HJOG 2021, 20 (2), 45-52 | doi: 10.33574/hjog.0045
Sofoklis Stavros1,2, Antonios Koutras2, Thomas Ntounis2, Konstantinos Koukoubanis2, Theodoros Papalios2, Despoina Mavrogianni1, Peter Drakakis1,2
1Molecular Biology Unit, Division of Human Reproduction and Recurrent Abortions, 1st Department of Obstetrics and Gynecology, National and Kapodistrian University of Athens, Athens, Greece
21st Department of Obstetrics and Gynecology, National and Kapodistrian University of Athens, Alexandra Hospital, Athens, Greece
Correspondence: Sofoklis Stavros MD, PhD, OB/GYN, Fellow of the 1st Department of Obstetrics and Gynecology, National and Kapodistrian University of Athens, Alexandra Hospital General Hospital of Athens “Alexandra”, Lourou and Vasilissis Sofias Ave, 11528 Athens, Greece, e-mail: email@example.com
Oxidative stress may play a role in implantation failure on multiple levels. Oxidative stress is found widely in several biological systems, as well as it acts on various molecular levels with different mechanisms. It has been shown that it is rather the disequilibrium between reactive oxygen species causing oxidative stress and antioxidant mechanisms counteracting their effects, than reactive oxygen species levels themselves. Reactive oxygen species play a role in implantation and fertilisation by acting on different levels of embryo-formation and endometrial changes. Additionally, it is widely abundant in the female reproductive tract including ovaries, oocytes, tubal as well as follicular fluid. Moreover, it has been shown that male fertility is affected by reactive oxygen species by determining sperm quality. Last but not least, oxidative stress may affect IVF indirectly through its actions on peritoneal fluid. As long as research studies on elucidating the development of oxidative stress markers on patients undergoing IVF continue, ever more new possibilities emerge on predicting the pregnancy outcome.
Keywords: Oxidative stress, abortion, recurrent pregnancy loss, reactive oxygen species, antioxidants, fetal development
The biological efficacy of human reproduction is not considered of highly efficacy. About 30-50% of first trimester conceptions will spontaneously abort, with the high majority of cases to be lost at the time of implantation. Furthermore, spontaneous abortions account for 15-20% of clinical pregnancies. 1
Oxidative stress is a clinical situation in which there is a disequilibrium between local antioxidant defense mechanisms and free radical production. This clinical entity causes un unhospitable hypoxic environment in the physiology of the female reproductive system, due to overproduction of reactive oxygen species (ROS), with or without diminished antioxidant system function. 2,3 Many hypotheses implicate oxidative stress and stress-induced placental dysfunction in idiopathic recurrent pregnancy loss, spontaneous abortion, defective embryogenesis, hydatidiform mole and drug-induced teratogenicity. Moreover, in the antiphosholipid syndrome there is a link between the creation of antiphospholipid antibodies with the modification of phospholipids caused by oxidative stress. 1
In order to achieve a successful pregnancy, it is appropriate to have a specific balance between the oxidative and inflammatory processes, as well as their interaction must be certain and precise. With the definition successful pregnancy, we order the pregnancy, which has the potentials to begin and being maintained. Impairment of these systems and their associations reportedly may lead to spontaneous abortion and implantation failure. 3,4 Problems upon which pathophysiology has yet to shed light, are recurrent pregnancy losses (RPL) or repeated implantation failures (RIF), for which a specific success cannot yet to be promised in the management process. RPL has an incidence of 0,5-3% in reproductive aged women, of which 50-60% are idiopathic, which creates an exasperating clinical problem. 1
RIF is associated with three repeated failed implantation attempts of good quality embryos in vitro fertilization (IVF). Causative factors of implantation failure in IVF may be of uterine, male, or embryo etiology, as well as the specific IVF protocol chosen. 5 Advanced maternal age, elevated body mass index, smoking, levels of stress, immunological factors, as well as any infectious organisms in the uterus causing chronic endometritis and anatomic anomalies, consist multiple risk factors for recurrent implantation failure. 6
In a lot of studies, although both the immune system and the oxidative processes, possessing each different pathophysiologic mechanisms, may implicate in RPL and RIF. New trends in studies include immune-modulatory treatment, anti-oxidant therapy and anti-inflammatory factors. 7,8
Moreover, oxidative stress not only does it play a key role in pregnancy success, but also it seems that it gets increased with repeated failures of reproduction. 1,5,9 This review aims to investigate the link between oxidative stress spontaneous abortion and recurrent pregnancy loss due to implantation failure in IVF. Additionally, the evaluation of the potential therapeutic targets concerning recurrent pregnancy loss and repeated implantation failure cases due to the inflammatory response are being examined in this review, as well as the prevention of preventable causes.
The role of antioxidant vitamins for primary prevention of oxidative stress-induced pathologies needs to be investigated further.
Oxidative stress causing implantation failure in IVF
Development abnormalities and embryo fragmentation and formation has been found to be implicated with reactive oxygen species, which may lead to recurrent miscarriage and spontaneous abortion. Additionally, the level of success in in vitro fertilization is depended on reactive oxygen species overproduction, which seems to play a key role. 10 Oxidative stress cause implantation failure in IVF by acting on different levels.
Concerning the role in endometrial changes, oxidative stress cause alterations in ROS and superoxide dismutase, which may cause endometrial break down in late secretory phase. Due to the reported endometrial reduced SOD concentrations and increased concentrations of lipid peroxide in the late secretory phase. 11 It has been reported that there is an increase in the production of F2a prostaglandins caused by reactive oxygen species, which takes part into the endometrial shedding. 12 It has been shown that increased reactive oxygen species concentration is due to reduction in superoxide dismutase caused by prostegeronoestrogen withdrawal taking part to endometrial sending. The mechanism by which endometrial sending occurs is the increase of prostaglandin F2a production as a result of cyclooxygenase-2 expression stimulation, induced by NF-KB activation from reactive oxygen species. 13 Although it is mid- secretory phase concentration of lipid peroxide in the endometrium in early pregnancy other than late secretory phase. 11
The location of ROS in the female reproductive tract is very diverse and significant and includes oocytes, embryos, tubal fluid as well as follicular fluid, as many animal and human studies are referred to it. 14–16 This is due to the fact that a number of different cells are in line of production of reactive oxygen species in the ovaries such as endothelial cells, phagocytic macrophages and steroeidogenic parenchymal cells. 17 Moreover, throughout the whole menstrual cycle, SOD presence is confirmed with an increased concentration especially in early pregnancy. 11 The mechanism by which, reactive oxygen species act, is by causing peroxidative damage in targeted cell macromolecules such as nucleic acids, lipids and proteins. 18
Essential for fertilization are the normal cellular mechanisms, which are disturbed by the lipid peroxidation. This is due to the fact that reactive oxygen species target the double bonds between carbon atoms in polyunsaturated fatty acids found in lipids, causing disturbances in the function of ion channels and membrane enzymes of biological membranes. 19
Mitochondria in oocytes and embryos are another domain that is prone to be affected by oxidative stress. 20 Mitochondria are essential for the normal function of cells, such as any disturbances to their metabolic activities, may cause problems (generation of ATP). Additionally, reactive oxygen species are mainly produced in mitochondria. Because histones are not found in mitochondrial DNA, this makes mitochondria susceptible to injury caused by oxidative stress, which leads to the leakage of ROS into the cytoplasm. 21
Follicular fluid has been found to contain many ROS and antioxidants, with the late protecting oocytes from damage induced by ROS. 22–26 Fertility and normal oocyte development as well as membranes, cytoskeleton and oocyte’s DNA are impaired by a disregulation in the anti-oxidant peroxidant system. In women undergoing IVF of patients with unexplained fertility, in contrast with those with male infertility factor and tubal infertility, has been found that there were lower selenium follicular concentrations. 22 Failed fertilization of oocytes follicles contained less GSHPx activity than those that they were apparently fertilized. Thus, this indicates fertilisability is depended on the presence of oxidative stress on the environment of oocytes as well as the reduction of the antioxidant activity. 22
In patients undergoing IVF there are many oxidative stress markers, such as lipid hydroperoxides, thiobarbituric acid-reactive substances and conjugated dienes, both found in serum and follicular fluid. Moreover, follicular fluid had less of these oxidative stress markers than serum indicating probably the protective mechanism of oocytes by higher concentrations of antioxidants. 14 Nevertheless, the pregnancy outcome is yet to be proven related with these oxidative stress markers. Another study indicates a positive relation between reactive oxygen species and pregnancy outcome in IVF patients, although the reference value of ROS concentration in the follicular fluid is yet to be established. 15 According to Oyawoye et al., who measured the non-enzymatic antioxidant role in follicular fluid of women undergoing IVF, successfully fertilized oocytes have higher baseline concentrations of TAC. Embryos that had the possibility of surviving transfer, obtained lower TAC concentrations in their follicles. 23
Sperm function could be negatively influenced by peritoneal fluid. Thus, making peritoneal fluid essential for early embryonic development and normal fertilization. 24 There is a balance between antioxidants and reactive oxygen species concentrations in the peritoneal fluid, keeping ROS within physiologic range. Spermatozoa, ova as well as zygote/embryo are damaged by higher concentrations of ROS in the peritoneal fluid. Although, lower concentrations of ROS are essential for the fusion process between sperm and oocyte. 18 The above mentioned is a key element due to the fact that peritoneal fluid is in continuous contact with tubal fluid, thus an imbalance between peroxidant and antioxidant levels, may damage early stage embryos within the fallopian tubes.
Fragmentation and unequal division may lead to abnormally appeared embryos, providing only a limited number of good quality embryos in IV conditions. 25 There is a hypothesis that high levels of oxygen concentration in in vitro culture conditions may lead to defective embryos. Because embryonic development may be retarded or locked by ROS acting on embryo’s cellular molecules. 26 It is evident supported that embryos themselves produce ROS from their embryo metabolism as well as from the surrounding environment. 27,28
Embryo cells and the external environment may be the source of reactive oxygen species. ROS could be generated by a preimplantation embryo and involves procedures like NADPH oxidase, xanthine oxidase and oxidative phosphorylation. Assisted reproduction is depended on exogenous sources of ROS. There has been reported that in in vitro conditions concentrations of ROS are bigger than those in in vivo conditions. 28 ROS concentrations can be affected by many environmental conditions, such as metallic ions, visible light, oxygen concentration and amine oxidases from dead spermatozoa, from which spermine and spermidine are been catabolized by their latter into hydrogen peroxide and other products. The atmospheric oxygen concentration is higher than the oxygen concentration in the Fallopian tubes. Under the condition of atmospheric oxygen concentration in mouse embryos, the production of ROS is augmented. 28 When the oxygen concentration is diminished, there is an enhanced embryo development in the mouse.
In order to be protected from the oxidative stress and the surrounding environment, embryos have a lot of protective procedures. 29,30 Non-enzymatic antioxidants like Vitamin C, glutathione, hypotaurine and taurine are being contained into the environment surrounding the oocyte and the embryo and they protect it from external sources of ROS. SOD, catalase and glutathione peroxidase are internal antioxidants protecting against ROS. 31
The key cellular organelle, which is demanded for rapid division of cells, is affected by ROS, delaying embryo development. Microtubules and membrane fluidity are the main factors in which embryo cleavage depends on and any enquiries in these factors could delay or stop embryo development. 21 ROS affects embryos with another procedure called apoptosis. In non-fragmented embryos, hydrogen peroxide concentrations are lower than in fragmented embryos (72,21 versus 31,30). Embryo fragmentation could be caused by higher doses of hydrogen peroxide, while apoptosis has been seen only in fragmented embryos. 32
The role of ROS in embryo development has been studied, indicating that in patients undergoing ICSI, fertilization and blastocyst development have shown an inverse correlation in ROS concentrations in day 1 culture media. 33 Lower pregnancy rates in IVF and ICSI cycles were connected with augmented concentrations of ROS in day 1 culture media. A predicting factor for fertilization, embryo development and pregnancy, would be ROS concentrations in day 1 culture media. In contrast to other studies, there has been remarked that the true outcome of a patient is influenced by ROS and not only fertilization and embryo development. Abundant number of cell mas, oocytes as well as spermatozoa used in IVF may be ROS sources. 34 On the other hand, onan ICSI, which is free of cumulus cells, only spermatozoa as well as oocytes are ROS potential cellular sources, leading to the conclusion that spermatozoa are the main source of exogenous ROS. Since though it is well known that ICSI is affected by ROS, even when a single spermatozoon used, indicates that may be potentially other sources of ROS. There are contradicting results over investigations, whether duration of insemination time used in IVF laboratories play a role in oxidative stress damage, due to the assumption that longer exposure time for spermatozoa and oocytes, the higher the risk of oxidative stress damage. Some studies indicate that more beneficial effects in IVF have been shown, with reduced gamete insemination time 32, whilst other studies indicate no correlation between them. 27
Female reproductive tract and oxidative stress management
It has been reported by several studies, both in-vivo and in-vitro, that the quality of semen as well as male fertility show significant improvement with the use of antioxidants in infertile patients 34. This leads to the assumption that beneficial effects of antioxidant use may be shown in female infertility as well, although only few studies are found in this filed. Supplementation of vitamin C with 750mg per day was evaluated in a multi-center randomized controlled study of luteal phase defected patients. It was reported that in the treatment group, compared to the control group, were a significant elevation of serum progesterone concentration, in addition to higher pregnancy rate [25% (19/76) versus 11% (5/46)]. 21 Another study reporting that lower antioxidant concentrations were found in healthy women with luteal phase defect and recurrent miscarriages history, support the above-mentioned findings. Moreover, an additional study indicated that vitamin C supplemented patients, compared to that of the controls, reported to have higher follicular fluid concentrations of vitamin C. In the same study, although without statistical significance, the supplemented group of patients was also reported to achieve higher pregnancy rates. 35 In contrast, it should be outlined that vitamin C as well as other antioxidants may be considered as pro-oxidants in higher concentrations. Animal studies shown impaired uterine and ovarian female function as well as reproductive fitness side effects after oral administration of antioxidants, even though other studies show improvement on the quality and the number of oocytes in addition to repel of female aging effects. 36
Female infertility and the use of antioxidants, as well as the type, dose and duration of antioxidants usage, is still a field which needs further investigation. Primarily before implicating any treatment plan, ROS source should be identified and eliminated, either medically or surgically, soon after the diagnosis of oxidative stress is made. It should be noted that more often it is the increase in the production of ROS rather than antioxidant elimination that cause oxidative stress.
Failure of implantation in IVF due to oxidative stress is a challenging and complex problem for both clinicians and researchers, because it does not enquire a holistic and standardized approach. Maybe the best option is personalized medicine depending on both the etiology and the special characteristics of each patient, due to the fact that it plays a key role in several biological and molecular mechanisms on patients undergoing IVF. Not only do reactive oxygen species provide a field for treatment options, but also a preliminary evaluation of every individual couple, which can be implemented by designing new studies and establishing new IVF protocols.
Conflict of interest
The authors declare that they have no conflict of interest.
All authors contributed equally to the preparation and drafting of manuscript.
The authors are grateful to all who provided assistance during the preparation of this manuscript.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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