-Ayala and Sanz2017). However, during times of environmental stress such as through UV radiation, heat exposure, and ionizing radiation, ROS levels can increase. At high concentrations, ROS react readily with lipids, proteins, carbohydrates and nucleic acids and may result in significant damage to cell structures (Valko et al., Reference Valko, Leibfritz, Moncol, Cronin, Mazur and Telser2007; Liu et al., Reference Liu, Ren, Zhang, Chuang, Kandaswamy, Zhou and Zuo2018). In the process of embryo development, ROS can cause lipid peroxidation, which affects cell division, metabolite transport and mitochondrial dysfunction. In addition, it causes a break in the nuclear DNA strand that is involved in inhibiting embryo development. Typically, the production of lipid peroxide formation is usually considered as an indirect indicator of free radical markers (Li et al., Reference Li, Wu, Zhuo, Mao, Lan, Zhang and Hua2015).

Under normal conditions, ROS and antioxidants keep a stable ratio. Excess ROS can create a negative environment, affecting fertilization, impairing embryo development, inducing apoptosis and resulting in embryo death (Paszkowski and Clarke, Reference Paszkowski and Clarke1996). Optimizing the composition of embryo culture medium is necessary for increasing embryo quality in vitro. There are metallic ions such as Fe²⁺ and Cu²⁺ in culture medium that have the potential to accelerate ROS production within the cell. In addition, some sera that are commonly added to culture medium, contain amine oxidase, which leads to enhanced H₂O₂ production. Moreover, ROS in the culture medium may be created from embryo metabolism, therefore it seems that ROS can play an essential role in IVF success (Agarwal et al., Reference Agarwal, Said, Bedaiwy, Banerjee and Alvarez2006). Concentrations of ROS in embryo culture medium correlate with the degree of embryo fragmentation or blastocyst formation (Lee et al., Reference Lee, Lee, Liu, Tsao, Huang and Yang2012). Embryo culture medium are often supplemented with antioxidants, therefore keeping an oxidant and antioxidant equilibrium in embryos (Agarwal et al., Reference Agarwal, Said, Bedaiwy, Banerjee and Alvarez2006). It has been demonstrated that embryos in culture medium produce ROS at various rates, depending on the compound of medium (Shih et al., Reference Shih, Lee, Liu, Tsao, Huang and Lee2014). However, Lan et al. (Reference Lan, Lin, Chang, Lin, Tsai and Kang2019) have reported that the relationship between ROS levels and early human embryo development in vitro is limited, such that ROS levels in culture medium have no significant relationship with embryo quality and blastocyst formation. These issues have shown that the exact role of ROS in early embryo development is not yet fully distinguished.

Mitochondria are organelles for ATP production that are important for controlling cell growth, dynamic response, signalling and apoptosis in most mammalian cells. In oocytes and embryos, a high level of ATP production is necessary for maturation of oocytes, fertilization, and early embryo development in vivo and in vitro. Mitochondria in oocyte and early embryo are spherical organelles with short cristae that surround the high-electron density matrix. Despite their simple appearance, they are active in oxidative phosphorylation and are the primary source of ATP in the human oocyte and early embryo (Van Blerkom et al., Reference Van Blerkom, Davis and Lee1995; Van Blerkom, Reference Van Blerkom2011). During ATP production, various types of ROS such as superoxide, hydrogen peroxide and hydroxyl radicals are produced by oxidative phosphorylation in mitochondria. This production of ROS is related to oocyte maturation, fertilization and embryo development, so that ROS accumulation decreases embryo development and blastocyst quality. In addition, severe oxidative stress resulting from enhancing ROS causes mitochondrial fission that leads to the mitochondrion dynamic response, therefore enhancing mitochondrial fission by the accumulation of ROS decreases ATP production (Yang et al., Reference Yang, Park, Kim, Jung, Kim, Jegal, Kim, Kang, Wee, Yang, Lee, Seo, Kim and Koo2018). During embryo culture, ROS levels enhance compared with in vivo embryos at similar stages. Hajian et al. (Reference Hajian, Hosseini, Ostadhosseini and Nasr-Esfahani2017) reported that ROS production in embryos decreased from fertilization to about 8 to 16 cell stage and increased from compaction to blastocyst stage. This increase in ROS production is likely to be related to the change from anaerobic to aerobic glycolysis, because ATP production at this stage depends on the Krebs’ cycle, whereas prior to this stage ATP production is mainly dependent on glycolysis.

For ROS measurement, fluorescent probes are superior sensors due to high sensitivity, ease in data collection and high resolution in microscopy imaging techniques. The fluorescent probe for detecting each type of ROS is different (Gomes et al., Reference Gomes, Fernandes and Lima2005). However, 2,7-dichlorodihydrofluorescein diacetate (H₂DCFDA) is used to detect overall ROS production and in culture medium and is used in IVF (Martin-Romero et al., Reference Martin-Romero, Miguel-Lasobras, Dominguez-Arroyo, Gonzalez-Carrera and Alvarez2008). Although in general, detection of ROS in human early embryos using sensitive fluorescent probes, rather than exact identification of species and quantification of level, has been generally observed as a negative factor (Betts and Madan, Reference Betts and Madan2008; Van Blerkom, Reference Van Blerkom2009). Due to the toxic effects of these fluorescent probes on embryo development, detection of ROS cannot be carried out at the same time in cultured embryos for transfer (Kohler et al., Reference Kohler, Kundig, Reist and Michel1994; Yang et al., Reference Yang, Hwang, Kwon, Kim, Choi and Oh1998).

In the embryo culture process, ROS are generated both endogenously and exogenously. Endogenous ROS are produced by embryo metabolism, while exogenous ROS are produced spontaneously by buffers and enriched culture medium. The most important ROS produced in this manner are superoxide anions, H₂O₂, hydroxyl radicals, and alkyl hydroperoxide. Given that extracellular oxidation rates of ROS-sensitive dyes are higher than those measured within the intracellular environment, these data should not be interpreted as a higher rate of total ROS generation in the extracellular environment. Buffers and culture media containing cupric or ferric salts may lead to the generation of significant amounts of superoxide anions. Moreover, in the composition of serum and serum synthetic replacements (SSR), oxidase activities exist that may speed up the generation of ROS in these buffers. Also, it seems that ROS production is higher in more complex culture media compared with simple media (Martin-Romero et al., Reference Martin-Romero, Miguel-Lasobras, Dominguez-Arroyo, Gonzalez-Carrera and Alvarez2008; Menezo et al., Reference Menezo, Dale and Cohen2010).

There are several types of ROS including peroxide, superoxide anions, hydrogen peroxide, hydroxyl radicals and nitric oxide.

Peroxide is a compound that possesses one or more oxygen–oxygen bonds. The most common peroxide is hydrogen peroxide. Hughes et al. (Reference Hughes, Morbeck, Hudson, Fredrickson, Walker and Coddington2010) showed that the presence of peroxide in culture medium affected mouse embryo development, such that 1-cell embryos had the highest sensitivity for peroxides in mineral oil (Hughes et al., Reference Hughes, Morbeck, Hudson, Fredrickson, Walker and Coddington2010). The sources of peroxide in embryo culture media are embryonic mitochondria and mineral oil, which is used to cover the culture media (Burton et al., Reference Burton, Hempstock and Jauniaux2003; Otsuki et al., Reference Otsuki, Nagai and Chiba2007).

Superoxide anions (O₂ ^•−) are the most common type of ROS that is generated in mitochondria. In aerobic organisms, most oxygen is converted to water through the mitochondrial respiratory chain, however a small proportion of the oxygen molecules (about 1–2%) is converted to superoxide anion radicals. Increased mitochondrial activity is directly related to increased levels of superoxide anion production, which could adversely affect mitochondrial respiration. The half-life of superoxide anions is about 10⁻⁹ to 10⁻¹¹ s, but in the presence of superoxide dismutase (SOD), this reduces to 10⁻¹⁵ s. (Taverne et al., Reference Taverne, Bogers, Duncker and Merkus2013; Chen et al., Reference Chen, Lai, Zhu, Singh, Ahmed and Forsyth2018). Joo et al. (Reference Joo, Kim, Na, Moon, Lee and Kim2001) have shown that increasing superoxide anion concentrations in embryo culture media reduces embryo development, and Nonogaki et al. (Reference Nonogaki, Noda, Narimoto, Umaoka and Mori1992) showed that adding SOD into the culture medium has a protective effect on embryo development against oxidative stress. For detecting superoxide anions, dihydroethidium (or hydroethidine) (DHE) is used as a fluorescent probe; when DHE is oxidized by superoxide anion, it is converted to ethidium which is a fluorescent compound (Benov et al., Reference Benov, Sztejnberg and Fridovich1998).

Hydrogen peroxide (H₂O₂) is a neutral molecule, which is the least reactive molecule among types of ROS and is stable under physiological pH and temperature in the absence of metal ions. It is highly diffusible and crosses the plasma membrane easily. H₂O₂ can be produced from superoxide anions by SOD. Moreover, in the presence of metal ions and superoxide anions, H₂O₂ can produce hydroxyl radicals. H₂O₂ is oxidized by catalase and peroxidase (Dickinson and Chang, Reference Dickinson and Chang2011; Taverne et al., Reference Taverne, Bogers, Duncker and Merkus2013). Catalase is a ROS-scavenging enzymes found in all adult organs and the embryo, although the activity of embryonic catalase is only about 5% that of adult activity, it may enhance the risk of injury during embryo development due to increased ROS. If catalase is not effective, H₂O₂ may initiate signal transduction pathways or react with iron to create highly reactive hydroxyl radicals, which can damage cells. The protective role for catalase against teratogenesis has been demonstrated in embryo culture, such that it is a specified exogenous catalase that increases embryonic antioxidant activity and protects against DNA oxidation (Abramov and Wells, Reference Abramov and Wells2011; Miller-Pinsler and Wells, Reference Miller-Pinsler and Wells2015). The half-life of H₂O₂ is about 10⁻³ s in the absence of catalase and 10⁻⁸ s in its presence (Taverne et al., Reference Taverne, Bogers, Duncker and Merkus2013). H₂O₂ is one of the major ROS produced in culture medium (Martin-Romero et al., Reference Martin-Romero, Miguel-Lasobras, Dominguez-Arroyo, Gonzalez-Carrera and Alvarez2008). Several studies have shown that H₂O₂ damages embryo development in vitro and adding antioxidants such as l-carnitine, apigenin, and quercetin into culture medium can protect the embryos against it (Abdelrazik et al., Reference Abdelrazik, Sharma, Mahfouz and Agarwal2009; Yu et al., Reference Yu, Long, Lyu, Zhang, Yan, Liang, Chai, Yan, Kuang and Qi2014; Safari et al., Reference Safari, Parsaie, Sameni, Aldaghi and Zarbakhsh2018). For detecting H₂O₂, H₂DCFDA and Amplex-red/horseradish peroxidase are used (Wang and Joseph, Reference Wang and Joseph1999; Martin-Romero et al., Reference Martin-Romero, Miguel-Lasobras, Dominguez-Arroyo, Gonzalez-Carrera and Alvarez2008).

The hydroxyl radical (^•OH) is the neutral form of the hydroxide ion (OH⁻). Hydroxyl radicals are the most reactive and dangerous radicals that can be formed from superoxide anions and H₂O₂ in the presence of metal ions. In vivo, the half-life of hydroxyl radicals is only about 10⁻⁹ s, therefore when hydroxyl radicals are produced in vivo, they react close to their site of formation (Dickinson and Chang, Reference Dickinson and Chang2011). Due to their features, the presence of hydroxyl radicals is very harmful to the embryo development in vitro (Dumoulin et al., Reference Dumoulin, Vanvuchelen, Land, Pieters, Geraedts and Evers1995). Fluorescein is used as a fluorescent compound to detect hydroxyl radicals. Fluorescein is oxidized by hydroxyl radicals to a non-fluorescent product. This reactivity is useful to assess antioxidant activity in an assay using hydroxyl radical averting capacity (Ou et al., Reference Ou, Hampsch-Woodill, Flanagan, Deemer, Prior and Huang2002).

Nitric oxide (NO) is an uncharged lipophilic molecule containing a single unpaired electron, which causes it to be reactive with other molecules such as oxygen, superoxide radicals and glutathione. While NO is not a very reactive free radical, it is able to form other reactive intermediates that have an effect on protein function and on the function of the entire organism. NO is removed within seconds by diffusion from tissues and enters the red blood cells and reacts with oxyhaemoglobin. The direct toxicity of NO is modest, but is greatly increased by reacting with superoxide anion. These reactive intermediates can trigger nitrosative damage in biomolecules. Conversely, NO can act as an antioxidant. NO is a neurotransmitter and blood pressure regulator. At physiological concentrations, the half-life of NO due to its reaction with oxygen is in the range 9–900 min. In aqueous solution, the half-life of NO decrease to between 6.2 and 3.8 s (Beckman and Koppenol, Reference Beckman and Koppenol1996; Kelm, Reference Kelm1999). In relating the effect of NO on embryo development in vitro, it seems that NO is useful and acts as a regulator in preimplantation embryo development (Chen et al., Reference Chen, Jiang and Tzeng2001; Tranguch et al., Reference Tranguch, Steuerwald and Huet-Hudson2003). NO has regulatory functions in modulating oxidative respiration by binding to the same site as oxygen in the electron transport chain. Such normal functions have been described for oocytes during their maturation and for embryos, in which for some species, endogenous NO synthase has been detected (Tranguch et al., Reference Tranguch, Steuerwald and Huet-Hudson2003; Feng, Reference Feng2012; Tengan and Moraes, Reference Tengan and Moraes2017). Multi-component Hantzsch ester synthesis of 1,4-dihydropyridines (DHPs) compounds can be used via the fluorescent probes to detect NO (Wang et al., Reference Wang, Liu, Ding, Ma, He, Lin and Lu2016).

Types of antioxidants

Antioxidants are divided into enzymatic and non-enzymatic antioxidants based on their catalytic activity (Haida and Hakiman, Reference Haida and Hakiman2019). Enzymatic antioxidants are produced in cells and protect the body against free radicals via some enzymes that form a distinctive group, with detoxification. Glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) are the key enzyme antioxidants of this defence system by which free radicals that are generated during metabolic reactions are removed (Jeeva et al., Reference Jeeva, Sunitha, Ananthalakshmi, Rajkumari, Ramesh and Krishnan2015). Non-enzymatic antioxidants mainly include polyphenols (flavonoids, phenolic acids, and anthocyanins), carotenoids (carotenes, xanthophylls) and vitamins (vitamins A and C) (Kumar et al., Reference Kumar, Sharma and Vasudeva2017; Xu et al., Reference Xu, Li, Meng, Zhou, Zhou, Zheng, Zhang and Li2017). These types of antioxidants are found naturally in fruits, vegetables and foods such as orange, tomato, carrot and fish. To obtain these antioxidants, their separation and purification are needed. This means that they must be separated from feedstock to enhance their purity and antioxidant capacity (Rozoy et al., Reference Rozoy, Simard, Liu, Kitts, Lessard and Bazinet2012; Jeeva et al., Reference Jeeva, Sunitha, Ananthalakshmi, Rajkumari, Ramesh and Krishnan2015). These types of antioxidants scavenge free radicals by donating hydrogen ions to stabilize the free radicals (Parveen et al., Reference Parveen, Akash, Rehman and Kyunn2016; Maarman, Reference Maarman2017; Xu et al., Reference Xu, Li, Meng, Zhou, Zhou, Zheng, Zhang and Li2017; Haida and Hakiman, Reference Haida and Hakiman2019).

Genes involved with antioxidants

Antioxidants usually activate certain genes to neutralize free radicals and protect cells. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a key transcription factor that is able to activate anti-oxidative reactions; it is known as the master regulator of the antioxidant response and modulates the expression of various antioxidant genes. Nrf2 plays an important role in immune and inflammatory responses and tissue remodelling (Hybertson et al., Reference Hybertson, Gao, Bose and McCord2011). Moreover, Nrf2 signalling is a key pathway by which enzymatic antioxidants remove ROS and other harmful free radicals to protect cells from oxidative stress. Upregulation of many enzymatic antioxidants or inhibition of lipid peroxidation is mediated by Nrf2 (Chen et al., Reference Chen, Lu, Chen and Cheng2015; Canella et al., Reference Canella, Benedusi, Martini, Cervellati, Cavicchio and Valacchi2018).

In addition to Nrf2, Kelch-like ECH-associated protein 1 (Keap1) and antioxidant response element (ARE) or electrophile response element (EpRE) are also important genes involved in protecting cells, therefore the Nrf2/Keap1/ARE signalling pathway is one of the most important cellular defence mechanisms against oxidative stress. When oxidative stress injures cells, Nrf2 expression levels are significantly increased, while Keap1, a signalling molecule that binds to motifs in the N-terminal region of Nrf2, is decreased. Subsequent stabilization and nuclear localization of ARE/EpRE binding leads to protection of cells (Kundu and Surh, Reference Kundu and Surh2010; Yan et al., Reference Yan, Fu, Jia, Ma, Tao, Yang, Ma, Liang, Liu, Yang and Wei2019).

NAD(P)H/quinone oxidoreductase 1 (NQO1) is an antioxidant gene that is overexpressed under certain conditions such as in cancer tumours, to suppress it. For this disease, NQO1 activates Nrf2 to initiate cellular defence mechanisms against the tumour (Pey et al., Reference Pey, Megarity, Medina-Carmona and Timson2016; Osman et al., Reference Osman, Abd El-Maqsoud and El Gelany2015).

Effect of antioxidants on apoptosis

Apoptosis or programmed cell death is essential for the normal functioning and survival of most multicellular organisms. Apoptosis is important for removing damaged or infected cells, however excess apoptosis can cause adverse biological consequences (Kannan and Jain, Reference Kannan and Jain2000). Apoptosis plays an important role in embryo development, If apoptosis is increased in embryonic cells, the blastocyst expands, decreasing the zona pellucida thickness, such that hatching and implantation may not occur correctly. Apoptosis could eventually lead to embryo death (Abdelrazik et al., Reference Abdelrazik, Sharma, Mahfouz and Agarwal2009; Yu et al., Reference Yu, Long, Lyu, Zhang, Yan, Liang, Chai, Yan, Kuang and Qi2014; Safari et al., Reference Safari, Parsaie, Sameni, Aldaghi and Zarbakhsh2018).

Maintaining the integrity of the mitochondrial membrane is an important process to prevent apoptosis. Under oxidative stress, the permeability of mitochondria increases and leads to uncoupling of the respiratory chain, resulting in hyperproduction of ROS, cessation of ATP synthesis, and depletion of glutathione (GSH) (Kannan and Jain, Reference Kannan and Jain2000; Susin et al., Reference Susin, Zamzami and Kroemer1998; Morel and Barouki, Reference Morel and Barouki1999). Under these conditions, to establish osmotic balance, diffusion of H₂O leads to swelling of mitochondria. This change in mitochondrial membrane potential predisposes the cells to oxidative damage by impairing the endogenous antioxidant defence mechanisms (Marzo et al., Reference Marzo, Susin, Petit, Ravagnan, Brenner, Larochette, Zamzami and Kroemer1998; Yang and Cortopassi, Reference Yang and Cortopassi1998). This antioxidant defence system has two main ROS degrading pathways that involve GSH and thioredoxin (Trx) (Tonissen and Di Trapani, Reference Tonissen and Di Trapani2009; Handy and Loscalzo, Reference Handy and Loscalzo2012).

In the GSH pathway, GSH is one of the most abundant molecules among endogenous antioxidants. GSH directly reacts with ROS or indirectly scavenges ROS by revitalizing other antioxidants. Many antioxidants used to scavenge oxidative stress are converted chemically into oxidation products, such that they react with GSH to form GSH adducts during protection against free radicals such as ROS and H₂O₂ (Espinosa-Diez et al., Reference Espinosa-Diez, Miguel, Mennerich, Kietzmann, Sanchez-Perez, Cadenas and Lamas2015; Kwon et al., Reference Kwon, Cha, Lee, Hong, Park, Park, Kim, Kim, Kim, Hwang and Choi2019). Mitochondria are the main intracellular sites of oxygen consumption and the chief sources of ROS production, most of which originate from the respiratory chain in mitochondria. Mitochondrial GSH (mGSH), an antioxidant enzyme existing in mitochondria, acts as the primary line of defence against oxidative modifications (Mari et al., Reference Mari, Morales, Colell, Garcia-Ruiz and Fernandez-Checa2009). The importance of mGSH is based on its abundance and its versatility to counteract with H₂O₂, mainly as a cofactor of enzymes such as GPx (Dannenmann et al., Reference Dannenmann, Lehle, Hildebrand, Kubler, Grondona, Schmid, Holzer, Froschl, Essmann, Rothfuss and Schulze-Osthoff2015). GPx is an enzyme antioxidant that is expressed in many cells and tissues during embryo formation and it protects embryos against oxidative stress. As GPx removes H₂O₂, the rate of apoptosis in embryonic cells decreases (Yu et al., Reference Yu, Long, Lyu, Zhang, Yan, Liang, Chai, Yan, Kuang and Qi2014). In embryogenesis, cytosolic GPx is highly expressed in most cells to protect embryos from oxidative stress (Baek et al., Reference Baek, Yon, Lee, Yun, Yu, Hong, Ahn, Kim, Kim, Kang and Nam2005). It seems that some flavonoid antioxidants can affect GPx activity. Yu et al. (Reference Yu, Long, Lyu, Zhang, Yan, Liang, Chai, Yan, Kuang and Qi2014) showed that quercetin reduced apoptosis in mouse zygotes by maintaining the activity of GPx, preventing mitochondrial dysfunction, and decreasing intracellular ROS levels (Yu et al., Reference Yu, Long, Lyu, Zhang, Yan, Liang, Chai, Yan, Kuang and Qi2014). Other studies have shown that these antioxidants, such as apigenin and quercetin, can decrease the rate of apoptosis in mouse embryonic blastomeres by decreasing the destructive effects of H₂O₂ (Safari et al., Reference Safari, Parsaie, Sameni, Aldaghi and Zarbakhsh2018; Sameni et al., Reference Sameni, Javadinia, Safari, Tabrizi Amjad, Khanmohammadi, Parsaie and Zarbakhsh2018). Lagoa et al. (Reference Lagoa, Graziani, Lopez-Sanchez, Garcia-Martinez and Gutierrez-Merino2011) have shown that flavonoid antioxidants inhibit H₂O₂ production by increasing mitochondrial activity. Boadi et al. (Reference Boadi, Amartey and Lo2016) have shown that flavonoid antioxidants, such as quercetin, genistein, and kaempferol, sustained intracellular GSH levels in the cells. Therefore, it seems that flavonoid antioxidants may improve embryo development in vitro by affecting GSH activity and decreasing the destructive effects of H₂O₂ and ROS.

In the Trx pathway, Trx is a major antioxidant for maintaining the intracellular reduction–oxidation (redox) state. Trx acts as a redox-active protein to regulate the activity of different enzymes within the cell. Also, Trx acts as a ROS scavenger and directly inhibits pro-apoptotic proteins such as apoptosis signal-regulating kinase 1 (ASK1). Mitochondria and cytoplasm contain Trx systems and inhibition of either system can lead to activation of apoptotic pathways (Miranda-Vizuete et al., Reference Miranda-Vizuete, Damdimopoulos and Spyrou2000; Tonissen and Di Trapani, Reference Tonissen and Di Trapani2009). Bing et al. (Reference Bing, Hirao, Takenouchi, Che, Nakamura, Yodoi and Nagai2003) showed that Trx is involved in the improvement of the development of bovine embryos in vitro. Thioredoxin-2 (Trx2), is a mitochondrial protein that reduces oxidative stress, regulates apoptosis and is essential for the control of cell survival during mammalian embryonic development (Patenaude et al., Reference Patenaude, Ven Murthy and Mirault2004; Hansen, Reference Hansen2012). Moreover, Trx2 may contribute to the development of the embryonic nervous system, as Pirson et al. (Reference Pirson, Debrulle, Clippe, Clotman and Knoops2015) reported that Trx2 modulated apoptosis of neurons during embryonic development of the chick spinal cord. It seems that flavonoid antioxidants can affect Trx (s) expression. Park et al. (Reference Park, Kang, Shah, Jin and Koh2020) showed that quercetin prevents the decrease in Trx expression following neuronal cell damage. Sharma et al. (Reference Sharma, Mishra, Ghosh, Tewari, Basu, Seth and Sen2007) reported that flavonoids significantly decreased the release of ROS from astrocytes stimulated with IL-1β. This decrease caused an increase in SOD and Trx1 expression levels and protection against oxidative stress. In this regard, Khera et al. (Reference Khera, Vanderlelie and Perkins2013, Reference Khera, Vanderlelie, Holland and Perkins2017) showed that overexpression of endogenous antioxidants such as GPx and Trx following supplementation with selenium, an exogenous antioxidant, could protect embryo trophoblast cells from mitochondrial oxidative stress.

Action mechanisms of antioxidants on embryo development

Culture conditions during early developmental stages affect metabolic activity and the ability of embryos to adapt to the environment. In embryo development in vitro, oxidative stress generates excessive ROS free radicals, and leads to increased apoptosis, changes in gene expression, and reduced embryo quality (Ullah et al., Reference Ullah, Li, Ali, Xu, Liu, Shah and Fang2019). Oxidative stress occurs as a result of an imbalance between antioxidants and ROS production which is induced by endogenous and exogenous factors during embryo culture in vitro. Enhanced embryo development under lower oxygen conditions is probably due to improved embryo metabolism and reduced ROS production (Guerin et al., Reference Guerin, El Mouatassim and Menezo2001). There are some mechanisms in embryo culture that lead to a decrease in levels of ROS and protect against oxidative stress. Under oxidative stress conditions, the Nrf2 transcription factor binds to ARE to induce antioxidant and detoxification enzymes. ROS generation in the culture medium, in particular, increases in the blastocyst stage. The Nrf2-mediated oxidative stress response pathway is the main pathway in the blastocyst for which upregulation for most antioxidant-related genes is controlled by transcription factor Nrf2 (Gad et al., Reference Gad, Hoelker, Besenfelder, Havlicek, Cinar, Rings, Held, Dufort, Sirard, Schellander and Tesfaye2012). Indeed, Nrf2 signalling has the most effect on embryo development in vitro (Ullah et al., Reference Ullah, Li, Ali, Xu, Liu, Shah and Fang2019). Expression of the Nrf2 gene also increases during embryo cryopreservation to protect embryonic cells (Mehaisen et al., Reference Mehaisen, Saeed, Gad, Abass, Arafa and El-Sayed2015). Nrf2 activates enzyme antioxidants such as GPx, SOD, and CAT against excessive ROS in embryos and therefore protects embryonic cells in vitro (Ullah et al., Reference Ullah, Li, Ali, Xu, Liu, Shah and Fang2019). Moreover, antioxidants can increase the expression of the anti-apoptotic gene Bcl-2 and reduce the expression of pro-apoptotic genes, Bax and caspase-3 in blastocysts, indicating the protective effects of antioxidants on embryo development (Mishra et al., Reference Mishra, Reddy, Gupta and Mondal2017; Ullah et al., Reference Ullah, Li, Ali, Xu, Liu, Shah and Fang2019). Pterostilbene is an antioxidant that acts by the same mechanism in embryonic cells or adult cells. Sireesh et al. (Reference Sireesh, Ganesh, Dhamodharan, Sakthivadivel, Sivasubramanian, Gunasekaran and Ramkumar2017) have reported that pterostilbene increases Bcl-2 expression and reduces the expression of Bax and caspase-3 in pancreatic β-cells of diabetic animals through Nrf2 activation. For embryo culture, pterostilbene protects embryonic cells by increasing expression levels of Bcl-2, Nrf2, CAT, Heme oxygenase1 (HMOX1), GPx and SOD, and by decreasing expression levels of Bax and caspase-3 (Ullah et al., Reference Ullah, Li, Ali, Xu, Liu, Shah and Fang2019).

The oviduct and the uterine environments contain many substances that maintain embryo development or remove toxic agents. To mimic these conditions in vitro, various substances that decrease ROS concentrations in embryos are added to culture media. For example, adding SOD or CAT improves embryo development by upregulating GSH synthesis as an effective antioxidant for the developmental potential of embryos (Orsi and Leese, Reference Orsi and Leese2001; Ali et al., Reference Ali, Liu, Zhong-Shu, Dong-Xue, Xu, Shah, Ullah and Nan-Zhu2018).

Conversely, intracellular lipid contents may affect embryo quality and developmental potential. Lipid metabolism and oxidative stress response-related genes are the most affected via embryonic genes in vitro (Jeong et al., Reference Jeong, Cho, Lee, Deb, Lee, Kwon and Kong2009). Moreover, inhibition of fatty acid oxidation during IVM impairs embryo development and indicates the importance of lipid metabolism in embryonic development (Somfai et al., Reference Somfai, Kaneda, Akagi, Watanabe, Haraguchi, Mizutani, Dang-Nguyen, Geshi, Kikuchi and Nagai2011). During preimplantation embryo development, lipid may be sequestered within cells and used by mitochondria to increase the production of ATP required for compaction and blastocyst formation or differentiation of cell lineages (Jeong et al., Reference Jeong, Cho, Lee, Deb, Lee, Kwon and Kong2009). Excess lipids may be accumulated in the embryo by uptake from the culture environment and may impair mitochondrial activity to metabolize complex lipids (Tarazona et al., Reference Tarazona, Rodriguez, Restrepo and Olivera-Angel2006; Krisher and Prather, Reference Krisher and Prather2012; Walther and Farese, Reference Walther and Farese2012). Some antioxidants such as l-carnitine, quercetin, and apigenin play a primary role in fatty acid transportation from the cytosol into mitochondria, increase mitochondrial activity, enhance lipid metabolism and improve cleavage rates in embryos, which indicates the importance of mitochondria and lipid metabolism in embryo development (Khanmohammadi et al., Reference Khanmohammadi, Movahedin, Safari, Sameni, Yousefi, Jafari and Zarbakhsh2016; Safari et al., Reference Safari, Parsaie, Sameni, Aldaghi and Zarbakhsh2018; Sameni et al., Reference Sameni, Javadinia, Safari, Tabrizi Amjad, Khanmohammadi, Parsaie and Zarbakhsh2018; Talebi et al., Reference Talebi, Hayati Roodbari, Reza Sameni and Zarbakhsh2020).

Optimizing embryo culture medium

Optimizing embryo culture media is essential to obtain high-quality embryos. Various factors added to culture media can improve the oxidative state of early embryos. Weathersbee et al. (Reference Weathersbee, Pool and Ord1995) reported that synthetic serum substitute was a suitable, standardized means of adding protein to embryo culture medium. Synthetic serum substitute is primarily a globulin-enriched protein preparation containing mostly human serum albumin (HSA). HSA scavenges ROS and protect embryos against DNA damage (Lan et al., Reference Lan, Lin, Chang, Lin, Tsai and Kang2019). Albumin is one of the most important substances used to optimize embryo culture media. Albumin is a small protein that is present in human plasma. Its functions consist of regulation of osmotic pressure and transport of some substances (Otsuki et al., Reference Otsuki, Nagai, Matsuyama, Terada and Era2013). Albumin acts as a surfactant that facilitates the handling of gametes and preimplantation embryos in vitro (Bungum et al., Reference Bungum, Humaidan and Bungum2002). Albumin has specific binding sites for copper ions. Copper ions can accelerate the destruction of free radical reactions. Most plasma copper is bound to the protein ceruloplasmin, which has antioxidant properties. In this regard, cytosolic SOD (SOD1) functions more than mitochondrial SOD (SOD2) because it works to scavenge ROS with the aid of metal cofactors including copper and zinc. The mechanism of scavenging ROS by SOD1 involves alternate reduction and reoxidation of the copper at the active site of the enzyme, and zinc participates in proper protein folding and stability (Fukai and Ushio-Fukai, Reference Fukai and Ushio-Fukai2011). Albumin is able to inhibit copper-stimulated peroxidation and inhibits the production of free radicals from systems containing copper ions and H₂O₂. Another antioxidant activity of albumin may be to scavenge peroxy radicals and decrease lipoxygenase activity (Halliwell, Reference Halliwell1988). Albumin has different functions in the growth of embryos in vitro. It acts as a regulator of pH and osmotic pressure, a scavenger of toxins and free radicals, a stabilizer of cell membranes, a carrier of growth-promoting substances, and a nutrient (Otsuki et al., Reference Otsuki, Nagai, Matsuyama, Terada and Era2013). Three different types of albumin are used to optimize embryo culture medium: HSA, bovine serum albumin (BSA), and recombinant HSA (rHSA):

1. 1. HSA is known to be a multifunctional protein in the intravascular compartment (Maciazek-Jurczyk et al., Reference Maciazek-Jurczyk, Szkudlarek, Chudzik, Pozycka and Sulkowska2018). HSA was used in human embryo culture media for the first time by Pool and Martin (Reference Pool and Martin1994), who showed that albumin accelerated the growth of embryos. HSA is the primary protein supplement used in clinical embryo culture media. HSA as a macromolecule supplementation can act as a surfactant, as a nitrogen source, as a carrier molecule for other compounds, modulate the physical microenvironment, and stabilize membranes (Swain et al., Reference Swain, Carrell, Cobo, Meseguer, Rubio and Smith2016).

2. 2. BSA is the most abundant protein in bovine blood plasma. Due to its unique characteristics and known structure, is commonly used as a model protein for the culture of animal oocyte and embryo and is effective in their development (Ledesma-Osuna et al., Reference Ledesma-Osuna, Ramos-Clamont and Vazquez-Moreno2008; Nasrollahzadeh et al., Reference Nasrollahzadeh, Varidi, Koocheki and Hadizadeh2017).

3. 3. rHSA has been shown to be as efficient as HSA for fertilization and embryo development. In addition, using rHSA for IVF may decrease the risk of contamination and the transmission of plasma-derived impurities. However, rHSA has not been widely applied in human embryo culture media because of the high cost of production (Bungum et al., Reference Bungum, Humaidan and Bungum2002; Otsuki et al., Reference Otsuki, Nagai, Matsuyama, Terada and Era2013).

In addition to the role of albumin in optimizing embryo culture medium, studies have shown that albumin can also optimize culture media used for sperm and oocytes through its antioxidant properties. Some studies have shown that centrifugation during sperm preparation using density gradient centrifugation or swim-up without serum albumin is associated with iatrogenic damage to sperm. Free radicals produced by mitochondria during centrifugation cause membrane lipid peroxidation and DNA damage in the absence of albumin (Aitken and Clarkson, Reference Aitken and Clarkson1988; Twigg et al., Reference Twigg, Irvine, Houston, Fulton, Michael and Aitken1998; Aitken et al., Reference Aitken, Finnie, Muscio, Whiting, Connaughton, Kuczera, Rothkirch and De Iuliis2014; Muratori et al., Reference Muratori, Tarozzi, Carpentiero, Danti, Perrone, Cambi, Casini, Azzari, Boni, Maggi, Borini and Baldi2019). This damage may result from tightly packed sperm pellets through peroxide formed from superoxide radicals by MnSOD. It is released from damaged mitochondria and induces lipid peroxidation of the plasma membrane, depolarizes mitochondria, affects sperm motility and reduces ATP generation (De Iuliis et al., Reference De Iuliis, Wingate, Koppers, McLaughlin and Aitken2006; Uribe et al., Reference Uribe, Boguen, Treulen, Sanchez and Villegas2015; Barbonetti et al., Reference Barbonetti, Castellini, Di Giammarco, Santilli, Francavilla and Francavilla2016; Kotwicka et al., Reference Kotwicka, Skibinska, Jendraszak and Jedrzejczak2016). Another study showed that BSA protected spermatozoa against cool storage-induced DNA damage through its antioxidant properties. (Sariozkan et al., Reference Sariozkan, Turk, Canturk, Yay, Eken and Akcay2013). Conversely, it has been shown that increasing serum albumin improved the viability of bovine oocytes in vitro (Hamman et al., Reference Hamman, Thompson, Smuts, Tshuma and Holm2019).

Despite all the benefits of using albumin in embryo culture media, Otsuki et al. (Reference Otsuki, Nagai and Chiba2009) reported that when peroxide in mineral oil (used to maintain embryos in the culture medium) is more than 0.02 mEq/kg, albumin present in culture media allows entry of free radicals into the zona pellucida, causing damage to the human embryos. In other studies, Martinez et al. (Reference Martinez, Nohalez, Ceron, Rubio, Roca, Cuello, Rodriguez-Martinez, Martinez and Gil2017) showed that peroxidized mineral oil enhanced the oxidant status of culture media and inhibited porcine embryo development in vitro. Otsuki et al. (Reference Otsuki, Nagai and Chiba2007) demonstrated that peroxidation of mineral oil applied in culture was harmful to fertilization and human embryo development. In this regard, Ainsworth et al. (Reference Ainsworth, Fredrickson and Morbeck2017) reported that the standard embryo assays used by manufacturers did not detect the potential toxicity of peroxides in mineral oil. It seems that the use of mineral oil requires further studies and would depend on various factors such as brand, purity, starting material, method of production and storage. These issues indicated that optimization of human embryo culture media requires more comprehensive, complete study.

Some studies have shown that when purified serum albumin cannot be used, organic compounds such as polyvinyl alcohol (PVA) or certain dextran polymers can be adequate substitutes for IVF and preimplantation embryogenesis. PVA is a polymer and a suitable replacement for BSA in embryo culture media that supports the development of preimplantation embryos. Dextran is also a polymer chain consisting of a non-toxic branch of glucose. These polymers with their surfactant properties facilitate the handling of the embryos, probably by influencing the physico-chemical attributes of the media. Moreover, they have been shown to protect embryos against cryoinjury by avoiding the mechanical pressure that occurs during cryopreservation (Dumoulin et al., Reference Dumoulin, Bergers-Janssen, Pieters, Enginsu, Geraedts and Evers1994; Biggers et al., Reference Biggers, Summers and McGinnis1997).

Comparison of the effect of different antioxidants on embryo development

Some studies have shown that different antioxidants have various differing effects on embryo development in vitro, therefore comparing their effects would help future studies to select the most suitable candidates. Conversely, it seems that combined and concomitant use of antioxidants can be even more effective than their separate use. In this regard, Sovernigo et al. compared the effects of five well known antioxidants (quercetin, cysteamine, carnitine, vitamin C and resveratrol) by the levels of ROS and GSH in bovine embryos. The results showed that quercetin, vitamin C and resveratrol significantly reduced ROS levels compared with cysteamine and carnitine. GSH levels increased in cysteamine and carnitine compared with quercetin, vitamin C and resveratrol (Sovernigo et al., Reference Sovernigo, Adona, Monzani, Guemra, Barros, Lopes and Leal2017).

Perez-Pasten et al. (Reference Perez-Pasten, Martinez-Galero and Chamorro-Cevallos2010) compared the effects of quercetin and naringenin on the development of mouse embryos in vitro. The results showed that quercetin and naringenin both reduced the abnormal development of embryos produced by hydroxyurea at doses less than 30 µM. Lee et al. (Reference Lee, Jin, Taweechaipaisankul, Kim and Lee2018) showed that the combination of resveratrol and melatonin supported a synergistic increase in blastocyst formation rates and total cell numbers of blastocysts and improved the development of porcine embryos. Truong et al. (2-16) showed that the combination of different antioxidants (acetyl-l-carnitine, N-acetyl-l-cysteine and α-lipoic acid) in culture media had a more beneficial effect on mouse embryo development in vitro (Truong et al., Reference Truong, Soh and Gardner2016).

Antioxidants in the clinic

The use of antioxidants clinically in IVF is still very limited. Currently, lipoic acid is the most common used antioxidant in human embryo culture media (Truong and Gardner, Reference Truong and Gardner2017), but more antioxidants are expected to be used in human embryo culture media in the future. More recently, there have been some investigations into the effects of antioxidants on human embryo development in vitro. Kim et al. (Reference Kim, Park, Paek, Kim, Kwak, Lee, Lyu and Lee2018) reported that adding l-carnitine to culture media improved human embryo quality and pregnancy outcomes. l-Carnitine may improve embryo development in vitro by increasing mitochondrial activity and β-oxidation processes (Arenas et al., Reference Arenas, Rubio, Martin and Campos1998; Abdelrazik et al., Reference Abdelrazik, Sharma, Mahfouz and Agarwal2009; Kepka et al., Reference Kepka, Chojnowska, Okungbowa and Zwierz2014; Khanmohammadi et al., Reference Khanmohammadi, Movahedin, Safari, Sameni, Yousefi, Jafari and Zarbakhsh2016). Truong et al. (Reference Truong, Soh and Gardner2016) suggested that the combination of some specific antioxidants may improve human embryo culture media, however before the use of human embryos proper evaluation is required (Truong et al., Reference Truong, Soh and Gardner2016; Truong and Gardner, Reference Truong and Gardner2017).

Prescribing the proper dose of antioxidants is one of the most important aspects of their effectiveness. Many antioxidants are dose dependent and may be harmful if consumed in too large quantities (Halliwell, Reference Halliwell2012). Quercetin is a flavonoid antioxidant that, at appropriate doses, has favourable effects in vitro on the development of different species of embryos (Perez-Pasten et al., Reference Perez-Pasten, Martinez-Galero and Chamorro-Cevallos2010; Lee et al., Reference Lee, Oh, Chung, Park, Lee, Kwon and Choi2015; Fan et al., Reference Fan, Feng, Liu, Zhang, Liu, Zhou and Xu2017; Sameni et al., Reference Sameni, Javadinia, Safari, Tabrizi Amjad, Khanmohammadi, Parsaie and Zarbakhsh2018). Perez-Pasten et al. (Reference Perez-Pasten, Martinez-Galero and Chamorro-Cevallos2010) showed that quercetin at doses above 100 µM caused significant increase abnormalities such as oedema, rotation failure, neural tube defects, somite dysmorphology, and telencephalic hypoplasia for developing mouse embryos in vitro. l-Carnitine is a useful antioxidant for ovarian regeneration (Zarbakhsh et al., Reference Zarbakhsh, Safari, Sameni, Yousefi, Safari, Khanmohammadi and Hayat2019) and embryo development in vitro, but it is toxic at high doses, (Abdelrazik et al., Reference Abdelrazik, Sharma, Mahfouz and Agarwal2009; Khanmohammadi et al., Reference Khanmohammadi, Movahedin, Safari, Sameni, Yousefi, Jafari and Zarbakhsh2016). Perez-Pasten et al. (Reference Perez-Pasten, Martinez-Galero and Chamorro-Cevallos2010) compared different doses of pure naringenin on developing mouse embryos in vitro. They reported that 30 µM pure naringenin with antioxidant and free radical scavenging activities had a protective effect against hydroxyurea-induced embryonic damage, while at doses above 100 µM it produced growth retardation, developmental defects and reduced viability in cultured mouse embryos.

As many antioxidants have beneficial effects on embryo development in vitro, to achieve proper supplement levels for human embryo culture media, a study on antioxidants is needed because they constitute a promising therapeutic approach.