Metformin (MF), a first-line drug to treat type 2 diabetes mellitus (T2DM), alone and in combination with other drugs, restores the ovarian function in women with polycystic ovary syndrome (PCOS) and improves fetal development, pregnancy outcomes and offspring health in gestational diabetes mellitus (GDM) and T2DM. MF treatment is demonstrated to improve the efficiency of in vitro fertilization and is considered a supplementary drug in assisted reproductive technologies. MF lacks teratogenic effects and has positive health effect in newborns. The entry is focused on use of MF therapy for restoration of female reproductive functions and improvement of pregnancy outcomes in metabolic and endocrine disorders.
The signaling pathways of metformin (MF) in cells are still not fully understood, they seem to be dependent on species and cell type, as well as doses and routes of administration, along with metabolic and hormonal status of subjects [1][2][3][4][5][6].
The molecule of MF, a small hydrophilic cation, is transported from the extracellular space to the cytoplasm of the target cell through organic cation transporters-1 and -2 (OCT1, OCT2), multidrug and toxin extrusion transporters (MATE), and ATM (ataxia telangiectasia mutated) transporter, and OCT1 and OCT2 are considered as the main functional units of MF transmembrane transport[7]. The transfer of MF across the placental barrier during pregnancy is largely dependent on the transporter OCT3[8]. The ultimate intracellular target for MF is the 5′-adenosine monophosphate-activated protein kinase (AMPK), the key energy sensor of the cell, although MF does not interact directly with the enzyme[1] [9][10][11]. In pathological conditions, like type 2 diabetes mellitus (T2DM) and metabolic syndrome (MetS), the activity of AMPK is reduced. MF’s action increases the activity of AMPK, and consequently normalizes the energy metabolism of the target cell. The AMPK consists of a catalytic α-subunit and the regulatory β- and γ-subunits that form a functionally active αβγ-heterotrimeric complex, and is widely distributed in all subcellular compartments (cytoplasmic, lysosomal, mitochondrial, and nuclear). AMPK is activated by increasing levels of AMP, a positive allosteric regulator of the enzyme[10][11][12][13]. The interaction of AMP with the adenine nucleotides-binding sites located in the γ subunit leads to stabilization of the αβγ heterotrimeric complex and enables phosphorylation of the α-subunit by liver kinase B1 (LKB1), which leads to the increase in AMPK activity [11][12][14] (Figure 1). Activating phosphorylation of AMPK may be also mediated by Ca2+-calmodulin-dependent protein kinase kinase 2 (CaMKK2)[15][16] and transforming growth factor β activated kinase-1 (TAK1) [17][18][19][20], but LKB1 is most important for AMPK activation[10][13][21][22][23]. Allosteric binding of AMP and ADP to γ-subunit of AMPK increases the ability of LKB1 and CaMKK2 to phosphorylate AMPK α-subunit at the Thr172 [24][25][26]. In the lysosomes, the “non-canonical” pathway of LKB1-mediated AMPK activation is carried out through dissociation of fructose 1,6-bisphosphate from aldolase. At the lysosomal surface, free aldolase promotes the formation of a multiprotein complex, including the vacuolar H+-ATPase and the scaffold protein AXIN, and this complex ensures the effective binding between AMPK and LKB1, thereby activating AMPK[27][28]. A negative regulator of AMPK is the protein phosphatase 2C (PP2C), which dephosphorylates and inactivates the α-subunit of AMPK, causing the dissociation of the αβγ-heterotrimeric complex. Elevated levels of AMP lead to an inhibition of PP2C activity, which allows AMPK to remain stable in the active Thr172-phosphorylated state[28][29].
Figure 1. The cellular mechanisms of metformin action which are carried out by activation of the AMP-activated protein kinase and inhibition of the mitochondrial electron transport chain complex I. Abbreviations: AC, adenylyl cyclase; ACC1/2, acetyl-CoA carboxylases 1 and 2; AMPD, AMP deaminase; AMPK, the heterotrimeric AMP-activated protein kinase consisting of the α1/2 (the target for activation phosphorylation at the Thr172), β1/2 and γ1/2/3 subunits; CREB, cAMP-activated transcription factor (cAMP response element-binding protein); ETC complex I, the mitochondrial NADH-dehydrogenase complex, the first complex of the respiratory electron transport chain; FA, fatty acids; LKB1, liver kinase B1; mG3PDH, mitochondrial glycerol-3-phosphate dehydrogenase; mTORC2, the mTOR complex 2; NFκB, nuclear factor κB; OCT1/2, the organic cations transporters 1 and 2; pCBP, the Ser436-phosphorylated form of CREB-binding protein with acetyltransferase activity, a co-activator of the factor CREB; PDE4B, cAMP-specific 3′,5′-cyclic phosphodiesterase 4B; PKA, cAMP-dependent protein kinase; PP2C, protein phosphatase 2C; ROS, reactive oxygen species.
MF penetrates into the mitochondria through intracellular space and accumulates in them. While in the mitochondria, MF inhibits the mitochondrial ETC complex I, which leads to decrease in ATP production and increase in the [AMP]i/[ATP]i and [ADP]i/[ATP]i ratios[30][31][32][33]. Moreover, MF decreases the activity of the enzyme AMP-deaminase (AMPD), which converts AMP to inosine monophosphate, inducing the accumulation of AMP within the cell [34]. The MF-induced increase in the intracellular AMP level leads to the activation of AMPK as described above[21][35]. The MF effect on AMPK activity is observed at drug concentrations below 80 μM, which are achieved with oral administration of therapeutic doses of MF [57]. The MF-induced activation of AMPK results in the stimulation of energy-producing catabolic pathways that mediate the increased glucose uptake by cells, the increased expression and activity of the membrane glucose transporters, the activated metabolic processes such as glycolysis and oxidative phosphorylation, and the normalization of mitochondrial biogenesis[36][3][37][38]. The MF-induced AMPK stimulation leads to phosphorylation of types 1 and 2 acetyl-CoA carboxylases (ACC1 and ACC2), inducing an inhibition of lipogenesis and stimulation of the β-oxidation of free fatty acids [39][40][41] (Figure 1). The ultimate results of this metabolic cascade is the decrease of T2DM- and MetS-produced dyslipidemia, and the normalization of lipid metabolism. In addition, the AMPK activation induces a plethora of cellular events, including regulation of autophagy and apoptotic processes, a decrease in the activity of inflammatory factors, including nuclear factor κB (NF-κB) and interleukin 1β, an inhibition of the ROS production, a decrease in the ER stress, as well as a decrease in insulin/IGF-1-induced activation of the mTORC1/2 complexes and a decrease in the protein synthesis[42][41][43][44][45][46].
The MF is a functional antagonist of cAMP-dependent signaling cascades, which are stimulated by hormones, glucagon in particular, through the Gs protein-coupled receptors and the membrane-bound forms of adenylyl cyclase (AC)[47][48]. The stimulation of AC results in an increase in the intracellular cAMP level and the activation of the protein kinase A (PKA) and the cAMP-activated transcription factor CREB (cAMP response element-binding protein). The MF-induced activation of AMPK promotes phosphorylation and activation of cAMP-specific 3′,5′-cyclic phosphodiesterase 4B (PDE4B), thereby reducing the intracellular level of cAMP[48] Moreover, MF causes an increase in the intracellular level of AMP, a negative regulator of the catalytic site of AC, which leads to inhibition of AC activity and a decrease in cAMP production. An increase in the level of AMP can be the result of both inhibition of the mitochondrial ETC complex I, and suppression of the activity of AMP deaminase [34][49] (Figure 1). A decrease in the activity of cAMP-dependent pathways in the liver, like activation of AMPK, leads to the inhibition of glucose synthesis in hepatocytes. Furthermore, MF-induced AMPK activation induces the protein kinase ι/λ-mediated phosphorylation of cyclic AMP response element binding (CREB)-binding protein (CBP or CREBBP) at the Ser436, which leads to the inability of the phospho-CBP to form a functionally active complex with the factor CREB and thereby inhibits the cAMP-dependent gene transcription[50]
Along with AMPK-dependent, there are also AMPK-independent pathways of MF action on the intracellular effector systems and gene expression. High-dose MF inhibits the activity of the mitochondrial glycerol-3-phosphate dehydrogenase (mG3PDH)[51]. The inhibition of mG3PDH leads to an increase in NADH levels and decreases NAD+ levels, and this causes a deficiency in NAD+, which is involved in the conversion of lactate to pyruvate (Figure 1). Since a decrease in mG3PDH activity inhibits the conversion of lactate to glucose, the result of impaired gluconeogenesis in hepatocytes is an accumulation of lactate, which can cause lactic acidosis in the conditions of high-dose MF treatment[51][52]. Another target of MF is the enzyme H3K27me3-demethylase KDM6A/UTX, which is responsible for the transcriptional activity of a large number of genes[53].
The antidiabetic effects of MF may be due to the changes in the gut microbiota, due to stimulation of the growth of bacteria that produce short-chain fatty acids[54]. By modulating the composition of the microbiota in rodents with T2DM and MetS, MF reduces the levels of bacterial lipopolysaccharides in the blood[55], and activates AMPK-dependent pathways in the mucosal layer of the intestine, reducing glucose absorption[56].
The most important mechanism of action of MF on target cells is the enhancement of the insulin signaling pathways and the decrease in insulin resistance (IR). This may be due to inhibition of hyperactivated nuclear factor κB (NF-κB), a transcription factor that provokes the development of IR, as well as a decrease in the expression of the phosphatase and tensin homolog (PTEN), which dephosphorylates phosphatidylinositol-3,4,5-triphosphate and thereby prevents insulin-induced stimulation of Akt kinase, a key effector component in the 3-phosphoinositide signaling pathway. The inhibitory effect of MF on the activity of NF-κB-dependent signaling pathways is carried out mainly through the stimulation of AMPK[34][57][58]. Since NF-κB plays a key role in inflammatory reactions, its inhibition by MF promotes the weakening of inflammation and increases the cell survival, and these effects of MF are prevented by AMPK inhibitors[34][59][60].
The polycystic ovary syndrome (PCOS) occurs in average from 9% to 18% of women of reproductive age and includes a number of metabolic and endocrine dysfunctions[61]. Some of them are: (i) the ovarian dysfunction, characterized by irregular or no ovulation (oligo- or amenorrhea), the increased secretion of androgens (hyperandrogenism, HA) and estrogens, the endometrial hyperplasia and the increased size of the ovaries, (ii) the pancreatic dysfunction leading to insulin hypersecretion and, as a result, to insulin resistance (IR) development, (iii) the adrenal dysfunction, which leads to hyperproduction of androgens, and (iv) the functional changes in the hypothalamic and pituitary links of the female hypothalamic-pituitary-gonadal (HPG) axis[62][63][64][65]. Since these dysfunctions and changes are usually associated with obesity, MetS and T2DM, the PCOS is much more common in women with these metabolic disorders (on average in 30% of cases), with a significant proportion of PCOS patients having IR with accompanying compensatory hyperinsulinemia[66][67][68][69][70][71]. According to the Rotterdam criteria (2003), the main diagnostic criteria for PCOS are clinical or biochemical HA, oligo- or amenorrhea associated with chronic anovulation, and morphological features of PCOS, which include 12 or more follicles (2 to 9 mm) in each ovary and/or an increase in ovarian volume over 10 mL[72][73][74]. It should be noted that about 80% of women with anovulatory infertility have typical signs of PCOS[61].
The etiology and clinical manifestations of PCOS depend on many factors, as well as combinations and interactions between them. The genetic predisposition[75][76][77][78] and epigenetic factors, including an increased level of gene methylation, histone modification, and microRNA pattern variation [79][80][81], are important for the development of PCOS. Environmental and socioeconomic factors are also of great importance, including ethnic characteristics, nutrition, and adverse environmental factors (toxins, xenobiotics, chemical mutagens, and ionizing radiation)[82][83][84]., The development of PCOS in women largely depends on the effects of maternal hormones during the prenatal period, as well as on their metabolic and hormonal status in the early childhood[79][85][86][87].
In recent years, MF therapy has become widely used for correction of the metabolic and hormonal impairments in women with PCOS and for restoration of their reproductive functions[80][88][89], including the improvement of IVF/ICSI outcomes in PCOS [90][91][92][93]. MF is most effective in treating PCOS patients with the metabolic disorders such as T2DM, obesity, dyslipidemia, and severe IR [94][95][96]. This is majorly attributed to the alleviation of negative effects of these disorders on the female reproduction by MF, increased tissues sensitivity to insulin, improved lipid and glucose metabolism and cell metabolism, and reduced inflammation and oxidative stress in the ovaries as well as in other tissues. In cases where significant metabolic changes in PCOS patients are not observed during treatment, MF therapy can lead to energy and hormonal imbalance. The outcomes may be the opposite of improvement, but a further deterioration in reproductive functions. This possibility is supported by the data from clinical trials on metabolic changes, including an increase in fasting glucose clearance and endogenous glucose production[97][98], as well as changes in the microbiota in non-diabetic individuals[99], as well as data on metabolic and hormonal dysfunctions in normal rodents, for a long time receiving MF[100].
There is a lot of clinical evidence of the high efficacy of MF in PCOS, which makes it feasible to consider MF as a second-line drug for ovulation induction in women with PCOS[101][102][103][104][105][106][107][108]. MF is recommended for the induction of ovulation in PCOS women who are either resistant to clomiphene citrate (CC) or require antiandrogen therapy without the use of contraceptives[105], as well as in PCOS patients with severe obesity and impaired lipid metabolism [109][114]. One very important consideration during PCOS treatment with MF is that drug has no or little adverse effects on the outcomes of pregnancy as well as the health of fetus and newborn, which indicates the safety of MF therapy[106][107] [126,127]. The gastrointestinal side effects of MF have been reported in a number of cases, but these effects did not significantly affect the health of PCOS women [92][110][108,112].
The MF treatment of PCOS women normalizes the frequency and regularity of ovulation, including when co-administered with exogenous gonadotropins[92][111][112]. This suggests that MF can also affect the sensitivity of ovarian cells to gonadotropins, which is important for the ART. As a result, during the ART, the most promising approach is the combined use of MF with gonadotropins[113][93]. In PCOS, MF improves clinical pregnancy rates and live birth rates[92] [113][92][93][114][115][116][117][118][119], and also reduces the number of miscarriages and increases the rate of embryo implantation[120][121].
There is evidence of a positive effect of MF on the effectiveness of IVF and IVF/ICSI in PCOS women [94]. It is believed to be due to the normalization of metabolic and hormonal parameters and the androgen levels in PCOS, which leads to an improvement of embryo implantation, an increase in the ovarian response to gonadotropins and a decrease in the rates of miscarriage[92][118][122][123][124][125]. The increased gonadotropin sensitivity allows avoiding the use of high-dose gonadotropins and, thereby, preventing the ovarian hyperstimulation syndrome (OHSS), a severe complication of gonadotropin-induced ovulation induction. However, it should be noted that some data on the use of MF in the ART technology in PCOS women are not so unambiguous, and there are results that do not support the efficacy of MF in IVF/ICSI. The clinical studies carried out by Egyptian group of physicians showed no improvement in IVF rates in PCOS women who received MF[126]. However, in this study, overweight or obese PCOS women received short-term courses of low-dose MF (1000 mg/day), from the start of ovarian stimulation with gonadotropins until proof of clinical pregnancy. As a result, in this case, the period of time for the manifestation of the restorative effects of MF on the ovaries and folliculogenesis in PCOS patients may not have been long enough. Potentially, for an adequate estimation of MF effectiveness in PCOS patients it is necessary to separate them in the groups, based on the severity and duration of the disease and in the body mass index [92][94], as well as the severity of IR, dyslipidemia and hyperglycemia.
A promising approach to treat PCOS is the use of combination of MF with the other drugs that improve the ovarian function and metabolic parameters in PCOS, with the best candidates for co-administration are CC, a mild nonsteroidal estrogen antagonist belonging to the family of selective estrogen receptor modulators, and letrozole, a non-steroidal aromatase inhibitor that prevents the conversion of androgens to estrogens [96][114][127][128][129][130][131][132][116,131,143,144,145,146,147,148].
The CC is the main drug of choice for treatment of PCOS, yet a significant proportion of PCOS women have weak or no response to CC therapy. Therefore, a search is underway for drugs that can potentiate the therapeutic effects of CC in PCOS, and MF is one of the most promising candidates [108,131,134,143,144,146]. Combined use of MF plus CC in PCOS showed significant improvement in clinical indices of pregnancy and the combination therapy is more effective than the use of CC alone. However, a number of studies reported no effect [149] or relatively weak potentiating effect of MF for CC therapy [134]. One of the possible reasons for these contradictory results may be the difference in the sensitivity to CC and MF in PCOS patients. The most profound potentiating effect of MF on the induction of ovulation and pregnancy rates is found in patients with a pronounced resistance to CC [144,146,150]. However, some PCOS patients may be also insensitive to MF, which is due to many factors, including the polymorphisms and inactivating mutations in the transmembrane proteins facilitating intracellular transport MF [151]. As a result, the combined therapy is expected to benefit mainly PCOS patients with reduced sensitivity to CC, pronounced obesity, IR and dyslipidemia, and high sensitivity to MF.
In recent years, the data have been obtained for the effectiveness of the combined use of MF and letrozole, an aromatase inhibitor that is widely used to restore the ovarian cycle and induction of ovulation and improves oocyte implantation and pregnancy rates in women with PCOS, primarily those with reduced sensitivity to CC [144,148,150]. The combined therapy with MF demonstrated enhancement for the improving effects of letrozole on the pregnancy and live birth rates. Moreover, there are clinical results showing that the combined use of letrozole and MF is more effective than the combined use of CC and MF [145,147].
In PCOS patients, the efficiency of MF therapy is increased when MF is used with oral estrogen-progestin contraceptives, both acting similar, by suppressing ovarian androgen overproduction and normalizing menstrual cycle, most noticeably in obese PCOS women [152,153]. On a contrary, when the same combined treatment (MF and oral contraceptives) is used for PCOS women with normal or reduced body weight, it results in a decrease in their muscles mass, leads to the water retention and the formation of an “osteosarcopenic” phenotype [154]. Two main reasons are behind the decrease in the muscles mass during combined therapy. First, MF and oral contraceptives reduce the blood androgen levels. It is known that in PCOS there is a significant positive correlation between the blood level of androgens and the muscles mass [155]. Second, MF-induced activation of AMPK and changes in mitochondrial energy status stimulate catabolic processes in the muscles tissue, which leads to muscles atrophy, as shown in patients with T2DM [156]. In this regard, it should be noted that MF treatment of T2DM patients leads to an increase in the blood level of fibroblast growth factor 21 (FGF21), which is one of the specific markers of muscles damage and degeneration [157]. Thus, it is highly recommended to take into account the proportion of the muscle tissue and body mass index in PCOS women, as well as the severity of HA when considering the option of using the combined therapy of MF and oral contraceptives [154].
The agonists of glucagon-like peptide-1 (GLP-1) receptor and the inhibitors of dipeptidyl peptidase-4 are widely used to treat T2DM and MetS [158,159,160,161], but they can also be used to correct the metabolic alterations and IR in PCOS women, as well as in pregnant women with GDM and T2DM [162,163]. It is shown that MF enhances the beneficial effect of liraglutide, a selective GLP-1 receptor agonist, on insulin sensitivity and glucose homeostasis. The 12-week treatment of 30 obese PCOS women with a combination of MF (1000 mg twice a day) and liraglutide (1.2 mg/day) causes a decrease in IR and normalizes the sensitivity of patients to glucose, and the combined therapy was more effective than monotherapy [164]. The treatment of premenopausal PCOS women with MF (2000 mg/day), saxagliptin (5 mg/day), an inhibitor of dipeptidyl peptidase-4, or a combination of MF and saxagliptin leads to normalization of glucose tolerance on average of 56% of patients [165]. Moreover, in the group treated with MF alone or saxagliptin alone, the improvement of glycemic control is demonstrated only in 25 and 55% of patients, respectively, while the combined therapy restores glucose tolerance in 91% of women with PCOS [165]. A high efficacy of the combined therapy was shown by other group of authors who monitored the 16-week treatment of 38 women with pre-diabetes and PCOS using the MF plus saxagliptin [166]. Weight loss and decrease in hyperglycemia and IR, which are induced by treatment of obese PCOS patients with GLP-1 receptor agonists, lead to a decrease in HA [167,168,169] and an improvement in menstrual frequency [167,169]. Liraglutide, an analogue of GLP-1, normalizes the menstrual cycle and fertility in women with HAIR-AN syndrome, which is due to a decrease in the levels of androgens and insulin [170]. Consequently, in PCOS patients, MF-induced potentiation of the metabolic-improving effects of GLP-1 agonists may also increase their restorative effects on the menstrual cycle and fertility.
5. Conclusions
The data presented in the review convincingly prove that MF has an improving effect on reproductive functions in women with PCOS, GDM and T2DM. At the same time, the effectiveness of MF therapy is due to a large number of different factors that must be taken into account when choosing this therapy and also when developing a strategy for using MF. Firstly, it is necessary to assess the efficiency of MF transport into the cell, which depends on the functional activity of the organic cation transporters and can be disrupted by inactivating mutations in their genes. The presence of certain mutations leads to a loss of responsiveness to MF and makes the use of MF therapy meaningless. Secondly, MF therapy is more effective in the severely overweight and obese patients with IR, compensatory hyperinsulinemia, impaired glucose tolerance, as well as with dyslipidemia, which is due to a decrease in the blood levels of HDL-C. This is not surprising, since the clinical effect of MF therapy is due to an improvement in insulin sensitivity, a decrease in the adipose tissue mass, and restoration of the glucose and lipid metabolism.
In addition, as demonstrated in a number of clinical studies in PCOS women, the effectiveness of MF is largely determined by the hormonal status of the ovaries and the functioning of the HPG axis. The MF therapy may be most effective in PCOS women who have: (1) severe HA, which may be due to hyperactivation of ovarian insulin/IGF-1-regulated signaling pathways that stimulate androgen synthesis, as well as an increase in LH levels and the LH/FSH ratio and a decrease in the blood levels of IGFBP-1 and SHBG; (2) an increase in the AMH production; and (3) an increased aromatase expression and FSH-induced estrogen synthesis in the ovaries. There is reason to believe that various combinations of these factors, including those with IR and metabolic disorders, may become reliable indications for prescribing MF alone and in combinations with other drugs, diet or exercises to correct the reproductive functions in PCOS. This can be helpful when using MF to treat the pregnant women with GDM and T2DM.
Since some of MF targets overlap well with those of leptin, the assessment of leptin status in patients with reproductive disorders may also be important. As a result, leptin resistance, both systemic and in the ovaries, as well as the changes in the hypothalamic leptin signaling pathways, negatively affecting the production of GnRH, can become factors that will determine the effectiveness of MF therapy. In this regard, it should be noted that the central mechanisms of action of MF, which easily penetrates the CNS and improves the metabolism of the neuronal and glial cells, still remain underestimated. By acting on the CNS, MF restores the signaling networks of the hypothalamus and the other brain regions that are involved in the control of reproductive functions and undergo significant compensatory and pathological changes in metabolic and endocrine disorders, including PCOS, GDM, T2DM, and MetS.
A unique feature of MF is the multiplicity of molecular mechanisms of its action on target cells, which include direct or indirect regulation of the AMPK-, calcium- and cAMP-dependent signaling pathways, as well as the MAPK cascade and the IRS/PI 3-K/Akt pathway. As a result, MF controls not only energy and metabolic processes in the cell, but also the processes of growth, differentiation, apoptosis, inflammation, and ER stress. At the same time, most of the regulatory effects of MF are based largely on its modulating and normalizing influence on intracellular signaling cascades than on their prolonged stimulation or suppression. Depending on the functional state of the IRS/PI 3-K/Akt pathway, MF can either prevent its hyperactivation, which is especially important for its antitumor effect, or, on the contrary, restore its reduced activity, improving the survival of target cells and their sensitivity to insulin and leptin. As expected, MF therapy affects the responsiveness of hypothalamic neurons, pituitary gonadotrophs, and ovarian cells to the hormones, growth factors, adipokines and cytokines, but more studies are required for complete elucidation of all regulatory mechanisms involved.
The use of MF in combination with the other drugs has great potential. This is supported by the encouraging results of clinical trials of the combined therapy with MF and insulin in pregnant women with GDM and T2DM. In metabolic and endocrine disorders, the combined therapy not only allows to increase the efficiency and pattern of the effects of MF on the HPG axis, but also to reduce the pharmacological doses of drugs, including MF, thus avoiding possible side effects of high-dose drug administration, including the undesirable effect of MF on the functioning of the gastrointestinal tract.
The presented results indicate a significant and not yet fully understood potential of MF therapy for the correction of reproductive dysfunctions in women. Significantly, of great importance are the absence of a teratogenic effect of MF and the low risks of MF therapy on the health of the mother and child. It should be taken into account that the unjustified use of MF for the treatment of patients lacking profoundly manifesting metabolic and endocrine disorders can lead to energy and metabolic imbalance and further deterioration of the functional state of their reproductive system.
This entry is adapted from the peer-reviewed paper 10.3390/ph14010042