Introduction
Preimplantation embryonic development occurs right after oocyte fertilization and is a process of division and cell differentiation of the embryo that, under optimum conditions, results in successful embryo implantation. Whether occurring in vivo or in vitro, embryonic development requires several factors that maintain the homeostasis of signalling pathways and cellular metabolism at each stage of the preimplantation process.
Considerable numbers of studies have focused on the different preimplantation embryo cell signalling pathways in vivo, and more importantly on the regulatory factors that maintain their physiological equilibrium (Tranguch et al., Reference Tranguch, Steuerwald and Huet-Hudson2003; Thisse and Thisse, Reference Thisse and Thisse2005; Kawamura et al., Reference Kawamura, Kawamura, Fukuda, Kumagai, Hsueh and Tanaka2007). Among the regulatory factors that have attracted the most interest due to their complexity are the reactive oxygen species (ROS). These are highly reactive molecules that result from the intermediate steps of oxygen reduction by electrons during cellular metabolism (Apel and Hirt, Reference Apel and Hirt2004; Murphy, Reference Murphy2009; Schieber and Chandel, Reference Schieber and Chandel2014). ROS act as second messengers in physiological cell signalling and control pathways, therefore they have an important role in embryonic development, cell proliferation and differentiation (Dröge, Reference Dröge2002; Dennery, Reference Dennery2007; Dumollard et al., Reference Dumollard, Ward, Carroll and Duchen2007; Finkel, Reference Finkel2011; Ray et al., Reference Ray, Huang and Tsuji2012).
Embryos are known for three free radicals during their normal aerobic metabolism: hydrogen peroxide (H2O2), superoxide anion (O2−) and hydroxyl radical (OH−) (Thannickal and Fanburg, Reference Thannickal and Fanburg2000; Guérin et al., Reference Guérin, El Mouatassim and Ménézo2001; Apel and Hirt, Reference Apel and Hirt2004). However, free radicals are more often associated with the principle of oxidative stress, which results from an imbalance in the level of oxidants and antioxidants, in favour of oxidants during cell metabolism (Hensley et al., Reference Hensley, Robinson, Gabbita, Salsman and Floyd2000; Jones, Reference Jones2008). This causes significant cellular damage in protein, lipid structures and DNA (Betteridge, Reference Betteridge2000; Blokhina et al., Reference Blokhina, Virolainen and Fagerstedt2003; Khansari et al., Reference Khansari, Shakiba and Mahmoudi2009). Precisely, the delicate influence of oxidative stress on embryonic development has been the subject of several studies, highlighting the negative effect that a high rate of oxidants can have during each phase of development (Tatemoto et al., Reference Tatemoto, Sakurai and Muto2000; Combelles et al., Reference Combelles, Gupta and Agarwal2009; Lee et al., Reference Lee, Lee, Liu, Tsao, Huang and Yang2012). It can be said that the complexity of the effect of free radicals on embryonic development lies in the fact that free radicals can have various effects, ranging from deterioration to cell promotion, depending on the rate of free radicals, the stage of development (fertilization, cell division, blastulation) and the nature of environment (in vivo or in vitro).
This review examines the available evidence that dwells on the importance and the deleterious effects of ROS in the embryonic development process. To understand the complexity of the essential oxidation balance required for the embryo development process, we will focus on the cellular and environmental conditions that influence the embryo’s redox status during its early development, in physiological and pathological profiles, in vitro and in vivo.
Oxidative stress in fertilization and early embryo development
Oocyte maturation
The meiotic cycle of oocytes stops at the diplotene stage of prophase 1 during birth. For fertilization to take place, the maturation of oocytes is characterized by the meiotic recovery that results in the passage of the oocyte to the metaphase I stage, then the first polar body excursion and metaphase II arrest (Mehlmann et al., Reference Mehlmann, Kalinowski, Ross, Parlow, Hewlett and Jaffe2006; DiLuigi et al., Reference DiLuigi, Weitzman, Pace, Siano, Maier and Mehlmann2008; Kubiak et al., Reference Kubiak, Ciemerych, Hupalowska, Sikora-Polaczek and Polanski2008). The oocyte maturation process, in response to the surge of luteinizing hormone (LH), consists of regulatory signalling pathways that involve protein phosphorylation, cyclic adenosine monophosphate (cAMP), calcium (Ca2+), hydrogen peroxide (H2O2) and nitric oxide (NO) (Pandey et al., Reference Pandey, Tripathi, Premkumar, Shrivastav and Chaube2010; Tripathi et al., Reference Tripathi, Kumar and Chaube2010; Tiwari et al., Reference Tiwari, Prasad, Shrivastav and Chaube2017).
Cyclic adenosine monophosphate (cAMP) is a second essential messenger in the extracellular transduction pathway cascade. It is produced by adenosine triphosphate (ATP) via adenylate cyclase (AC). In the oocyte, an increase in cAMP concentration is associated with meiotic arrest at the diplotene stage (Sirard et al., Reference Sirard, Richard and Mayes1998; Conti et al., Reference Conti, Andersen, Richard, Shitsukawa and Tsafriri2002; Downs et al., Reference Downs, Hudson and Hardie2002). In response to LH surge, cAMP and cyclic guanosine monophosphate (cGMP), are produced by granulosa cells and transferred to the oocyte via gap junctions. cGMP, an important inhibitory second messenger, inactivates phosphodiesterase 3P (PDE3A) and inhibits oocytes cAMP hydrolysis. cAMP binds to the regulatory chain of protein kinase A and leads to the release of the catalytic chain and therefore allows the phosphorylation of target proteins, CD25b phosphatase (CDC25-P) and kinase Wee1/MYT1 (Nebreda and Ferby, Reference Nebreda and Ferby2000; Norris et al., Reference Norris, Ratzan, Freudzon, Mehlmann, Krall, Movsesian, Wang, Ke, Nikolaev and Jaffe2009; Vaccari et al., Reference Vaccari, Weeks, Hsieh, Menniti and Conti2009). Activation of target proteins affects the activity of the CDK/cyclin B complex also known as meiosis, mitosis or maturation promoting factor (MPF). Cyclin-dependent kinase-1 (CDK1) is phosphorylated on Thr14 and Tyr15 residues, then the MPF complex is maintained inactive. Through this process, a constitutive concentration of cAMP, above a threshold level, maintains meiotic arrest at the diplotene stage of prophase I (Motlík and Kubelka, Reference Motlík and Kubelka1990; Chaube, Reference Chaube2001; Mehlmann et al., Reference Mehlmann, Kalinowski, Ross, Parlow, Hewlett and Jaffe2006). For maturation from germinal vesicle to metaphase I stage, cAMP levels decrease with the association of decrease in intra-oocyte ROS levels. Calcium levels from either the extracellular medium or the endoplasmic reticulum induce mitochondrial H2O2 generation (Tripathi et al., Reference Tripathi, Kumar and Chaube2010; Latham, Reference Latham2015; Zeeshan et al., Reference Zeeshan, Lee, Kim and Chae2016) and calcium/calmodulin-dependent protein kinase II (CAMKII) activation. In physiological conditions, 1–2% of oxygen molecules from the mitochondrial respiratory chain are converted into superoxide anion (Cadenas and Davies, Reference Cadenas and Davies2000). Because of its low membrane permeability, superoxide anion is rapidly converted into H2O2 by superoxide dismutase (SOD) (Cejas et al., Reference Cejas, Casado, Belda-Iniesta, De Castro, Espinosa, Redondo, Sereno, García-Cabezas, Vara, Domínguez-Cáceres, Perona and González-Barón2004). For oocyte meiotic resumption, H2O2 can be upregulated, which is known to cause lipid peroxidation that increases inner membrane permeability, mitochondrial swelling and loss of matrix components (Almeida et al., Reference Almeida, Bertoncini, Borecký, Souza-Pinto and Vercesi2006; Murphy, Reference Murphy2016). However, in the oocyte, mitochondria are transcriptionally silent and have a slow ATP production (Babayev and Seli, Reference Babayev and Seli2015). Additionally, in growing follicles, there is a distance between the oocyte and a diffusible source of oxygen due to the surrounding cumulus and coronal cells, as it has been shown in bovines oocytes (Redding et al., Reference Redding, Bronlund and Hart2007). This observation might lead to the conclusion that the metabolism in the developing follicles is anaerobic. Nevertheless, it has been reported that a minimum level of oxygen still can reach the oocyte in the preantral follicles, which spares oxygen from the oxidative phosphorylation by the oocyte (Clark et al., Reference Clark, Stokes, Lane and Thompson2006). As far as we know, no study has investigated the role of the oocyte slow metabolism in ROS regulation during in vitro maturation. However, quiescent state of oocyte mitochondria and the low oxygen metabolism during oocyte maturation can play a role in the control of H2O2 generation and upregulation during oocyte maturation, therefore leading to lower risk of DNA mutations that can be transferred to embryo.
Regarding cAMP, the EGF-like factor activates MAPK that interrupts communications between cumulus cells and oocyte, and therefore the transfer of cAMP to the oocyte. cAMP level in the oocyte is then reduced (Su and Eppig, Reference Su and Eppig2002; Mehlmann et al., Reference Mehlmann, Kalinowski, Ross, Parlow, Hewlett and Jaffe2006; Zhang et al., Reference Zhang, Ouyang and Xia2009; Tiwari et al., Reference Tiwari, Prasad, Shrivastav and Chaube2017). The decrease of cAMP level, associated with the increase of intra-oocyte H2O2 level and CAMKII activity activates Cdc25b phosphatase and Wee1. The MPF complex is then destabilized by cyclin B degradation and CDK1 phosphorylation in residues Thr14/Tyr15. MPF destabilization finally induces meiotic resumption (Lincoln et al., Reference Lincoln, Wickramasinghe, Stein, Schultz, Palko, De Miguel, Tessarollo and Donovan2002; Kubiak et al., Reference Kubiak, Ciemerych, Hupalowska, Sikora-Polaczek and Polanski2008).
Oocyte development
After meiotic resumption from prophase I to metaphase II stage, the oocyte becomes fully competent for the fertilization process. Oocyte fertilization with spermatozoa is a process that allows exit from metaphase II arrest, known as ‘egg activation’. A universal second messenger of the oocytes fertilization process is intracellular Ca2+. Ca2+ oscillations are responsible for polyspermy blocking by cortical granule exocytosis, zona pellucida hardening, second polar body emission, cytoskeletal rearrangements and specific maternal mRNAs recruitment (Kline and Kline, Reference Kline and Kline1992; Schultz and Kopf, Reference Schultz and Kopf1995; Kono et al., Reference Kono, Jones, Bos-Mikich, Whittingham and Carroll1996; Deguchi et al., Reference Deguchi, Shirakawa, Oda, Mohri and Miyazaki2000; Nixon et al., Reference Nixon, McDougall and Jones2000; Ducibella et al., Reference Ducibella, Huneau, Angelichio, Xu, Schultz, Kopf, Fissore, Madoux and Ozil2002; Berridge et al., Reference Berridge, Bootman and Roderick2003; Ozil et al., Reference Ozil, Markoulaki, Toth, Matson, Banrezes, Knott, Schultz, Huneau and Ducibella2005; Whitaker, Reference Whitaker2006).
Several studies have demonstrated the involvement of calcium oscillations in the regulation of various cell signalling pathways in animal and plant cells (Landolfi et al., Reference Landolfi, Curci, Debellis, Pozzan and Hofer1998; Li et al., Reference Li, Llopis, Whitney, Zlokarnik and Tsien1998; Stricker, Reference Stricker1999; Berridge et al., Reference Berridge, Lipp and Bootman2000; Allen et al., Reference Allen, Chu, Harrington, Schumacher, Hoffmann, Tang, Grill and Schroeder2001; Wallingford et al., Reference Wallingford, Ewald, Harland and Fraser2001; Roderick and Bootman, Reference Roderick and Bootman2003; Uhlén and Fritz, Reference Uhlén and Fritz2010). The precise mechanism of calcium oscillation stimulations during fertilization is not fully clear. A few hypotheses have been proposed, such as ‘the fusion hypothesis’ involving activation of the phosphoinositide (PI) pathway by gamete interaction, and ‘the receptor hypothesis’ involving the protein tyrosine kinase signalling cascade (PTKs) (Santella et al., Reference Santella, Lim and Moccia2004; Miyazaki, Reference Miyazaki2006; Swann and Yu, Reference Swann and Yu2008). These stimulus hypotheses all lead to phospholipase C activation. Phospholipase C activation results in phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis into two signalling molecules, IP3 and diacylglycerol (DAG). An increase in intracellular IP3 concentration initiates Ca2+ release from the endoplasmic reticulum (ER) by binding to its receptor inositol triphosphate receptor (IP3R). Plus, DAG production plays a role in calcium influx regulation for the intracellular store refilling which makes the oscillations possible (Jones, Reference Jones1998; Jellerette et al., Reference Jellerette, He, Wu, Parys and Fissore2000; Runft et al., Reference Runft, Jaffe and Mehlmann2002; Malcuit et al., Reference Malcuit, Kurokawa and Fissore2006; Miyazaki, Reference Miyazaki2006; Ducibella and Fissore, Reference Ducibella and Fissore2008; Berridge, Reference Berridge2009).
Homeostasis of cellular calcium is regulated by mitochondria, in addition to the endoplasmic and sarcoplasmic reticulum (Hansford, Reference Hansford1994; Boitier et al., Reference Boitier, Rea and Duchen1999; Duchen, Reference Duchen2000; Gunter et al., Reference Gunter, Buntinas, Sparagna, Eliseev and Gunter2000; Simpson, Reference Simpson2000; Camello-Almaraz et al., Reference Camello-Almaraz, Salido, Pariente and Camello2002; Patergnani et al., Reference Patergnani, Suski, Agnoletto, Bononi, Bonora, De Marchi, Giorgi, Marchi, Missiroli, Poletti, Rimessi, Duszynski, Wieckowski and Pinton2011). During fertilization, the cytosolic concentration of calcium increases and is absorbed by active mitochondria to regulate calcium oscillations (Dumollard et al., Reference Dumollard, Hammar, Porterfield, Smith, Cibert, Rouvière and Sardet2003; Gunter et al., Reference Gunter, Yule, Gunter, Eliseev and Salter2004).
In several cell types, mitochondria, nearby ER, detect the calcium microdomains of IP3 receptors in ER to control and facilitate the diffusion of calcium signals, in addition to the delivery of ATP to calcium pumps of ER according to the cellular needs (Hajnóczky et al., Reference Hajnóczky, Robb-Gaspers, Seitz and Thomas1995; Landolfi et al., Reference Landolfi, Curci, Debellis, Pozzan and Hofer1998; Hoth et al., Reference Hoth, Fanger and Lewis1997; Park et al., Reference Park, Ashby, Erdemli, Petersen and Tepikin2001; Roderick and Bootman, Reference Roderick and Bootman2003; Dumollard et al., Reference Dumollard, Duchen and Sardet2006).
The role of mitochondria in calcium signalling of cells also involves the regulatory factors that are the mitochondrial ROS (Camello-Almaraz et al., Reference Camello-Almaraz, Gomez-Pinilla, Pozo and Camello2006a; Camello-Almaraz et al., Reference Camello-Almaraz, Pozo, Murphy and Camello2006b; Feissner et al., Reference Feissner, Skalska, Gaum and Sheu2009). ROS, such as H2O2, superoxide anion (O2−) and NO− act as second messengers (Chen et al., Reference Chen, Vazquez, Moghaddas, Hoppel and Lesnefsky2003; Luberda, Reference Luberda2005). During fertilization in mammals, an increase in the ROS level acts directly on the IP3R, the calcium pumps of the ER and the plasma membrane. Also, ROS act on the thiol residue of the ryanodine receptor. This action activates the calcium channel of the sarcoplasmic reticulum and therefore stimulates calcium release (Favero et al., Reference Favero, Zable and Abramson1995; Zhang et al., Reference Zhang, Wang, Vikash, Ye, Wu, Liu and Dong2016). Mitochondrial ROS can not only induce the release of internal calcium reserves, but also inhibit calcium accumulation in sarcoplasmic reticulum and mitochondria (Kaminishi and Kako, Reference Kaminishi and Kako1989). Conversely, calcium also modulates ROS generation and elimination by mitochondria via regulation of ATP synthesis, stimulation of Krebs’ cycle enzymes and oxidative phosphorylation (Brookes et al., Reference Brookes, Yoon, Robotham, Anders and Sheu2004; Lewis et al., Reference Lewis, Hayashi, Su and Betenbaugh2014). ATP, calcium, and ROS influence each other along a triangular network. This highlights the importance of these signalling molecules in the fertilization process, among many other calcium-dependent cellular processes. As long as these signalling pathways are positively regulated, ROS levels contribute to oocyte development and fertilization competence. Conversely, in the case of excess ROS and signalling disregulation, it is well established that it can result in a profound development effect that leads to spindle instability, chromosomal abnormalities and oocytes ageing due to reduced development competence (Sasaki et al., Reference Sasaki, Hamatani, Kamijo, Iwai and Kobanawa2019).
Embryo development
After fertilization, embryo development implies cleavage, rapid cell division, compaction and differentiation into inner cell mass and trophectoderm at the blastocyst stage (Paria and Dey, Reference Paria and Dey1990; Iwata et al., Reference Iwata, Yumoto, Sugishima, Mizoguchi, Kai, Iba and Mio2014). During cell division, a fast and regulated process, preimplantation embryos have high energy demands, provided by ATP through mitochondrial oxidative phosphorylation that produces ROS, namely H2O2. H2O2 generation by the hydroxyl radical (OH−), via the Fenton reactions, is involved in the normal circumstances of the mitochondrial respiratory chain and is managed by the enzymatic defence system that includes metalloenzymes like SOD and catalase (CAT) (Guérin et al., Reference Guérin, El Mouatassim and Ménézo2001; Dumollard et al., Reference Dumollard, Ward, Carroll and Duchen2007; Levine and Puzio-Kuter, Reference Levine and Puzio-Kuter2010; Migdal and Serres, Reference Migdal and Serres2011; Moreira da Silva et al., Reference Moreira da Silva, Marques and Chaveiro2014). The secretory mechanism of H2O2 is well determined in mammalian cells, it is produced as a by-product of many oxidative reactions, such as peroxisomal enzyme activities, the respiratory chain cascade and oxidative protein folding in the ER. H2O2 is also produced in neutrophils, to act against microbial infections, but our interest will be limited to its second messenger role during cell proliferation and differentiation (Rhee et al., Reference Rhee, Kang, Jeong, Chang, Yang and Woo2005; Rhee, Reference Rhee2006; Lennicke et al., Reference Lennicke, Rahn, Lichtenfels, Wessjohann and Seliger2015).
Early embryogenesis involves initial cell divisions, and cleavage. Several factors are known to influence the division cell cycle, namely plasma membrane ion transport, pH changes, redox potential, and intracellular ROS levels. Several studies have demonstrated the effect of physiological ROS concentration, namely H2O2, in cell proliferation by the regulation of key transcription factors, such as nuclear factor κB (NF-κB) (Liou and Storz, Reference Liou and Storz2010; Diaz Vivancos et al., Reference Diaz Vivancos, Wolff, Markovic, Pallardó and Foyer2010; Lennicke et al., Reference Lennicke, Rahn, Lichtenfels, Wessjohann and Seliger2015).
NF-κB is a ubiquitous, multisubunit, eukaryotic transcription factor composed of two subunits, and is sequestered in cytoplasm (Gius et al., Reference Gius, Botero, Shah and Curry1999; Mercurio and Manning, Reference Mercurio and Manning1999; Sun and Andersson, Reference Sun and Andersson2002). In mouse embryos, it has been shown that NF-κB is involved in cell division from the first cleavage stage (Nishikimi et al., Reference Nishikimi, Mukai and Yamada1999), as it has been suggested that its signalling pathways are also implicated in meiotic–mitotic transition and activation of the embryonic genome (Paciolla et al., Reference Paciolla, Boni, Fusco, Pescatore, Poeta, Ursini, Lioi and Miano2011). NF-κB regulation and activation involves different stimuli, such as cytokines like tumour necrosis factor alpha (TNFα), lipopolysaccharide (LPS) and, in some cell types, by oxidants like H2O2 (Meyer et al., Reference Meyer, Pahl and Baeuerle1994; Brennan and O’Neill, Reference Brennan and O’Neill1995; Karin et al., Reference Karin, Takahashi, Kapahi, Delhase, Chen, Makris, Rothwarf, Baud, Natoli, Guido and Li2001). As far as we know, there is no direct evidence linking NF-κB signalling to redox mechanism during early embryo development. Conversely, it is known that NF-κB and its regulation by ROS play a crucial role in the process of cell proliferation, as has been established in many cell lines, whether under physiological or pathological conditions such as cancer. As the basic mechanisms of cell proliferation and cleavage are similar throughout different cell populations, NF-κB and its regulation by ROS regulation might also be involved in rapid cell division during the process of early embryo development. (Schreck et al., Reference Schreck, Rieber and Baeuerle1991; Meyer et al., Reference Meyer, Schreck and Baeuerle1993; Sauer et al., Reference Sauer, Wartenberg and Hescheler2001).
NF-κB, when inactive, is composed of three subunits p50, p65, and an inhibitory molecule nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα). In response to oxidative stress, IκBα is phosphorylated on serines 32 and 36 by IκB kinase, which results in its rapid ubiquitination and degradation in the proteasome. IκBα subunit dissociation uncovers the nuclear localization signals on the p65, p50 complex which leads to NF-κB translocation in the nucleus when it binds to the promoter region of the target genes (Beg et al., Reference Beg, T. S. Finco, Nantermet and Baldwin1993; Schoonbroodt et al., Reference Schoonbroodt, Ferreira, Best-Belpomme, Boelaert, Legrand-Poels, Korner and Piette2000; Van den Berg et al., Reference van den Berg, Haenen, van den Berg and Bast2001; Takada et al., Reference Takada, Mukhopadhyay, Kundu, Mahabeleshwar, Singh and Aggarwal2003; Häcker and Karin, Reference Häcker and Karin2006; Liou and Storz, Reference Liou and Storz2010; Zhang et al., Reference Zhang, Wang, Vikash, Ye, Wu, Liu and Dong2016).
H2O2 is also involved in the activation and regulation of the transcription factor hypoxia-inducible factor (HIF-1). From normoxia to hypoxia, this transcription factor is upregulated by various growth factors such as insulin-like growth factor-1 (IGF-1), IGF-2 and vascular endothelial growth factor (Ke and Costa, Reference Ke and Costa2006). HIF-1 activity is also known to be stimulated by follicle-stimulating hormone (FSH) and expressed in granulosa cells and endometrium during implantation (Critchley et al., Reference Critchley, Osei, Henderson, Boswell, Sales, Jabbour and Hirani2006; Alam et al., Reference Alam, Weck, Maizels, Park, Lee, Ashcroft and Hunzicker-Dunn2009), as this pathway regulates target genes that include those involved in angiogenesis and cell survival (Ke and Costa, Reference Ke and Costa2006).
These findings can explain why ROS have a positive role to play in the regulation of mitotic divisions during early embryonic development. Several studies have highlighted the complex relationship between intracellular ROS levels and preimplantation embryo development. H2O2 has been shown to increase during the first cell divisions, the 8-cell stage to increase blastulation capacity and hatching in mouse and bovine embryos (Nasr-Esfahani and Johnson, Reference Nasr-Esfahani and Johnson1991; Thomas et al., Reference Thomas, Jain, Kumar and Laloraya1997; Morales et al., Reference Morales, Tilquin, Rees, Massip, Dessy and Van Langendonckt1999; Lopes et al., Reference Lopes, Lane and Thompson2010). Oxidative fluctuations are variable depending on the stage of embryo development and necessary for the control and regulation of each of these stages (Rhee, Reference Rhee2006; Veal et al., Reference Veal, Day and Morgan2007). While a high ROS level leads to oxidative stress that causes cell death, especially in the early stages of embryo development (Karagenc et al., Reference Karagenc, Sertkaya, Ciray, Ulug and Bahçeci2004), cell proliferation might require a reduced oxidation state, followed by a moderate oxidative state that promotes cell differentiation (Dennery, Reference Dennery2007).
The complex link between ROS and embryo development has been further supported by embryonic oxidant supplementation. Interestingly, when adding PEG-catalase to embryo culture, the 2–4-cell stage failure was significantly reduced, while the 5–8-cell embryo arrest increased. All these results show H2O2 requirements in the early embryo development process (Bain et al., Reference Bain, Madan and Betts2011). However, a recent study showed that ROS levels, while measured in culture medium, are not necessarily associated with embryo quality during preimplantation development, as fertilized, polyspermic and unfertilized zygote showed similar ROS levels and were not associated with damaged embryos (Lan et al., Reference Lan, Lin, Chang, Lin, Tsai and Kang2019). In conclusion, further research is needed to have a clearer idea view on the relationship between ROS levels and preimplantation embryo development.
Oxidative stress in pathologic profiles: polycystic ovary syndrome (PCOS) endometriosis and ovarian stimulation
Polycystic ovary syndrome (PCOS)
PCOS occurs in 10% of women of reproductive age. It is a pro-inflammatory process that involves obesity, insulin resistance and hyperglycaemia (Kelly et al., Reference Kelly, Lyall, Petrie, Gould, Connell and Sattar2001; Diamanti-Kandarakis et al., Reference Diamanti-Kandarakis, Paterakis, Alexandraki, Piperi, Aessopos, Katsikis, Katsilambros, Kreatsas and Panidis2006; Escobar-Morreale et al., Reference Escobar-Morreale, Luque-Ramírez and González2011; Layegh et al., Reference Layegh, Mousavi, Farrokh Tehrani, Parizadeh and Khajedaluee2016; Huang et al., Reference Huang, Wang, Liu, Pei, Liu, Zhu and Huang2019). It has been shown that hyperglycaemia and insulin resistance are associated with inflammation, which can be mediated by NF-κB activation (González et al., Reference González, Rote, Minium and Kirwan2006). Obesity is also linked to the PCOS inflammatory process. Many studies have indeed identified abnormal levels of inflammatory markers in overweight PCOS women (Escobar-Morreale et al., Reference Escobar-Morreale, Luque-Ramírez and González2011; Duleba and Dokras, Reference Duleba and Dokras2012). Chronic inflammation is known to be closely associated with oxidative stress, as oxidative stress can activate various inflammatory cytokines and result in chronic inflammation (Reuter et al., Reference Reuter, Gupta, Chaturvedi and Aggarwal2010). Other studies showed that inflammatory cytokines are the source of ROS as O2− and H2O2 via NADPH oxidase (Zalba et al., Reference Zalba, San José, Moreno, Fortuño, Fortuño, Beaumont and Díez2001; Dröge, Reference Dröge2002), which can support the concept of vicious cycle between inflammation and oxidative stress (Fenkci et al., Reference Fenkci, Fenkci, Yilmazer and Serteser2003) in which oxidative stress enhances inflammation while inflammation generates ROS as well. Many studies have reported abnormal levels of inflammatory markers that are regulated by pro-inflammatory cytokines, in overweight PCOS women. Interleukin-6 (IL-6), IL-8 and TNFα have been identified in the follicular fluid (FF) of PCOS patients and has been suggested to originate from granulosa cells (Amato et al., Reference Amato, Conte, Mazziotti, Lalli, Vitolo, Tucker, Bellastella, Carella and Izzo2003; Ojeda-Ojeda et al., Reference Ojeda-Ojeda, Murri, Insenser and Escobar-Morreale2013; Pawelczak et al., Reference Pawelczak, Rosenthal, Milla, Liu and Shah2014; Artimani et al., Reference Artimani, Karimi, Mehdizadeh, Yavangi, Khanlarzadeh, Ghorbani, Asadi and Kheiripour2018).
In PCOS women, it has also been shown that obesity and hyperglycaemia promotes oxidative stress by enhancing ROS generation (González et al., Reference González, Rote, Minium and Kirwan2006; Victor et al., Reference Victor, Rocha, Bañuls, Sanchez-Serrano, Sola, Gomez and Hernandez-Mijares2009). Inflammation, oxidative stress and PCOS co-morbidities such as obesity and insulin resistance are all linked and have deleterious effects on female fertility.
In addition to cytokines, growth factors and macrophages are also a source of ROS in FF. Several studies have highlighted the excess of oxidative stress in FF of PCOS women and its effect on in vitro fertilization (IVF) outcomes (Arya et al., Reference Arya, Haq and Chaudhury2012; Yilmaz et al., Reference Yilmaz, Inal, Gorkem, Sargin Oruc, Yilmaz and Turkkani2016). It has been demonstrated that when ROS levels are superior to 100 counted photons per second (cps), there are less oocytes with meiotic spindle visualization which can lead to the disruption of chromosome segregation and therefore poor oocyte and embryo quality (Chattopadhayay et al., Reference Chattopadhayay, Ganesh, Samanta, Jana, Chakravarty and Chaudhury2010; Rajani et al., Reference Rajani, Chattopadhyay, Goswami, Ghosh, Sharma and Chakravarty2012). According to another study, the percentage of ROS in granulosa cells would also increase 20-fold in PCOS patients, compared with the normal rate. Intact granulosa cells already produce a large amount of ROS by electron transport. Their physiological role is mostly maintained by enzymatic antioxidants such as SOD and CAT. As SOD reduces O2− to H2O2, CAT removes H2O2 to maintain homeostasis (Behl and Pandey, Reference Behl and Pandey2002; Migdal and Serres, Reference Migdal and Serres2011). In contrast, in PCOS women, hyperandrogenism in FF can reduce aquaporin-9 (AQP9) mRNA expression in granulosa cells membranes through the PI3K pathway. Knowing that the PI3K pathway is regulated by ROS, the reduction of AQP9 mRNA levels can be one of the sources of excess ROS in granulosa cells in PCOS women (Qu et al., Reference Qu, Wang, Lu, Dong, Sheng, Lv, Ding, Shi, Zhang and Huang2010; Zhao et al., Reference Zhao, Zhang, Huang, Yu, Li, Li, Liu, Liu and Qiao2015). This high rate of ROS would alter DNA and mitochondrial metabolism, which results in a high rate of immature oocytes, as well as a significantly low rate of fertilization, embryonic development, and implantation (Karuputhula et al., Reference Karuputhula, Chattopadhyay, Chakravarty and Chaudhury2013).
Endometriosis
Endometriosis is one of the most common causes of infertility in women of reproductive age (Gupta et al., Reference Gupta, Agarwal, Krajcir and Alvarez2006; Giudice, Reference Giudice2010; Carvalho et al., Reference Carvalho, Samadder, Agarwal, Fernandes and Abrão2012; Gupta et al., Reference Gupta, Ghulmiyyah, Sharma, Halabi and Agarwal2014). As for PCOS, endometriosis is also associated with chronic inflammation (Van Langendonckt et al., Reference Van Langendonckt, Casanas-Roux and Donnez2002; Halis and Arici, Reference Halis and Arici2004; Augoulea et al., Reference Augoulea, Mastorakos, Lambrinoudaki, Christodoulakos and Creatsas2009; De Ziegler et al., Reference de Ziegler, Borghese and Chapron2010; Maybin et al., Reference Maybin, Critchley and Jabbour2011; Augoulea et al., Reference Augoulea, Alexandrou, Creatsa, Vrachnis and Lambrinoudaki2012) It has been shown that there is a complex interaction between the immune system failure, inflammation, and oxidative stress. The increased angiogenesis in peritoneal fluid (PF) involved in endometriosis can lead to chronic inflammation by the recruitment of monocytes, which lead to ROS production. This cascade of events is responsible for antioxidative system failure, cell damage and then the proliferation of endometriotic cells. (Nothnick, Reference Nothnick2001; Van Langendonckt et al., Reference Van Langendonckt, Casanas-Roux and Donnez2002; Augoulea et al., Reference Augoulea, Mastorakos, Lambrinoudaki, Christodoulakos and Creatsas2009; Harlev et al., Reference Harlev, Gupta and Agarwal2015). Several studies have investigated oxidative stress markers to understand its effect on IVF outcomes. The pro-oxidative markers include malondialdehyde (MDA) (Mier-Cabrera et al., Reference Mier-Cabrera, Jiménez-Zamudio, García-Latorre, Cruz-Orozco and Hernández-Guerrero2011), NO in macrophage and peritoneal fluid, thiobarbituric acid reactive substances (TBARS) in serum (Jackson et al., Reference Jackson, Schisterman, Dey-Rao, Browne and Armstrong2005), heat shock protein 70 (HSP70) (Lambrinoudaki et al., Reference Lambrinoudaki, Augoulea, Christodoulakos, Economou, Kaparos, Kontoravdis, Papadias and Creatsas2009) and PF 8-OHdG in granulosa cells (Saito et al., Reference Saito, Seino, Kaneko, Nakahara, Toya and Kurachi2002). According to all this research, endometriosis patients are exposed to a higher rate of oxidative stress, which explains the low rate of antioxidants such as vitamins C and E (Prieto et al., Reference Prieto, Quesada, Cambero, Pacheco, Pellicer, Codoceo and Garcia-Velasco2012; Singh et al., Reference Singh, Chattopadhyay, Chakravarty and Chaudhury2013), SOD and glutathione peroxidase (GPx) in FF (Szczepańska et al., Reference Szczepańska, Koźlik, Skrzypczak and Mikołajczyk2003; Prieto et al., Reference Prieto, Quesada, Cambero, Pacheco, Pellicer, Codoceo and Garcia-Velasco2012; Singh et al., Reference Singh, Chattopadhyay, Chakravarty and Chaudhury2013) CAT (Singh et al., Reference Singh, Chattopadhyay, Chakravarty and Chaudhury2013) and serum paraoxonase-1 (Verit et al., Reference Verit, Erel and Celik2008).
In the context of endometriosis, adenomyosis is defined by the presence of endometrial glands and cytogenic stroma inside the myometrium. This condition, as an internal form of endometriosis, can also be related to oxidative stress. Gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) has been suggested to cause apoptosis and inhibit cell growth in endometrial tissue from patients with adenomyosis. Also known as NDUFA13, GRIM-19 is a subunit of mitochondrial complex I that enhances mitochondrial respiration, therefore leading to ROS production (Chen et al., Reference Chen, Lu, Liu, Huang, Lim, Zhang, Hao and Cao2012; Shulga and Pastorino, Reference Shulga and Pastorino2012; Dixon and Stockwell, Reference Dixon and Stockwell2014; Wang et al., Reference Wang, Deng, Yang, Yang, Kong and Chao2016). This oxidative stress may lead to oocyte DNA damage leading to failure of fertilization, embryo development or implantation (Barnhart et al., Reference Barnhart, Dunsmoor-Su and Coutifaris2002; Saito et al., Reference Saito, Seino, Kaneko, Nakahara, Toya and Kurachi2002).
Ovarian stimulation
Ovarian stimulation has a very important place in IVF cycles. Therefore, high exogenous doses of oestrogen (E2) are administered to stimulate the development of multiple oocytes. To understand the effect of ovarian stimulation on IVF outcomes, many studies have evaluated its effect on ROS and antioxidant levels. It has been shown that ovarian stimulation can sensitize lipoproteins to in vitro oxidation by a decreased serum level of hydrophilic antioxidants and vitamin E (Aurrekoetxea et al., Reference Aurrekoetxea, Ruiz-Sanz, del Agua, Navarro, Hernández, Matorras, Prieto and Ruiz-Larrea2010). Conversely, Appasamy et al. (Reference Appasamy, Jauniaux, Serhal, Al-Qahtani, Groome and Muttukrishna2008), found a positive association between FF E2 during ovarian stimulation and total antioxidant capacity, suggesting that E2 may play a key role in the ovarian antioxidant–oxidant balance. Also similarly, Younis et al. (Reference Younis, Clower, Nelsen, Butler, Carvalho, Hok and Garelnabi2012), who observed an increase in SOD serum levels after ovarian oestrogen stimulation. Plus, in a comparative study between agonist and antagonist treatments, it was shown that serum GSH levels of metaphase II stage oocytes and embryos of two pronuclei were lower in the GnRH antagonist group (Celik et al., Reference Celik, Celik, Kumbak, Yilmaz, Turkcuoglu, Simsek, Karaer, Minareci, Ozerol and Tanbek2012).
These results were agreeable with Seino et al. (Reference Seino, Saito, Kaneko, Takahashi, Kawachiya and Kurachi2002), who found a negative correlation between the oxidative stress marker 8-hydroxy-20-deoxyguanosine in FF E2. A positive correlation between E2 peaks and antioxidant levels does not always have a positive effect on IVF outcomes. It has been suggested that the high SOD levels on FF after ovarian stimulation can impair the follicular microenvironment of oocytes, their maturation, and fertilization (Sabatini et al., Reference Sabatini, Wilson, Lower, Al-Shawaf and Grudzinskas1999; Celik et al., Reference Celik, Celik, Kumbak, Yilmaz, Turkcuoglu, Simsek, Karaer, Minareci, Ozerol and Tanbek2012). When SOD levels are high, a corresponding increase of CAT and GPx is lacking, which can imbalance the oxidation–reduction system. O2− is then highly converted to H2O2, which is known to be membrane permeable and causes lipoperoxidative damage to the cellular membrane. Plus, in the presence of heavy metals, SOD and H2O2 together can contribute to Fenton and Habber–Weiss reactions, to produce OH− and lead to further cellular damage (Sabatini et al., Reference Sabatini, Wilson, Lower, Al-Shawaf and Grudzinskas1999). The cycle number of stimulation also seems to harm the pro/antioxidant balance. According to Chao et al. (Reference Chao, Lee, Lee, Liao, Wei and Kao2005), an increase in the number of cycles increases oxidative damage including lipoperoxides and carbonyl proteins in the ovaries. It has also been reported that embryonic quality and in vitro blastulation significantly deteriorates starting from the third stimulation cycle (Chao et al., Reference Chao, Lee, Lee, Liao, Wei and Kao2005).
According to these studies, we can only understand that the more intense is the ovarian stimulation protocol, whether in the number of cycles or the administrated doses of oestrogen, the greater the risk of imbalance between pro-oxidant and antioxidant levels. This may have a negative effect on preimplantation embryo development and endometrial receptivity (Ertzeid, Reference Ertzeid2001; Edwards et al., Reference Edwards, Kind, Armstrong and Thompson2005; Santos et al., Reference Santos, Kuijk and Macklon2010).
Oxidative stress in assisted reproduction techniques (ART)
As mentioned before, ROS are beneficial for oocyte maturation and embryo development in vivo, at an optimal level that is maintained by enzymatic antioxidants in the female reproductive system (Combelles et al., Reference Combelles, Gupta and Agarwal2009; Opuwari and Henkel, Reference Opuwari and Henkel2016). The oxidant/antioxidant balance is maintained by SOD, GPx and CAT that play a key role in minimizing the deleterious effect of ROS in follicles, oocyte and embryos. As GSH is oxidized to glutathione disulfate (GSSG) by GPx, CAT reduces H2O2 to water (Fujii et al., Reference Fujii, Iuchi and Okada2005; Rizzo et al., Reference Rizzo, Roscino, Binetti and Sciorsci2012).
In contrast, in ART, the environment surrounding gametes lacks the antioxidant system that is normally present in vivo, and accentuates their exposure to oxidative stress. Several studies have demonstrated the link between oxidative stress and the poor embryo quality in the in vitro culture system (Choi et al., Reference Choi, Banerjee, Agarwal, Falcone and Sharma2005; Tamura et al., Reference Tamura, Takasaki, Miwa, Taniguchi, Maekawa, Asada, Taketani, Matsuoka, Yamagata, Shimamura, Morioka, Ishikawa, Reiter and Sugino2008; Ahelik et al., Reference Ahelik, Mändar, Korrovits, Karits, Talving, Rosenstein, Jaagura, Salumets and Kullisaar2015; Prasad et al., Reference Prasad, Tiwari, Pandey, Shrivastav and Chaube2016). ROS concentration can be even greater due to a few external factors, namely culture medium, microfertilization techniques, oxygen levels and cryopreservation (Agarwal et al., Reference Agarwal, Said, Bedaiwy, Banerjee and Alvarez2006b; Leoni et al., Reference Leoni, Rosati, Succu, Bogliolo, Bebbere, Berlinguer, Ledda and Naitana2007; Pomeroy and Reed, Reference Pomeroy, Reed, Elder, van den Bergh and Woodward2015).
Oxidative stress in ART techniques: intracytoplasmic sperm injection (ICSI) and conventional IVF
IVF involves two main microfertilization techniques; conventional IVF and ICSI. Conventional IVF involves incubation of oocytes surrounded with cumulus cells and treated sperm in culture medium for fertilization, while ICSI consists in microinjection of one denuded oocyte with one spermatozoon to increase the chances of fertilization (Aboulghar et al., Reference Aboulghar, Mansour, Serour, Amin and Kamal1996; Yang et al., Reference Yang, Shahata, al-Bader, al-Natsha, al-Flamerzia and al-Shawaf1996; Dumoulin, Reference Dumoulin2000; van der Westerlaken et al., Reference van der Westerlaken, Helmerhorst, Dieben and Naaktgeboren2005).
The choice of the fertilization method (IVF or ICSI) is based on semen parameters, oocyte quality and the outcome of previous IVF attempt (Shuai et al., Reference Shuai, Ye, Huang and Xie2015). Conventional IVF is known as an effective technique for fertilization and successful embryo development in case of non-male infertility factors. Several studies have shown that conventional IVF is a better approach than ICSI when sperm concentration, motility and morphology are normal (Bhattacharya et al., Reference Bhattacharya, Hamilton, Shaaban, Khalaf, Seddler, Ghobara, Braude, Kennedy, Rutherford, Hartshorne and Templeton2001; Gennarelli et al., Reference Gennarelli, Carosso, Canosa, Filippini, Cesarano, Scarafia, Brunod, Revelli and Benedetto2019; Stimpfel et al., Reference Stimpfel, Jancar, Vrtacnik-Bokal and Virant-Klun2019). During the conventional IVF process, ROS can originate from dead spermatozoa and white blood cells of sperm. This is mainly why sperm preparation techniques and time of oocyte exposure to sperm are two key factors that minimize ROS damage during this process.
Sperm washing by centrifugation and swim-up allow the separation of mobile and normal spermatozoa from abnormal spermatozoa and allows a better selection before IVF (Sakkas et al., Reference Sakkas, Manicardi, Tomlinson, Mandrioli, Bizzaro, Bianchi and Bianchi2000; Hammadeh et al., Reference Hammadeh, Kühnen, Amer, Rosenbaum and Schmidt2001; Henkel and Schill, Reference Henkel and Schill2003; Ren et al., Reference Ren, Sun, Ku, Chen and Wu2004; Ricci et al., Reference Ricci, Perticarari, Boscolo, Montico, Guaschino and Presani2009; Jayaraman et al., Reference Jayaraman, Upadhya, Narayan and Adiga2012; Fácio et al., Reference Fácio, Previato, Machado-Paula, Matheus and Filho2016; Santiago-Moreno et al., Reference Santiago-Moreno, Esteso, Castaño, Toledano-Díaz, Delgadillo and López-Sebastián2017; Kocaman, Reference Kocaman2019; Rivera-Egea et al., Reference Rivera-Egea, Garrido, Varghese, Nagy, Varghese and Agarwal2019). Density gradient centrifugation was introduced to separate spermatozoa depending on chromosome packaging (Yamanaka et al., Reference Yamanaka, Tomita, Hashimoto, Matsumoto, Satoh, Kato, Hosoi, Inoue, Nakaoka and Morimoto2016) to select functional spermatozoa, while the swim-up method was designed to isolate highly motile sperm (Jayaraman et al., Reference Jayaraman, Upadhya, Narayan and Adiga2012; Beydola et al., Reference Beydola, Sharma, Agarwal, Rizk, Aziz, Agarwal and Sabanegh2014; Volpes et al., Reference Volpes, Sammartano, Rizzari, Gullo, Marino and Allegra2016). At first, centrifugation protocol was reported to increase ROS production in sperm and therefore alters its function (Shekarriz et al., Reference Shekarriz, DeWire, Thomas and Agarwal1995; Younglai et al., Reference Younglai, Holt, Brown, Jurisicova and Casper2001). However, many studies have improved sperm preparation techniques to identify which method has the best spermatozoa selection, with less iatrogenic consequences. Recently, research has revealed that density gradient centrifugation significantly ameliorated sperm quality in terms of morphology, motility and DNA fragmentation levels (Yu et al., Reference Yu, Zhou, Liu, Zheng, Jiang, Zhao, Xu and Huang2015; De Martin et al., Reference De Martin, Miranda, Cocuzza and Monteleone2019). It has also been suggested to combine both methods to minimize DNA fragmentation levels and give better results. Plus, iatrogenic damage can also be significantly reduced by adding albumin to the sperm pellet, after centrifugation (Volpes et al., Reference Volpes, Sammartano, Rizzari, Gullo, Marino and Allegra2016; Yamanaka et al., Reference Yamanaka, Tomita, Hashimoto, Matsumoto, Satoh, Kato, Hosoi, Inoue, Nakaoka and Morimoto2016).
Conventional IVF exposes oocytes to sperm in culture medium for fertilization. During the early stages, shortening the exposure time to a minimum was proposed to prevent oocyte damage caused by sperm toxicity. As gamete interaction occurs within 15 to 20 min in vitro, it has been established that the time of exposure gamete co-incubation can be reduced to at least 4 h to decrease ROS accumulation in culture medium during fertilization (Gianaroli et al., Reference Gianaroli, Fiorentino, Cristina Magli, Ferraretti and Montanaro1996; Coskun et al., Reference Coskun, Roca, Elnour, al Mayman, Hollanders and Jaroudi1998; Enkhmaa et al., Reference Enkhmaa, Kasai and Hoshi2009; Dai et al., Reference Dai, Qiao, Jin, Xin, Su, Sun and Chian2012; Zhang et al., Reference Zhang, Liu, Liu, Gao, Han, Xiong and Wu2013). However, other reports demonstrated that the duration of sperm–oocyte incubation does not significantly increase embryo viability (Swenson et al., Reference Swenson, Check, Summers-Chase, Choe and Check2000; Barraud-Lange et al., Reference Barraud-Lange, Sifer, Pocaté, Ziyyat, Martin-Pont, Porcher, Hugues and Wolf2008). These conflicting reports may be due to the continuous amelioration of sperm washing techniques that minimizes sperm toxicity levels and culture medium composition that prevents oocyte and embryo contamination (Lane and Gardner, Reference Lane and Gardner2007; Morbeck et al., Reference Morbeck, Krisher, Herrick, Baumann, Matern and Moyer2014; Fácio et al., Reference Fácio, Previato, Machado-Paula, Matheus and Filho2016; Morbeck et al., Reference Morbeck, Baumann and Oglesbee2017). Each IVF laboratory has its own manipulations techniques that can differently effect outcomes IVF.
ICSI was designed to overcome unexplained infertility, poor quality embryo retrieval, advanced maternal age, severe form of male infertility, (Feldman et al., Reference Feldman, Aizer, Brengauz, Dotan, Levron, Schiff and Orvieto2017), fertilization after in vitro maturation and fertilization of frozen–thawed oocytes (Luna et al., Reference Luna, Bigelow, Duke, Ruman, Sandler, Grunfeld and Copperman2011; Practice Committees of the American Society for Reproductive Medicine and Society for Assisted Reproductive Technology, 2012; Tannus et al., Reference Tannus, Son, Gilman, Younes, Shavit and Dahan2017). In this way, ICSI is an efficient technique that can prevent 30% of fertilization failures (Tournaye et al., Reference Tournaye, Verheyen, Albano, Camus, Van Landuyt, Devroey and Van Steirteghem2002; Gennarelli et al., Reference Gennarelli, Carosso, Canosa, Filippini, Cesarano, Scarafia, Brunod, Revelli and Benedetto2019). During ICSI procedures, cumulus cell removal can deprive oocytes from protection against irreversible oxidative damage, during maturation and fertilization. However, it is well established that naked oocytes have their own protective antioxidant system including SOD (Tatemoto et al., Reference Tatemoto, Sakurai and Muto2000; Cetica et al., Reference Cetica, Pintos, Dalvit and Beconi2001). Plus, culture medium supplementation with antioxidants largely contributes to oocyte protection against the deleterious effects of ROS accumulation in vitro (Deleuze and Goudet, Reference Deleuze and Goudet2010; Khazaei and Aghaz, Reference Khazaei and Aghaz2017; Truong and Gardner, Reference Truong and Gardner2017).
The shearing force resulting from spermatozoa microinjection into the plasma membrane can also be considered as stressful for the oocyte. Nevertheless, considering the very high frequency and success of ICSI (Suzuki and Perry, Reference Suzuki, Perry, Palermo and Sills2018; Fernández-González et al., Reference Fernández-González, Laguna, Ramos-Ibeas, Pericuesta, Alcalde-Lopez, Perez-Cerezales and Gutiérrez-Adan2019; Keating et al., Reference Keating, Cheung, Parrella, Xie, Rosenwaks and Palermo2019), this shearing force is negligible and seems to have no major effect on the microinjection outcomes. In fact, vigorous cytoplasmic aspiration can activate calcium release, which increases fertilization rate (Vanden Meerschaut et al., Reference Vanden Meerschaut, Nikiforaki, Heindryckx and De Sutter2014).
ART are routine practice of fertility centres worldwide. They are highly validated methods that ensure fertilization with minimum ROS damage, as studies continually propose updates to minimize the effect of ROS production on ART outcomes.
Oxidative stress in ART environment: culture medium and oxygen tension
The external environment that surrounds ART also plays an important role in the development of oxidative stress in vitro.
Oxygen tension
In the female reproductive tract, oxygen concentration varies between 2 and 5%. From the oocyte fertilization to the morula stage, the embryo metabolism is quiescent, which can explain the low oxygen tension requirement. However, as blastulation starts, a higher oxygen concentration is required, as has been shown that metabolic activity at that stage suddenly increases (Fischer and Bavister, Reference Fischer and Bavister1993; Ng et al., Reference Ng, Mingels, Morgan, Macklon and Cheong2018). Also, oxygen tension within the female reproductive tract has a considerable effect on endometrium receptivity that consequently interferes with implantation and pregnancy rates. For example, when the surface layer of the endometrium is thin, the implanting embryo is exposed to higher oxygen concentrations due to their close proximity to the spiral arteries (Nejat et al., Reference Nejat, Zapantis, Rybak and Meier2011).
In vitro, atmospheric oxygen tension is higher (20%), which is known to promote enzyme activity, leading to excess ROS production, namely O2−, H2O2 and hydroxyl radical (OH−) (Cetica et al., Reference Cetica, Pintos, Dalvit and Beconi2001). ROS accumulation in an environment that already lacks antioxidants can lead to oxidative stress and be responsible for DNA fragmentation and apoptotic cell death (Hockenbery et al., Reference Hockenbery, Milliman and Korsmeyer1994; Yang et al., Reference Yang, Hwang, Kwon, Kim, Choi and Oh1998; Agarwal et al., Reference Agarwal, Saleh and Bedaiwy2003; Agarwal et al., Reference Agarwal, Gupta and Sikka2006a). From that point of view, a few studies have reported various results by comparing the effects between high and low oxygen tension on oocyte maturation, embryo development and blastulation.
During in vitro maturation, it has been reported that high oxygen tension alters the expression of genes that are related to oocyte competence and glucose metabolism (Catt and Henman, Reference Catt and Henman2000). In contrast, it also has been established that atmospheric oxygen tension can be beneficial for porcine oocytes, leading to greater blastomere number and better blastulation (Park et al., Reference Park, Hong, Yong, Hwang, Lim and Lee2005). Other studies showed that lower oxygen tension (5%) is favourable for oocytes maturation in pig, mice, cow (Goto et al., Reference Goto, Noda, Narimoto, Umaoka and Mori1992; Dumoulin et al., Reference Dumoulin, Meijers, Bras, Coonen, Geraedts and Evers1999; Hashimoto et al., Reference Hashimoto, Minami, Yamada and Imai2000b; Iwamoto et al., Reference Iwamoto, Onishi, Fuchimoto, Somfai, Takeda, Tagami, Hanada, Noguchi, Kaneko, Nagai and Kikuchi2005) and humans (Catt and Henman, Reference Catt and Henman2000; Kovačič et al., Reference Kovačič, Sajko and Vlaisavljević2010; Kasterstein et al., Reference Kasterstein, Strassburger, Komarovsky, Bern, Komsky, Raziel, Friedler and Ron-El2013; Kirkegaard et al., Reference Kirkegaard, Hindkjaer and Ingerslev2013).
Experimental studies on oxygen tension and embryo development had similar observations and all reached the same conclusion: lower oxygen tension leads to lower ROS accumulation in the cytoplasm, namely O2− (Noda et al., Reference Noda, Matsumoto, Umaoka, Tatsumi, Kishi and Mori1991; Hashimoto et al., Reference Hashimoto, Minami, Takakura, Yamada, Imai and Kashima2000a) and then higher quality developing embryos (Goto et al., Reference Goto, Noda, Mori and Nakano1993; Noda et al., Reference Noda, Goto, Umaoka, Shiotani, Nakayama and Mori1994, Kwon et al., Reference Kwon, Yang, Hwang, Yoo, Kim, Lee, Ryu and Oh1999; Catt and Henman, Reference Catt and Henman2000; Karja et al., Reference Karja, Wongsrikeao, Murakami, Agung, Fahrudin, Nagai and Otoi2004; Corrêa et al., Reference Corrêa, Rumpf, Mundim, Franco and Dode2008). In many mammalian species, such as pig, mice (Goto et al., Reference Goto, Noda, Mori and Nakano1993; Kwon et al., Reference Kwon, Yang, Hwang, Yoo, Kim, Lee, Ryu and Oh1999) and humans (García-Martínez et al., Reference García-Martínez, Sánchez Hurtado, Gutiérrez, Sánchez Margallo, Romar, Latorre, Coy and López Albors2018; Van Montfoort et al., Reference Van Montfoort, Arts, Wijnandts, Sluijmer, Pelinck, Land and Van Echten-Arends2020), when preimplantation embryos are exposed to atmospheric oxygen, H2O2 concentration increases, releasing highly reactive OH− in the cytoplasm (Kitagawa et al., Reference Kitagawa, Suzuki, Yoneda and Watanabe2004). The resulting oxidative stress in embryos (Fukui et al., Reference Fukui, McGowan, James, Pugh and Tervit1991; Yuan et al., Reference Yuan, Van Soom, Coopman, Mintiens, Boerjan, Van Zeveren, De Kruif and Peelman2003) causes cell injury, and embryo fragmentation (Yang et al., Reference Yang, Hwang, Kwon, Kim, Choi and Oh1998). Oxidative stress also alters the expression of genes that are involved in embryo quality (Corrêa et al., Reference Corrêa, Rumpf, Mundim, Franco and Dode2008; Kovačič, Reference Kovačič2012) by affecting genes transcription (Adler et al., Reference Adler, Yin, Tew and Ronai1999; Lim and Luderer, Reference Lim and Luderer2011). At the blastocyst stage, embryos consume more oxygen than at the cell division stage, which might explain why some studies found no difference in bovine blastocyst quality, when cultured under 20%, 5% or 2% O2 (Harvey et al., Reference Harvey, Kind, Pantaleon, Armstrong and Thompson2004; Corrêa et al., Reference Corrêa, Rumpf, Mundim, Franco and Dode2008; Bermejo-Álvarez et al., Reference Bermejo-Álvarez, Lonergan, Rizos and Gutiérrez-Adan2010; Nanassy et al., Reference Nanassy, Peterson, Wilcox, Peterson, Hammoud and Carrell2010; De Munck et al., Reference De Munck, Janssens, Segers, Tournaye, Van De Velde and Verheyen2019). But ‘no difference’ does not necessarily mean that atmospheric oxygen tension is beneficial for IVF outcomes (Kea et al., Reference Kea, Gebhardt, Watt, Westphal, Lathi, Milki and Behr2007; Kovačič and Vlaisavljević, Reference Kovačič and Vlaisavljević2008; De Los Santos et al., Reference de los Santos, Gámiz, Albert, Galán, Viloria, Pérez, Romero and Remohï2013). The studies that have been done above the normal hypoxic conditions for oocyte and embryo development have no physiological relevance, but they still demonstrate the importance of low oxygen tension during incubation in IVF programmes.
According to laboratory experiments during these last years, it is well established that oxygen tension at low level (5%) is required to minimize oxidative stress damage in vitro. Still, the difference between the high oxygen tension effect on each of the preimplantation phase, as the fertilization, cell division, compaction and blastulation, remains to be established (Nastri et al., Reference Nastri, Nóbrega, Teixeira, Amorim, Diniz, Barbosa, Giorgi, Pileggi and Martins2016).
Culture medium
Embryonic culture medium is an important factor in IVF outcomes, given the continuous contact with oocytes, from fertilization to embryo transfer. ROS are present in culture medium, whether from the medium composition or the embryonic metabolism, resulting in oxidative stress in the culture environment (Goto et al., Reference Goto, Noda, Mori and Nakano1993; Kitagawa et al., Reference Kitagawa, Suzuki, Yoneda and Watanabe2004; Dalvit et al., Reference Dalvit, Cetica, Pintos and Beconi2005; Combelles et al., Reference Combelles, Gupta and Agarwal2009).
During embryonic metabolism in vitro, ROS levels can result from glucose produced by glycolysis and oxidative phosphorylation or from lipid peroxidation in the cell membrane (Agarwal et al., Reference Agarwal, Saleh and Bedaiwy2003; Martín-Romero et al., Reference Martín-Romero, Miguel-Lasobras, Domínguez-Arroyo, González-Carrera and Alvarez2008), leading to intracellular GSH deficiency (Hashimoto el al., Reference Hashimoto, Minami, Takakura, Yamada, Imai and Kashima2000a). Culture medium supplementation with antioxidants (SOD, CAT, serum albumin) (Agarwal et al., Reference Agarwal, Gupta and Sikka2006a) can contribute to the regulation of GSH synthesis in the redox environment, as has been shown in the bovine and murine oocytes models (Goto et al., Reference Goto, Noda, Narimoto, Umaoka and Mori1992; Lane et al., Reference Lane, O’Donovan, Squires, Seidel and Gardner2001). More interestingly, GSH originates from the precursor amino acids, cysteamine and cysteine, Cysteamine elevates intracellular cysteine levels that lead to GSH production. Therefore, culture medium supplementation with these precursor amino acids maintains the cellular GSH system and consequently contributes to ROS regulation (Sovernigo et al., Reference Sovernigo, Adona, Monzani, Guemra, Barros, Lopes and Leal2017). Antioxidant supplementation is also known to reduce ROS levels by increasing GSH concentration (Mukherjee et al., Reference Mukherjee, Malik, Saha, Dubey, Singhal, Boateng, Saugandhika, Kumar, De, Guha and Malakar2014; Jeong and Joo, Reference Jeong and Joo2016). However, in a recent study, while adding quercetin, resveratrol, cysteamine or vitamin C to culture medium during oocyte maturation, no increase in GSH levels was observed. According to the research, this may be due to oxidation of antioxidant that interacts with ROS (Sovernigo et al., Reference Sovernigo, Adona, Monzani, Guemra, Barros, Lopes and Leal2017). GSH regulation in oocytes and embryos mainly depends on the available precursor amino acids and also on the antioxidants supplementation. However, adding supplements to IVF culture medium is not always beneficial and their concentration needs to be carefully defined. For example, culture medium supplementation with proteins, such as albumin, acts as a potent antioxidant. Its binding sites catch toxic molecules (such as heavy metals, lipoprotein) and contribute to the inhibition of lipid peroxidation. Nevertheless, the high levels of amine oxidase in serum albumin can increase H2O2 concentration (Market-Velker et al., Reference Market-Velker, Denomme and Mann2012).
The heavy metals contained in culture medium (Fe2+, Ca2+) favour ROS generation via the Fenton reaction, consisting of H2O2 decomposition (Guérin et al., Reference Guérin, El Mouatassim and Ménézo2001; Agarwal et al., Reference Agarwal, Gupta and Sikka2006a). In this way, chelating molecules such as ethylenediaminetetraacetic acid (EDTA), EGTA, vitamin E, vitamin C and transferrin are added to culture medium to lower ROS production, namely H2O2 and OH− (Lane et al., Reference Lane, O’Donovan, Squires, Seidel and Gardner2001). EDTA acts as a ligand that isolates ‘metal ions’ and blocks the ion-driven reactions such as the Fenton reaction (Martín-Romero et al., Reference Martín-Romero, Miguel-Lasobras, Domínguez-Arroyo, González-Carrera and Alvarez2008; Gruber and Klein, Reference Gruber and Klein2011).
Growth factors have also been reported to moderate the deleterious effect of ROS (Hardy, Reference Hardy1999; Spanos et al., Reference Spanos, Becker, Winston and Hardy2000; Kurzawa et al., Reference Kurzawa, Glabowski, Baczkowski, Wiszniewska and Marchlewicz2004; Chronopoulou and Harper, Reference Chronopoulou and Harper2015; Goel et al., Reference Goel, Goel, Bhatia and Kharche2017). Adding EGF and IGF-1 to IVF culture medium moderates ROS accumulation, improves fertilization, oocyte fertilization and embryo development until the blastulation stage in cats (Thongkittidilok et al., Reference Thongkittidilok, Tharasanit, Songsasen, Sananmuang, Buarpung and Techakumphu2015), bovines (Koçyiğit et al., Reference Koçyiğit, Çevik, Sen and Kuran2015; Arat et al., Reference Arat, Caputcu, Çevik, Akkoc, Cetinkaya and Bagis2016) and humans (Spanos et al., Reference Spanos, Becker, Winston and Hardy2000; Kurzawa et al., Reference Kurzawa, Glabowski, Baczkowski, Wiszniewska and Marchlewicz2004).
According to these studies, it is not negligible to take into consideration the composition of the culture medium and their effects on embryonic oxidation. Indeed, under poor growing conditions, oxidative stress causes a change in the ratio of intracellular/extracellular ROS concentration (Martín-Romero et al., Reference Martín-Romero, Miguel-Lasobras, Domínguez-Arroyo, González-Carrera and Alvarez2008), which can damage key cellular organelles that are required for rapid cell division, cause embryo fragmentation (Agarwal et al., Reference Agarwal, Gupta and Sharma2005; Bedaiwy et al., Reference Bedaiwy, Mahfouz, Goldberg, Sharma, Falcone, Abdel Hafez and Agarwal2010), mitochondrial DNA disruption via lack of histones (Kowaltowski and Vercesi, Reference Kowaltowski and Vercesi1999), apoptosis and embryo failure (Kurzawa et al., Reference Kurzawa, Glabowski, Baczkowski, Wiszniewska and Marchlewicz2004). In this way, it has been found that when ROS levels in culture medium are high, embryo fragmentation levels increase accordingly, which is known to affect embryo development (Bedaiwy et al., Reference Bedaiwy, Falcone, Mohamed, Aleem, Sharma, Worley, Thornton and Agarwal2004; Bedaiwy et al., Reference Bedaiwy, Mahfouz, Goldberg, Sharma, Falcone, Abdel Hafez and Agarwal2010; Lee et al., Reference Lee, Lee, Liu, Tsao, Huang and Yang2012).
The embryonic metabolism and the composition of the culture medium combined, are a source of excess ROS, which can be destructive for embryonic quality and, consequently, the implantation rate in medically assisted procreation (PMA). This is why the culture medium must contain components that minimize the accumulation of ROS, while maintaining an optimal physiological rate.
Cryopreservation
Gametes and embryo cryopreservation are a part of the routine procedures in human assisted reproduction (Konc et al., Reference Konc, Kanyó, Kriston, Somoski and Cseh2014). The cryopreservation process can include a slow freezing protocol or vitrification. Successful gamete and embryo cryopreservation depends on many factors including the solution in which gametes are suspended, the cryoprotectant concentration, the rate at which gametes are cooled and the condition under which the cryoprotectants are removed after thawing (Singh et al., Reference Singh, Mal, Gautam and Mukesh2019).
Slow freezing techniques have been shown to preserve embryo viability but, more recently, vitrification is the method of choice of oocyte and embryo cryopreservation in clinical ART. (Levi-Setti et al., Reference Levi-Setti, Patrizio and Scaravelli2016). Vitrification is a rapid process of cooling oocytes and embryos to cryogenic temperatures (from 37°C to −196°C in less than 1 s) without a detrimental phase of transition of liquid to solid ice (Balaban et al., Reference Balaban, Urman, Ata, Isiklar, Larman, Hamilton and Gardner2008; Saragusty and Arav, Reference Saragusty and Arav2011). This method is known to preserve cellular viability by raising intracellular and extracellular viscosity, leading to the arrest of all molecular diffusion and chemical processes (Wong et al., Reference Wong, Mastenbroek and Repping2014).
During the last few years, cryopreservation procedures have shown positive results in improving IVF. However, it remains a process that disrupts the cellular homeostasis of gametes and embryos (Len et al., Reference Len, Koh and Tan2019). According to a few studies, gamete and embryo freezing is still likely to induce oxidative damage. Either through slow freezing or vitrification, cryopreservation has been associated with an increase in H2O2 in mouse embryos and pig oocytes, in addition to a concomitant decrease in GSH concentration (Somfai et al., Reference Somfai, Ozawa, Noguchi, Kaneko, Karja, Farhudin, Dinnyés, Nagai and Kikuchi2007; Gupta et al., Reference Gupta, Uhm and Lee2010). Sperm cryopreservation also showed significant spermatozoa damaging effects. A decrease in sperm motility, an elevated rate of DNA fragmentation and oxidative damage have been observed after sperm thawing. These deleterious effects have been justified by the elevated sensitivity of sperm to lipid peroxidation and the limited amounts of antioxidants available due to seminal plasma removal (Zribi et al., Reference Zribi, Feki Chakroun, El Euch, Gargouri, Bahloul and Ammar Keskes2010).
In conclusion, despite the revolution of cryopreservation techniques in clinical ART, more research is needed to have a better understanding of the cellular effects of freezing/thawing procedures and ameliorate post-thaw gamete and embryo intracellular viability.
Conclusion
Accumulation of intracellular/extracellular ROS (oxidative stress) during oocyte maturation and embryo development can be detrimental to fertilization and to implantation success rate. However, according to most studies carried out over recent years, it is clear that the effect of ROS on normal embryonic development varies according to the stage and the conditions of development. Indeed, whether in vivo or in vitro, variations in embryonic sensitivity to oxidative stress make an accurate ROS level have a positive effect at a specific stage and a negative effect at another stage of development. These observations can only highlight the complexity of the regulatory role of ROS in the process of embryonic development. Therefore it is necessary to analyze each phase of development, for a better understanding of the phases that are dependent on the regulating ROS and subsequently a better optimization of the conditions of embryonic development in vitro.
Financial Support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Conflicts of Interest
None.
Ethical Standards
Not applicable
