https://orcid.org/0000-0001-9183-6206
ABSTRACT
Over the last forty years, many trials have been performed using mammalian embryo cultures with reduced oxygen tension (O2) to encourage proper embryo development and increase the success rate for in vitro fertilization (IVF) outcome. Even if the use of atmospheric O2 (20%) affects in vitro embryo development and intracellular redox balance, the use of low (5% O2, physiologic) and ultra-low (close or less to 5% O2) O2 applied to in vitro embryo culture is still under debate. Numerous studies in various mammalian species have shown that embryo development improves when culturing embryos under low O2, although culture conditions are not the only factors involved in the success of IVF. This article reviews the literature data of the last four decades and discusses the current evidence on the use of low and ultra-low O2 in embryo culture, and examines the impact of multiple factors on IVF outcomes.
Abbreviations
O2: oxygen tension; IVF: in vitro fertilization; IVC: in vitro culture; ET: embryo transfer; ROS: reactive oxygen species; ARTs: assisted reproductive technologies.
In vitro fertilization (IVF) in past years: An approach of embryo transfer
Until 1978, women who had no functioning Fallopian tubes were considered sterile. These patients usually underwent reparative surgery or tuboplasty in order to re-establish the normal strait for gametes to transit. Unfortunately, these surgeries often failed (Steptoe and Edwards Citation1976). In the 1970s, the fertilization of oocytes outside the human body (a process known as in vitro fertilization or IVF) was considered experimental, and when attempted, resulted only in miscarriages or unsuccessful extrauterine pregnancies (Steptoe and Edwards Citation1976). In 1978, Lesley Brown, the first patient to undergo IVF, underwent a laparoscopic egg retrieval without medications to stimulate her ovaries. The single egg retrieved was fertilized in the laboratory, and then transferred into her uterus. Steptoe and Edwards (Citation1978) successfully obtained the first live birth from IVF.
From this success forwards, IVF became a medical treatment for infertility. The controlled ovarian hyperstimulation usually takes place by using a combined administration of gonadotropin-releasing hormone agonists or antagonists and gonadotropins. Oocytes are aspirated through transvaginal or transabdominal ultrasound-guided aspiration of follicles and fertilized in vitro. One or more embryo(s) are then transferred into the uterus. This procedure occurs over an approximately two-week interval of time and is called the IVF cycle (Glujovsky et al. Citation2012). The embryos obtained are usually transferred into the uterus at the second (E2) or third (E3) day of the in vitro culture (IVC), approximately corresponding to the 4–8 cell stage, i.e., when the receiving uterus can provide the best environment for embryo development (Laverge et al. Citation2001). However, recent evidence indicates that human embryos at the fifth (E5) or sixth (E6) day of culture (i.e., 64-cell or blastocyst stage) are at the best biological stage to be transferred to the uterus because the extended culture may give an improved chance to select high-quality embryos (Glujovsky et al. Citation2012).
In fresh human IVF, the transfer of E5 embryos improved the ongoing clinical pregnancy rates up to 43.1% from 24% for E3 transfer embryos in female patients of 35 years with optimal rates of fresh IVF clinical pregnancy, ongoing pregnancy and cumulative ongoing pregnancy (Fernández-Shaw et al. Citation2015). Furthermore, not only pregnancy, but also successful implantation rates, increased to 33.3% with E5 embryo transfer (ET) (Aziminekoo et al. Citation2015). Regarding vitrified/warmed embryos, an improvement in clinical outcomes resulted after E5 ET with delayed expansion or blastulation, when the prolonged culture was applied (Wirleitner et al. Citation2016). Embryologists are now concerned about whether it is more beneficial for women undergoing IVF to receive a fresh or frozen embryo based on if they are classified as low or non-responders (Acharya et al. Citation2018). Recently, Zhao et al. (Citation2018) showed that the extended culture of E3 embryos for 7–8 h reduced the risk of IVF-ET treatment if compared to E5 embryo culture through another 2–3 days, and improved the clinical outcomes and the efficiency of each transferred cycle and each transferred embryo.
However, it is not enough for the positive outcomes of IVF to only consider the above factors, such as the day, fresh or frozen embryo, and the benefits of the prolonged culture on IVF-ET. At present, many factors should be considered that could contribute to the success of IVF, including the importance of different oxygen tension (O2) usage in the culture media of IVC in the mammalian embryo, either in clinical or experimental IVF, combined with more strict conditions that can improve embryo viability, development, and quality. On one hand, De Munck et al. (Citation2019) did not observe any difference in embryo viability in embryos cultured under low O2 when levels were reduced from 5% to 2% O2 in human embryo extended culture after E5. On the other hand, Gelo et al. (Citation2019), when culturing embryos under low O2, a high number of blastocysts leading to increased pregnancy rates, thus providing a better alternative for human embryos selection was observed.
Low and ultra-low oxygen tensions in embryo culture: A crucial stage in the IVF process
In 1971, simultaneously with the development of IVF treatment, Patrick Steptoe and collaborators noticed the first efficient culture of a human embryo from E2 to E5 and clearly described the culture conditions facilitating this breakthrough. Specifically, the scientist underlined that the O2 in the culture system was not approximately 20% (atmospheric O2), but that the gas phase was 5% O2 (physiologic O2), 5% carbon dioxide, and 90% nitrogen (Steptoe et al. Citation1971). During this period, in human IVF treatments, both 20% and 5% O2 were widely used in embryo culture and showed similar successful developmental rates. However, culturing human embryos under physiologic conditions, in comparison to atmospheric O2, was not beneficial. Although different O2 levels did not show an improvement in the embryo culture and development, Bavister and Minami (Citation1986) used an IVC embryo approach, including oviductal mouse cells in a culture system. In particular, they cultured in vitro hamster zygotes within the explanted oviduct maintained in tissue culture. Zygotes incubated in this culture system did not show an arrest of development. However, they grew into vital blastocysts. (Bavister and Minami Citation1986). Using the same culture systems, Minami and collaborators showed that 21% of early 2-cell hamster embryos cultured under low O2 for 65–70 hours developed to the blastocyst stage within the explanted mouse oviduct (Minami et al. Citation1988). Additionally, IVC of embryos from several mammalian species (mouse, sheep, goat, and cattle) under reduced O2 tension showed improved development (Bavister Citation1995). Different culture systems might require different O2 to achieve optimal results (Dumoulin et al. Citation1999).
The improved development of mammalian embryos under physiologic O2 in vitro is based on the in vivo O2 tension in the female reproductive tract. In fact, after Steptoe and Edwards, numerous scientists described the O2 concentrations in the mammalian reproductive tract at a range from 2 to 8%. In particular, O2 in rhesus monkeys, rabbits, and hamsters ranged from high rates around 8% O2 in the oviduct, to low rates of 2% O2 in the uterus (Byatt-Smith et al. Citation1991; Fischer and Bavister Citation1993; Kasterstein et al. Citation2013).
Even if Edwards and Steptoe designed a culture system with reduced O2, for decades, most embryological laboratories used atmospheric O2, likely to avoid costs for laboratory equipment associated with the O2 reduction. The embryo culture with reduced O2 requires a nitrogen gas system, specialized incubators, and quality assurance related to oxygen sensors (Bontekoe et al. Citation2012). Currently, the use of a gas phase incubator in the culture of mammalian embryos can be successfully used either under atmospheric or physiological O2. However, some studies failed to show a significant difference in the clinical outcomes with low O2 in embryo culture (De Los Santos et al. Citation2013). Recently, multiple meta-analysis reviews demonstrated an increase in pregnancy and live birth rates at 5% O2 embryo culture (Nasri et al. Citation2016), especially after E3 ET (Glujovsky et al. Citation2012). As Morin (Citation2017) mentioned, if the goal of the embryo culture system is to recapitulate the in vivo environment, an essential question for clinicians performing E5 transfers is if the O2 concentration inside the female reproductive tracts is consistent throughout the embryo’s journey from the oviduct to the uterus or not (Morin Citation2017). This issue is worthy of discussion since the number of clinical IVF programs that are utilizing extended culture (up to 5 days) is increasing (Kissin et al. Citation2015).
The debate is regarding the effects of physiological and atmospheric O2 levels in embryo culture continues. Time-lapse monitoring studies on the developmental arrest in human and mouse preimplantation embryo culture found delayed/arrested embryo development under atmospheric O2 with respect to those using low O2 (Kirkegaard et al. Citation2013; Ma et al. Citation2017). Mouse embryos cultured from zygote to blastocysts under different O2 (3% and 20%) gave higher blastocyst developmental (92.3% vs. 79.4%) and hatching rates (80% vs. 70.4%) when embryos were cultured in 3% O2, suggesting that low O2 utilization in IVC may improve embryo viability with increased expression of antioxidant enzymes and glucose transporter activities (Ma et al. Citation2017). According to these findings, most of the modern IVF laboratories accepted the superiority of using physiologic O2, arguments regarding whether a further reduction in O2 after E3 of embryo development represents the most physiologic system still exists (Morin Citation2017). These arguments deserve two considerations: 1) O2 levels are lower in the uterus than in the oviduct, and 2) the embryo usually crosses the uterus-tubal junction on day 3. A few recent studies using ultra-low O2 (2%) in embryo culture after E3 of development, suggested that the best O2 level may be stage-dependent (Morin Citation2017; Crawford and Ledger Citation2019). However, the mechanism by which low O2 could improve in vitro embryo development is still not fully understood.
Up to now, the studies on the effects of low and ultra-low levels of O2 during in vitro embryo culture produced conflicting results. Discrepancies are present among studies regarding the quality of bovine embryo development cultured in physiologic and 2% O2 (Yuan et al. Citation2003; Harvey et al. Citation2004). The advantage of physiologic over atmospheric O2 was the object of extensive research (Thompson et al. Citation1990; Berthelot and Terqui Citation1996). The beneficial effects of O2 concentrations lower than the physiologic level are species-specific and stage-dependent (Li and Foote Citation1993; Yang et al. Citation2016). A study on mouse embryos cultured with 2% O2 showed, in fact, an increase in embryo loss rates at day 4 and a decrease in fetal weight at day 18 of pregnancy (Feil et al. Citation2006), although the use of 2% O2 at E5 was optimal for bovine embryo development. In cows specifically, embryos cultured in 2% O2 during the post-compaction phase, from E5 and on, produced high quality blastocysts and high developmental rates in around 40% of the total embryos (Thompson et al. Citation2000). These so-called ultra-low O2 concentrations in extended human embryo culture with sequential media may represent a more physiologic culture system. In humans, to our knowledge, only a few studies on this topic were present in the literature up to now. In one, the donated embryos cultured in atmospheric O2 underwent cryopreservation at E3 of development (Yang et al. Citation2016). Yang and collaborators showed that at warming, the embryos were then subjected to further culture until E5 at 2%, physiologic, or atmospheric O2. No significant differences were detected either in the number of preimplantation embryos reaching the blastocyst stage or in the number of high-quality blastocysts (Yang et al. Citation2016). In a second study, the ultra-low O2 environment was superior in respect to the continuous culture under physiologic O2 in human embryos cultured in physiologic (from day 1-to-3) and 2% (from day 3-to-5) O2 (Kaser et al. Citation2016). However, a more recent study demonstrated that a reduction in oxygen tension from 5 to 2% O2 in human embryo culture after day three did not improve embryo development, quality, and utilization rate (embryos transferred and cryopreserved) (De Munck et al. Citation2019).
Can the oxygen tension alter the embryo genetics, physiology, morphology, and development in vitro?
The study of the indicators of metabolic health required the evaluation of the performance of preimplantation embryos cultured at different O2 levels. In sheep, the O2 reduction from atmospheric to physiologic levels increased the catabolic utilization of glucose in preimplantation embryos (Harvey Citation2007). In mouse embryos cultured in low O2, pyruvate oxidation increased (Khurana and Wales Citation1989); high cell number blastocysts, associated with a reduction in the number of apoptotic ones, were also reported (Van Soom et al. Citation2002). Low O2 tension and pyruvate oxidation may collaborate to improve the energetic functionality of the preimplantation embryos by reducing the oxidative stress and ameliorating the biosynthetic activity. Reactive oxygen species (ROS) can cause serious damage to lipids (hampering the stability of cell membranes), DNA (fragmentation), and proteins (modifying protein function and cell signaling) (Catt and Henman Citation2000; Oliveira Citation2017). In pig embryos, physiologic O2 concentrations induced lower hydrogen peroxide production and, consequently, lower DNA fragmentation, with respect to the counterpart cultured under atmospheric O2 (Kitagawa et al. Citation2004). Also, the global gene expression pattern of mouse zygotes cultured in vitro showed more significant perturbations when atmospheric levels were used instead of the physiologic levels (Rinaudo et al. Citation2006). Others described an increase in global DNA methylation under high oxygen exposure in bovine preimplantation embryos, thus suggesting that oxidative stress can also alter the embryonic epigenome (Li et al. Citation2014). These alterations can be responsible for proteome alterations, as demonstrated by profiles of protein expression, obtained under reduced O2, that were similar to those from embryos developed in vivo (Katz-Jaffe et al. Citation2005). summarizes the indicators of the metabolic health of the preimplantation mammalian embryo cultured under different oxygen tension.
Table 1. Variations of the indicators of metabolic health of the pre-implantation embryo cultured under low (5%) and high (20%) oxygen tension with respect to the in vivo condition.
Our recently published data (Belli et al. Citation2019) showed that IVC of mouse embryo under physiological and atmospheric O2 affects mitochondrial morphology and numerical density by electron microscopy, as previously described (Palmerini et al. Citation2017; Zhurabekova et al. Citation2018; Bernardi et al. Citation2018). In mouse, morphometric analysis of EM micrographs showed a lower numerical density of healthy mitochondria and a higher numerical density of abnormal (i.e., hooded) mitochondria. Since mitochondria play a critical role in cellular metabolism (Iorio et al. Citation2015; Belli et al. Citation2018), these qualitative and quantitative findings indicate that IVF, and in particular high O2, significantly alters the metabolism and ultrastructure of the developing embryos (Crosier et al. Citation2000).
The impact of culture conditions on IVF success
During IVF, high production of ROS was detected in culture media due to the disturbances in embryonic culture, despite the presence of the endogenous defense mechanisms in both oocytes and embryos. Consequently, embryonic fragmentation, apoptosis, and even delay or interruption of embryo development was frequently seen (Oliveira Citation2017). ROS may stem directly from the environment in which the embryos are located (Cebral et al. Citation2007). The exposure of oocytes and embryos to xenobiotic agents, the changes in metabolic substrate concentrations, and the traces of transition elements are factors that favor the generation of ROS during in vitro culture/manipulation. Not only these factors but also the high levels of O2 were harmful for IVC (Feuer et al. Citation2014). The reduction of ROS generation, the improved air quality/reduced volatile organic compounds due to filtered nitrogen gas, and the mechanisms connected with gene expression, metabolism, or other cellular processes are potential factors that may contribute to an evaluated and efficient low O2 mechanism (Morin Citation2017).
Bontekoe et al. (Citation2012) suggested that culturing embryos under low O2 improved the success rates of IVF, resulting in the birth of healthy new-borns. Physiologic O2 also resulted in an increase of 43% in ongoing pregnancies/live birth and clinical outcome rates up to 47% compared to atmospheric O2 with 38% and 42%, respectively (Nasri et al. Citation2016). However, protocols used in clinical trials need to be improved, and additional effort is required to obtain a better-weighted overall view on the treatment effects of embryo culture under low O2 in IVF (Oliveira Citation2017). Therefore, the question of if the culture of oocytes and embryos under low O2 can enhance the positive clinical outcomes of IVF cycles still requires more data.
Other factors affecting positive IVF outcomes
The success of IVF relies on the quality of gametes and embryos and also the assisted reproductive technologies (ARTs) protocols (Bianchi et al. Citation2015; Coticchio et al. Citation2016; Khalili et al. Citation2017) as demonstrated by the importance of proper preservation of the cellular and organelle integrity (Nottola et al. Citation2011; Palmerini et al. Citation2018). However, several important factors can affect IVF outcome, e.g., oocyte retrieval of denuded oocytes without cumulus complex (COCs) showing an abnormal meiotic spindle morphology, as compared to oocytes with intact COCs that preserved a better specific-organelle structure (Cecconi et al. Citation2006). Other factors that may have an impact on IVF outcomes are the gamete or embryo cryopreservation with vitrification (Khalili et al. Citation2012), sperm selection, oocyte and embryo culture conditions, laboratory equipment, and the availability of an experienced embryologist (Rossi et al. Citation2006; Cecconi et al. Citation2010; Palmerini et al. Citation2016). Different culture systems close to the in vivo conditions could overcome the effects on oocyte quality and embryo yield. For example, using 5% O2 in IVC to improve embryo viability, and different antioxidant mixtures in the culture media during IVC to reduce oxidative stress could be used to improve embryo development (Silva et al. Citation2015; Belli et al. Citation2019). In fact, different studies demonstrated that the addition of antioxidants in the culture media, such as quercetin, taxifolin, and 7,8-dihydroxyflavone during IVC reduced ROS levels in oocytes and enhanced the developmental competence of the embryos (Choi et al. Citation2013; Kang et al. Citation2016; Von Mengden et al. Citation2020). It is evident that the IVF process and its success are a multi-factorial combination of the above factors in association with selected ARTs used in IVF may generate significant concerns for developmental trajectories.
Conclusions and future perspectives
In mammals, the issue of using low (less than the atmospheric O2) or ultra-low O2 (less or close to the physiologic O2) tension during embryo culture in clinical and experimental IVF has been the object of studies and debates for decades, since the role of oxygen, moreover species- and stage-dependence, in contributing to the success of IVF is well recognized. Ultra-low concentrations in human IVF are a recent acquisition, nevertheless, the use of conditions closer to the human oviduct environment in the extended embryo culture seems to have a beneficial impact on IVF outcome. Its application follows the development of advanced incubators allowing the maintenance of lower oxygen tension. In fact, in order to protect embryos from the oxidative stress correlated to the O2 and the resultant side-effects, in the last decade, there was an increased focus on approaches based on the supplementation of antioxidant mixtures and specific O2 during the in vitro embryo culture. The various combinations of antioxidants tested during in vitro maturation and in IVC media (Silva et al. Citation2015) showed an improvement in the quality of developing preimplantation embryos. However, many trials are still necessary in order to identify and clarify the most effective supplements combined with low and ultra-low O2 for optimizing IVC systems.
Author contributions
Conceived and designed the study: MB, SA, GM; Performed the literature research and drafting the ms: SBi, SBe, OD; Wrote the manuscript: MB, SA, MGP; Critically reviewed the ms: SAN, MAK, MGP, GM; Final approval of the ms: MB, SA, MGP, SBi, SBe, MAK, OD, SAN, GM.
Disclosure statement
No potential conflict of interest was reported by the authors.
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