Diverse Roles of Metformin During Peri-Implantation Development: Revisiting Novel Molecular Mechanisms Underlying Clinical Implications

Abstract

Metformin is not only a widely used oral antidiabetic drug, which acts as an insulin sensitizer and suppressor of hepatic gluconeogenesis, but it also exhibits antitumor properties. Besides, it has been utilized in the treatment of polycystic ovary syndrome (PCOS) for infertile women with glucose intolerance and as a component of combination therapy to reduce early (first trimester) pregnancy loss or spontaneous abortion (SAB). Based on recent studies demonstrating its beneficial effects on mothers and the fetus, metformin is even recommended for later stages of pregnancy. Probing into the mechanism of action revealed that it can activate a stress modulatory pathway, none other than the AMP-activated protein kinase (AMPK) via LKB 1. It is well accepted that AMPK signaling plays a crucial role during implantation by combating stress in multiple ways. Stress factors commonly encountered during pregnancy are malnutrition, diabetes, and hypoxia, which may result in SABs or other complications. For instance, the elevated levels of insulin, which are a typical characteristic of hyperinsulinemic, obese, or PCOS patients, can impair the development of the blastocyst and the preimplantation embryo. Further, a severe hypoxic environment prompts early and untimely differentiation of the embryonic cells leading to abnormal growth and development. Therefore, the modulation of stress-related pathways could be pivotal in ameliorating such stress responses during implantation. Here we hypothesize a putative noncanonical pathway underpinning the role of metformin in high-risk pregnancies to counteract stress by recreating an in vitro replica of human implantation, engaging embryonic stem cells, trophoblast stem cells, and endometrial stromal cells in a three-dimensional scaffold.

Background

M etformin (N′,N′-dimethylbiguanide) is a prominent antidiabetic drug. Metformin was first synthesized in the 1920s; however, it was first clinically used only in 1957. Metformin controls high blood sugar by decreasing hepatic gluconeogenesis, inhibiting the transcription of phosphoenolpyruvatecarboxykinase and glucose-6-phosphatase in the liver [1], and increasing insulin-stimulated glucose uptake in skeletal muscles and adipocytes [2,3]. Epidemiological studies have shown a decrease in cancer incidence in metformin-treated patients suggesting that it has antimetastatic effects [4]. It is found that metformin can activate one of the principal stress-associated pathways called AMP-activated protein kinase (AMPK). AMPK is a stress enzyme in eukaryotic cells, which plays a major role in sensing the AMP:ATP balance [5,6]. Accordingly, the AMPK pathway is activated by the increase in cellular AMP levels, which in turn decides if catabolic metabolic pathways would precede anabolic pathways in cases of cellular stress. There are also reports available on the administration of metformin to treat polycystic ovary syndrome (PCOS), one of the most common forms of female infertility and reduce the associated spontaneous abortion (SAB) [7]. Pilot studies on metformin have suggested that it can reduce gestational diabetes efficiently [8]. Therefore, in this article, we discuss the possible mechanistic action of metformin during implantation in a stressed environment caused by insulin resistance, excessive hypoxia, and malnutrition, which is occasionally fatal leading to fetal resorption.

Genesis of the Study

Most (70%) of the human embryos, which are lost as a result of SABs or failed pregnancies are at the preimplantation stage. There is enough evidence to reinforce that one of the contributing reasons underlying this problem is stress, wherein cells of the early conceptus, which includes embryonic and placental trophoblast stem cells (ESCs and TSCs, respectively), might face stressful situations like depletion of nutrients, severe hypoxic conditions, oxidative stress, heat shock (hyperthermia), high osmotic exposure, DNA damage caused by exposure to UV/radiation or anticancer agents, proinflammatory cytokines, and mechanical stress. These deleterious effects are carefully handled by a set of stress enzymes, namely, AMPK/SAPK. Additionally, there are reports from the Downs laboratory, which corroborate that a variety of stressors like heat shock proteins are mediated through the activation of AMPK; they stimulate meiosis in oocytes and are said to functionally act downstream of follicle-stimulating hormone to induce hormonal maturation of oocytes [9,10]. These stress proteins generally act by modulating cell cycle, repairing damaged proteins and reverting them back to their native state, thereby helping in regulating gene expression and behaving as antioxidants. Tosca et al. too emphasized that AMPK could be one of the pathways simultaneously controlling both metabolism as well as reproduction, and thus could emerge as a potential target for therapeutic purpose [11].

Thus, we postulate that it might be critical to have a tight control over the following two of the most important events or checkpoints during implantation. (1) Maintenance of pluripotency of ESCs, which become highly vulnerable and tend to differentiate prematurely once they encounter such a stressed environment, and (2) timely onset of differentiation that aids in proper organogenesis of the conceptus. ESCs need to maintain their integrity and numbers, and then differentiate to mediate other necessary functions downstream. Further, these two events need to synchronize effectively because the balance is very crucial for a healthy and successful pregnancy. So, stress enzymes need to be activated at the right time in such cases of high-risk pregnancy. We strongly foresee that metformin-induced triggering of stress-related signaling pathways will be instrumental in sustenance and survival of the conceptus.

Introduction

In the following section, we have listed the various functions of metformin (Table 1) at multiple levels and specifically during the preimplantation stage, such as the insulin sensitization of blastocysts, maintenance of the ESC pool during unfavorable conditions as well as its role in tackling oxidative stress.

Table 1.

Important Experimental Observations with Respect to Metformin Action

Serial no.	Major finding	Effector molecule	Cell line/model used	References
1.	Antagonistic effect on EMT	TGF beta	Madin–Darby canine kidney cells- MCF 7	15
2.	Regulates breast cancer stem cells by transcriptionally downregulating EMT inducers	TGF beta	MDA-MB-231; MDA-MB-468	16
3.	Activates AMPK in hepatocytes	AMPK	Primary hepatocytes isolated from rats (animal model)	17
4.	Arrests cell cycle in breast cancer by downregulating cyclin D1	Cyclin D	MCF 7	18
5.	Affects prostate and colon neoplasia involving activation of AMP-activated protein kinase	AMPK	PC-3; HT-29	19
6.	LKB 1 activates AMPK in response to metformin analogue phenformin treatment in skeletal muscle	LKB 1 and AMPK	Rat skeletal muscle (in vivo study)	20
7.	Activation of AMPK via mitochondrial oxidants	Mitochondrial reactive nitrogen species	C57BL6 (in vivo eNOS knockout model); bovine aortic endothelial cells	21
8.	Metformin exposure reduces AKT activation	AMPK, TSC 2/1 pathway, mTOR	MCF 7	22
9.	Decrease in endometrial cancer cell proliferation, indicates its role against endometrial cancer	AMPK, mTOR	Endometrial cancer cell line and Ishikawa cell line	23
10.	Combination therapy of metformin and 2-deoxyglucose trigger apoptosis in prostate cancer cells	p53, AMPK	LNCaP, P69, PC-3, and DU145 cells	24

EMT, epithelial-mesenchymal transition; TGF, transforming growth factor; MCF 7, Michigan Cancer Foundation-7; AMPK, AMP activated protein kinase; eNOS, endothelial nitric oxide synthase; TSC, trophoblast stem cell; mTOR, mammalian target of rapamycin.

Effect of metformin in maintenance of glucose level and modulation of AMPK activity

Metformin is administered during type II diabetes. It promotes insulin-dependent uptake of glucose as well as lowers hepatic glucose synthesis [3,12]. Metformin reduces fatty acid synthesis due to catecholamine-induced lipolysis [13] and can also activate the AMPK stress pathway, a serine/threonine protein kinase, via LKB 1 [14]. To be precise, the AMPK pathway is activated by the increase in cellular AMP levels, which decides if catabolism would precede anabolism at times of cellular stress. Evolutionarily conserved AMPK is a stress enzyme, which plays a major role in sensing the AMP:ATP balance. Activation of AMPK by metformin is reactive nitrogen species mediated; wherein inhibition of the mitochondrial chain is brought about by metformin generating O₂(−), which via a cascade of ONOO, c-Src-PI3K initiates the interaction of LKB 1 with AMPK resulting in a corresponding activation of AMPK [15]. Calcium/calmodulin-dependent protein kinase kinase B acts upstream of AMPK in mammalian cells. This mechanism of AMPK activation by metformin leads us to believe that stress enzymes could be manipulated, which can strengthen implantation and improve chances of pregnancy. Besides, there are reports suggesting that metformin can have some important AMPK-independent function. Saeedi et al. and Foretz et al. have established that metformin can act without activating AMPK (AMPK independent) in a dose-dependent manner in heart muscles and hepatocytes, respectively, via p38 MAPK- and PKC-dependent pathways [25,26]. A few studies show that metformin inhibits mTOR via the Rag GTPase-dependent mechanism, which is again AMPK independent [27].

Possible role of metformin during the onset of differentiation in preimplantation embryos

It is at this time (implantation) that the cells of the mammalian embryo segregate into two morphologically and functionally distinct cell lineages—one is the inner cell mass (ICM), and the other is the trophectoderm (TE). The TE containing TSCs should differentiate to trophoblastic giant cells, invade into the maternal tissue, and form the placenta so as to ensure proper nourishment of the developing fetus. It is noteworthy that these stem cells are under a constant stress of hypoxia, which sets up a compensatory differentiation at the same time [28]. It is anticipated that stem cells existing at this point sense this stress of hypoxia and adopt differentiation as their survival strategy, which in turn limits the resident stem cell pool expansion. Hence, the regulatory event that takes place as soon as the implantation occurs is placental differentiation, which mainly happens by inhibiting ID2 [29]. Intriguingly, downregulation of ID2 is again controlled by none other than the AMPK pathway [30]. This event facilitates secretion of the first placental hormone CSH1, implying maternal recognition of the pregnancy secretory phenotype by tissue-restricted basic helix loop helix transcription factors, such as heart and neural crest derivatives expressed 1, which directly interact with the CSH1 promoter and activate it. It has been shown that the abnormal AMPK activity is responsible for poor implantation [31]; thus, activation of the same by metformin could revert the outlook.

Another interesting observation is that when blastocysts are exposed to high insulin or during insulin resistance in diabetic women, insulin-like growth factor 1 receptor (IGFR) is desensitized and consequentially activates various apoptotic pathways resulting in fatal pregnancy termination [32]. This effect has been well studied in neurulation in a chick embryo and preimplantation blastocyst [32,33]. A few reports from investigators who represent a new school of thought have pointed out that activation of AMPK pathways by metformin can actually rescue the condition of insulin resistance during implantation [34]. Such treatment increased insulin-stimulated 2-deoxyglucose transport and cell survival and can potentially be helpful for management of complications during gestational diabetes. The failure of implantation in PCOS and other conditions could be due to nonutilization of glucose by the embryo as a consequence of insulin resistance. Therefore, metformin could play a decisive role in the initiation of differentiation via the modulating AMPK pathway.

Function of metformin in oxidative stress response during implantation

The ICM has to maintain its stem cell pool for a very short interval of time during implantation. Autonomous control helps the resident stem cells present in the niche to maintain the balance, which means that beyond a certain threshold, the cells would commit for differentiation [28]. One of the important factors underlying such a decision might be hypoxic stress. ICM gives rise to hypoblast and pluripotent epiblast. It is this epiblastic stage that harbors the requisite developmental potential to produce all embryonic lineages and has appropriately been described as “developmental ground state” by the Austin Smith's group [35]. In addition, reactive oxygen species (ROS) can be involved in depleting the existing stem cell pool, whereas the forkhead O (FOXO) transcription factors play a major role in upregulating genes involved in detoxification of ROS and its effects. A recent study showed that one of the downstream molecules of the AMPK pathway is FOXO; reports have also showed that FOXO1 is essential in maintaining pluripotency in human ESCs by a direct control on the promoter of Oct-4 and Sox-2 [36] and thereby helps in maintaining the pluripotency and self-renewal of stem cells (Fig. 1). Therefore, the essence of maintaining pluripotency although for a very narrow window of time during blastulation could not be ignored and that the equilibrium of stemness and onset of differentiation is to be stringently controlled. This decisive event is governed by stress enzymes like AMPK, which in turn can be modulated by metformin.

FIG. 1.

The master drug metformin can modulate the AMPK pathway, which marks the onset of differentiation by inhibiting ID2 via upregulation of CSH1. On the other hand, AMPK can also activate FOXO1, which has direct control over the promoter of Oct-4 and Sox-2, the hallmark of pluripotency. AMPK, AMP activated protein kinase; FOXO, forkhead O.

It is evident that AMPK has many adaptive effects in stem cells during peri-implantation development, which can improve maternal uptake and metabolism of the stem cells in the embryo, but it is important to adopt caution as toxicity is possible at higher (nonoptimal) concentrations of this drug that may activate too much AMPK and have negative effects on growth and potency. For example, high dose of metformin can lead to hyperactivation of AMPK, which may cause neurodegenerative disorders [37] and occasionally can even trigger cell death by corresponding overexpression of proapoptotic proteins [38]. Nevertheless, several clinical trials have concluded that when compared to other biguanines like phenformin and buformin, metformin has always shown beneficial effects in the long run [39]. However, Legro et al. ascertained that metformin alone causes accelerated loss of embryos during fertilization and Moll et al. suggest caution in using metformin for treating infertility associated with PCOS [40,41].

Rationale of the Hypothesis

There have been reports on metformin-induced activation of the AMPK pathway during implantation and it has been accepted as a significant pathway for fighting stress. On the other hand, metformin is a well-recognized and approved drug for the treatment of PCOS, which is characterized by hyperandrogenism, oligomenorrhea, and multiple ovarian cysts. Several pilot studies have shown that without metformin, SAB is common in women with PCOS (Table 2), occurring in as many as 42% of pregnancies. However, these studies have failed to explain the actual/potential reason accounting for such outcomes. In this article, we hypothesize the probable mechanism behind the action of metformin. A few reports have hinted that metformin only controls the blood sugar level, but we would suggest that it has got a much deeper role to play. We propose that efficient handling of stress during implantation (via the AMPK pathway) by maintaining the equilibrium of undifferentiated state and commitment for differentiation might play an imminent role behind metformin-mediated improvement/rescue of pregnancy rates in PCOS patients.

Table 2.

Pilot Clinical Studies Using Metformin

Serial no.	Clinical effect	Subject	References
1.	Reduces risk of cancer in diabetic patients	Type 2 diabetes patients	[4]
2.	Constant administration of metformin throughout pregnancy safely reduces first-trimester spontaneous abortions	PCOS patients (diabetic and nondiabetic)	[7]
3.	Significantly improves pregnancy outcome for women with PCOS undergoing IVF treatment	PCOS patients	[43]
4.	Improves pregnancy outcomes in PCOS patients with modulation of insulin-like growth factors	Patients with clomiphene-resistant PCOS	[44]
5.	During pregnancy reduces insulin, insulin resistance, insulin secretion, weight, testosterone, and development of gestational diabetes	PCOS patients	[45]
6.	Effect on noninsulin-dependent diabetes mellitus	Obese noninsulin-dependent diabetes mellitus patients	[46]
7.	Increases the rate of ovulation and pregnancy rate in patients with PCOS	PCOS patients	[47]
8.	Improvement of ovarian function (ovulation frequency)	Oligomenorrhea and PCOS patients	[48]
9.	Preventive effect of metformin on rectal aberrant crypt foci	Colorectal cancer patients	[49]
10.	Antiproliferative effect during preoperative stage in operable breast cancer	Nondiabetic invasive breast cancer patients	[50]
11.	Reduces systemic inflammation by decreasing levels of C reactive protein and IL6	Type 2 diabetic patients	[51]

PCOS, poly cystic ovary syndrome; IVF, in vitro fertilization; IL6, interleukin 6.

Therefore, we also suggest that ES/TS cultured on endometrial cells could be an eligible in vitro model system for testing our hypothesis as it closely mimics the in vivo situation and recapitulates events during early embryogenesis. The materno-fetal environment represented in this alternative model of implantation will allow us to acquire a better understanding of the molecular and biochemical changes within the ESC niche under stress followed by their rescue in response to metformin exposure.

The Hypothesis

We hypothesize that administration of metformin could be an appropriate option to maintain the glucose level and downplay stress for the developing fetus by regulating the AMPK pathway. The role of metformin has been well elucidated in cancer and diabetic models, but we propose that it could even play a crucial role during implantation in course of mammalian development by striking the right balance between maintenance of pluripotency and onset of differentiation.

How to Test the Hypothesis

In the following section, we have designed multiple models (in vitro), which we believe would help us in the experimentation of our hypothesis. To test compounds/drugs that may impair the blastocyst development, implantation, and cause infertility, it is necessary to develop suitable model systems that would recreate the initial steps of TS and ICM differentiation. Differentiation of the TS can be simulated through differentiation of hESCs and segregation of ICM into epiblasts and hypoblasts can be reproduced by embryoid body formation. Nevertheless, the major challenge is to develop a true replica of human implantation wherein one cannot only mimic the orchestration of events during implantation, but also represent the accurate cellular organization as well as architecture of the preimplantation embryo. We propose that this goal could be achieved in the following ways (Fig. 2). (1) First, TSCs could be grown in the form of spheroids followed by their attachment on monolayers of replication-inhibited primary endometrial stromal cells. The functional activity of these spheroids will be analyzed by human chorionic gonadotropin secretion, and then ESCs could be plated 24–48 h later. (2) Second, we would like to postulate an in vitro three-dimensional model, wherein commercially available TSCs could be impregnated on collagen scaffolds combined with heparin. This biodegradable scaffold would be designed in such a way that basic fibroblast growth factor (FGF-2), an important growth factor required during implantation could be loaded in the scaffold and the existing heparin would allow the sustained/slow release of this growth factor along the period of study. Collagen scaffolds soaked with TSCs will then be allowed to attach on the immortalized human stromal endometrial cell line [42] complemented by subsequent seeding of ESCs in a growth medium supplemented with other essential growth factors like epidermal growth factor, platelet-derived growth factor, and transforming growth factor. Thus, the aforesaid models would enable us to replicate/recapitulate the obligatory physical as well as biochemical interaction in the form of a crosstalk that takes place in utero. (3) Third, we propose to prepare Matrigel microspheres with ESCs immobilized on them; adherent TSCs grown on nonadherent tissue culture plates in the presence of fibroblast growth factor 2 (FGF-2), bone morphogenetic protein 4 (BMP-4), and insulin-like growth factor 2 (IGF-II) would encapsulate these Matrigel beads, which will again be allowed to flourish on growth inactivated endometrial stromal cells. In this way, the true cellular organization of the two cell types constituting the blastocyst, that is, ICM and TS can be reproduced in a more pragmatic manner. Thereafter, multiple forms of stress associated with pregnancy such as oxidative damage, malnutrition, and gestational diabetes can be tested upon either/both of the aforesaid cell types making it a true representation of the human scenario on an in vitro platform. IGFR-1 knockdown via shRNA-mediated gene silencing method, culturing feeder-free ESCs under hypoxia and high glucose using a culture medium devoid of key micro-/macronutrients like vitamin B6, B12, folic acid, and vitamin D could simulate gestational diabetes, oxidative stress, and malnutrition, respectively. Metformin will then be administered to this stress-induced in vitro implantation scene at different (relevant) concentrations. Then, the readouts in comparison to controls will be analyzed to determine the effects of metformin. The Oct/Sox/ROS/Cdx genes could be tagged with reporter genes to observe their expression once they are treated with metformin and their activity could be monitored by luciferase assay. These findings are likely to provide significant insights into comprehending the molecular mechanisms triggering metformin-mediated rescue of ESCs/TSCs. Whole genome microarray analysis could be performed on postmetformin-treated, stressed ESCs/TSCs, which will distinctly show the specific set of genes playing a role in this reversal. The specificity of AMPK activation in response to metformin exposure can be reconfirmed by suitable inhibitors or knockout studies.

FIG. 2.

Establishment of a surrogate model to understand metformin action during failed implantation. (A) Shows transient stress due to diabetes, malnutrition, and high oxygen at the time of implantation of blastocysts during early pregnancy. (B) Development of the in vitro implantation model employing three different strategies: (1) TSC spheroids followed by plating of ESCs on endometrial cells; (2) collagen scaffolds with heparin, impregnated with TSCs facilitating slow release of FGF-2 followed by coculture of ESCs grown on endometrial feeders; (3) Matrigel microbeads immobilized with TSCs and ESCs grown on endometrial cells. Inducible stress factors such as IGFR-1 knockdown, hypoxia, and nutrient-compromised media, were introduced in the microenvironment that closely imitates the in utero scenario. (C) Administration of metformin expecting a reversal of the impairment could be assessed by several parameters by morphology, gene, and protein levels to delineate the mechanism of metformin action in infertility. TSCs, trophoblast stem cells; ESCs, embryonic stem cells; FGF-2, fibroblast growth factor 2; IGFR-1, insulin-like growth factor 1 receptor.

Future Perspective

In summary, it appears that AMPK has a well-defined regulatory role in retaining the pluripotent stem cell pool for a very short time frame, regulating differentiation and maintaining glucose levels to overcome the stress inflicted during implantation. Rising levels of obesity and diabetes as well as a high prevalence of PCOS in reproductive-age women have led to an increased occurrence of insulin resistance during conception and pregnancy. Therefore, this strategy of employing a substantial in vitro model followed by the modulation of the AMPK pathway by metformin could be an effective approach to oppose these kinds of stress responses during implantation. However, we realize that it is quite difficult to create such a model; extensive literature search revealed that a good in vitro model, even for the implantation of an intact mouse embryo does not exist. Therefore, we claim that the success of this prototype will heavily depend on the behavior of ESCs and TSCs together within the scaffolds since it is unlikely that ESCs would implant by themselves because the TSCs induce a decimal reaction from the uterine epithelium during implantation. Nonetheless, we are highly optimistic about this concept and strongly believe that it would indeed be interesting to do such experiments in the laboratory to at least find out what happens.

Footnotes

Acknowledgments

The authors acknowledge the support of the vice chancellor and the registrar of Manipal University, Manipal, for extending the required facilities for carrying out the present study. We thank Sandhyaa Venkatachalam and Dr. Anjan Kumar Das for careful proof reading of this article.

Author Disclosure Statement

None declared.

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