Effects of resveratrol, granulocyte-macrophage colony-stimulating factor or dichloroacetic acid in the culture media on embryonic development and pregnancy rates in aged mice

Introduction

Due to recent lifestyle changes, many women are delaying childbearing and it is associated with an increased risk of infertility [1]. Previous studies have suggested that the reproductive capacity in women of advanced maternal age is significantly lower than that in women under 35 years of age [2–4]. The major factors in the reproductive capacity of older patients are the decrease in oocyte quality, low fertilization rate, poor embryonic development, low pregnancy rate, and increased rate of chromosomal aberrations. These can lead to increased aneuploidy, miscarriage, and birth defects [2, 5]. Reproductive aging pathologies are frequently associated with impaired DNA repair, metabolic disorders, genomic instability, telomeric shortening, apoptosis, and mitochondrial dysfunction [3, 6, 7]. However, various aspects of those pathologies require further studies because understanding the mechanisms of reproductive aging is essential to improving the pregnancy rates in advanced age women.

Resveratrol is a natural polyphenolic compound found in many plant species, such as grapes, peanuts, and berries [8, 9]. Resveratrol is known to possess biological activities, including antioxidant, anti-inflammatory, anti-carcinogenic, and telomerase-enhancing activities [8, 10]. In addition, several studies have reported that resveratrol can improve in vitro oocyte maturation (IVM) and embryonic developmental competence in various species [6, 11–13]. Furthermore, resveratrol can delay aging and prevent age-related diseases by improving mitochondrial function and decreasing reactive oxygen species (ROS) production [6, 8]. Liu et al. (2018) suggested that resveratrol can induce oocyte maturation and subsequent embryonic development in aged mice.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine expressed in the female genital tract during early pregnancy [14, 15]. The addition of GM-CSF to culture medium can promote preimplantation embryonic development and decrease apoptosis [14, 16]. GM-CSF supplementation can also improve pregnancy rates in patients with multiple unsuccessful in vitro fertilization (IVF) attempts and previous miscarriages [15, 17, 18]. Zhou et al. (2016) [19] reported that the addition of GM-CSF could decrease the occurrence of biochemical pregnancies in women aged over 35 years.

Dichloroacetic acid (DCA) is a stimulator of pyruvate dehydrogenase activity, thus accelerating the uptake of pyruvate into the tricarboxylic acid (TCA) cycle and increasing adenosine triphosphate (ATP) output [20, 21]. A previous study demonstrated that the addition of DCA to embryo culture media improved blastocyst development rates and mitochondrial output in embryos produced from aged mice [21].

With the recent trends of delaying marriage and childbirth, understanding the mechanisms occurring in oocytes and embryos in advanced maternal age is very important to improve pregnancy rates. However, only a limited number of studies have been conducted on pregnancy rates and embryonic development rates in older women. Therefore, the objective of this study was to determine the effects of resveratrol, GM-CSF or DCA in the culture media on embryonic development and pregnancy rates in aged mice.

Results

Discussion

The success rate of assisted reproductive technology is correlated with maternal age, which affects pregnancy and miscarriage rates [7, 22]. Studies have reported that reproductive capacity and oocytes were affected by old age, resulting in poor quality oocytes and mitochondrial dysfunction [2, 6, 23].

Oocyte aging is divided into two physiological processes, postovulatory aging and maternal aging [22]. Oocyte aging associated with maternal aging is a major problem in most clinical laboratories. Therefore, it is important to establish a protocol to improve both the developmental rate of oocytes and the pregnancy rate in response to oocyte aging due to maternal aging in in vitro fertilization programs. Since oocytes at ovum pick-up are already in the MII stage, studies on mitosis rather than meiosis may be more effective in improving the pregnancy rate in older patients. Mitochondrial dysfunction and ROS production during mitosis in aged embryos were reported to influence the developmental competence of pre-implantation embryos [22]. Therefore, this study was conducted to observe how selected compounds influenced the developmental and reproductive potentials of aged oocytes.

In contrast to GM-CSF or DCA, reports of the effective resveratrol concentrations for embryonic development have varied depending on the study group, animal species, and culture medium used [10, 12, 13, 24]. Therefore, a dose-response trial was conducted to determine the appropriate concentration of resveratrol. High concentration (2.0 μM) significantly reduced the total blastocyst formation rate compared to the control or low concentrations (Table 1). This was consistent with previous studies indicating that high concentrations of resveratrol might have negative effects on embryonic development [25, 26]. However, since different results have been reported, differences in the fertilization method, the culture medium used, and the mice species might be associated with such discrepancies [10].

In older organisms, increased ROS levels, decreased mitochondrial functions, and/or increased miscarriage rates are frequently observed. Thus, three biomolecules (resveratrol, GM-CSF, and DCA) selected based on previous reports were tested for their effects on embryonic development and pregnancy rates in aged mice (Tables 2, 3). Trials with human embryos have shown that the addition of GM-CSF to culture media positively influenced the blastocyst development rate and implantation competence [15, 16]. Particularly, it has been shown that GM-CSF supplementation can improve pregnancy rates in patients with multiple unsuccessful IVF attempts and previous miscarriages [15, 17, 18]. In contrast, GM-CSF did not show any significant influence on embryonic developmental competence and pregnancy rates in this study, consistent with previous mice studies [27, 28]. This conflict might be explained by the studies of Elaimi et al. [28], who reported high proportions of chromosomal abnormalities in mouse blastocysts cultured in GM-CSF-containing media. This result suggests a reason why GM-CSF had no significant impact in our study. Different mechanisms between patients with previous miscarriages and older patients with high miscarriage rates might also affect the results. In addition, GM-CSF may not have a positive effect to decrease the abortion rate caused by aneuploidy in older women. This is, in part, consistent with the results reported by Zhou et al. [19]. They suggested that low concentrations of GM-CSF supplementation (0.2 ng/ml) in the culture medium had no beneficial effect on embryo quality, pregnancy rate, implantation rate, and the take-home baby rate of women aged over 35. To clarify the controversial results of the effects of GM-CSF on the embryonic developmental competence and pregnancy potential of older mice and humans, comparative studies of the GM-CSF mechanism of action are in progress.

McPherson et al. [21] reported that DCA supplementation stimulated mitochondrial activity, which in turn, increased the blastocyst development rates, fetal development, and MMP in mice aged 26–28 weeks. They concluded that stimulation of the mitochondria by DCA could improve embryos from aged mothers. Similarly, the present study found that the addition of DCA to the culture media significantly increased blastocyst formation compared to the control. However, DCA did not significantly affect the implantation rate (Table 3). These results are inconsistent with the above study, which reported that the fetal development of the DCA group (46.3%) was significantly higher than that of the control group (35.2%). These differences may be attributed to differences in species or the age of the mice, fertilization method, or culture medium used. Thus, further experimentation is necessary to determine the exact effect of DCA on aged mice.

In contrast to the effects of GM-CSF and DCA, resveratrol treatment significantly increased the pregnancy and implantation rates (Table 3), although it did not affect the fertilization and blastocyst development rates (Table 2). Several reports have suggested that resveratrol can delay aging and prevent aging-related diseases by improving mitochondrial function and decreasing ROS [6, 8]. Therefore, to examine the mechanism behind these results, the effect of resveratrol on ROS expression and mitochondrial function in aged mice embryo was investigated.

ROS are normally generated through oxidative phosphorylation in the mitochondria [29, 30]. However, high levels of ROS can damage many cellular biomolecules, such as carbohydrates, lipids, proteins, and DNA, which is thought to be involved in aging-related pathologies [22]. Bentov et al. [23] and Silva et al. [29] reported that mitochondrial DNA mutations and mitochondrial dysfunction increased with maternal aging and that ROS levels in oocytes from older women were associated with declines in oocyte and embryo quality. Resveratrol has beneficial effects on embryonic development by decreasing ROS levels [25, 31–35]. Therefore, the present study investigated whether resveratrol could increase the pregnancy and implantation rates in aged mice by reducing ROS levels (Figure 1). During pre-implantation development, ROS levels in the embryos of old mice were about 1.2-fold higher than those of young mice. This indicates that elevated ROS levels in embryos from old mice might contribute to the low pregnancy rates. The addition of resveratrol significantly reduced ROS levels in the embryos of older mice to levels similar to those of young mice. This result is, in part, consistent with the results of Liang et al. [11]. They reported that the addition of resveratrol increased the resistance of post-ovulatory aging and decreased ROS levels. Abe et al. [25] also suggested that blastocysts developed after resveratrol treatment showed low ROS levels. These results imply the positive effects of resveratrol on embryonic development and pregnancies in aged mice.

Mitochondria are the major source of energy in all eukaryotic cells. It has been suggested that mitochondrial dysfunction results in chromosomal anomalies during mitosis, influencing the developmental competence of pre-implantation embryos [22, 29, 30]. Mitochondrial functions depend on the maintenance of membrane potential [2]. Thus, the effect of resveratrol on mitochondrial functions was evaluated by measuring MMP levels. The MMPs in young mice embryos were higher than those in old mice embryos (Figure 2). Resveratrol supplementation significantly increased MMPs throughout stages of pre-implantation embryos in old mice, compared to those in the embryos of young mice. These results suggest that the increased pregnancy and implantation rates after resveratrol supplementation were, in part, due to higher MMP.

Several studies have reported a correlation between increased MMPs in early-stage embryos (1~2 cell) and improved embryonic development [21, 36]. Particularly, Silva et al. [29] demonstrated that supplementation with antioxidants could increase membrane potentials in 2-cell embryos of aged mice and that this increase was correlated with embryonic development. In the present study, MMP increased by resveratrol treatment was 2.3-fold higher in the 2-cell stage than in any other stage (4-cell: 1.5-fold, morula: 1.6-fold, blastocyst: 1.7-fold, compared to old mice embryos). These results indicate that the large increase in MMP during the early embryonic stage might have affected the developmental competence of embryos and the pregnancy rate in aged mice.

In addition to the ROS levels and MMP, histone deacetylation can also explain how resveratrol upregulates pregnancy rates. Resveratrol is known to be a SIRT1 activator, which is involved in the aging process through histone deacetylation [35, 37]. Reports showing undetectable SIRT1 protein in old oocytes and increased maternal age-related aneuploidy by decreased histone deacetylation suggest that advanced maternal age is related to SIRT1 expression and histone deacetylation [4, 38, 39]. They also suggest the possibility that resveratrol may improve the embryonic development and pregnancy rates of aged mice by increasing SIRT1 and stabilizing histone deacetylation. This was supported by the results of Liu et al. demonstrating that resveratrol supplementation in IVM medium increased SIRT1 mRNA expression in oocytes from aged mice [8]. To investigate other mechanisms of action of resveratrol, experiments regarding SIRT1 expression and histone deacetylation are in progress.

In conclusion, the current study was the first to investigate the effects of physiological regulators (GM-CSF, DCA, and resveratrol) on the developmental competence and pregnancy potential of aged mice embryos. Among the three substances, resveratrol significantly improved pregnancy and implantation rates. These results are thought to be due to the biological activities of resveratrol as an anti-oxidant and/or a mitochondrial nutrient. The results from more extensive studies of the physiological and molecular mechanisms of resveratrol may increase the successful pregnancy rates in older patients.

Materials and Methods