**2. Autophagy and placenta**

The placental development requires multiples roles of autophagy. Experimental studies suggest that autophagy plays important functions in survival of neonates during nutritional deficiency at the early stage of birth [40]. Moreover, it was seen that the growth and remodeling of cervical fascia progress by autophagy regulation. Meanwhile, it has not been reported that autophagy affects differentiation

of trophoblasts in pregnant women by LC3-II and beclin-1, markers which are analyzed and compared between term placentas, and in their first trimester of gestation [39]. Autophagy is essential for placental development and for maintaining pregnancy, and the disruption of autophagy in extravillous trophoblast (EVT) contributes to hypoplastic placentation. Placentas of patients with preeclampsia present high levels of autophagy, with lower LC3 activation, and higher apoptosis than normal pregnancies. When induced by external factors such as hypoxia, autophagy directly affects trophoblast infiltration during normal placental development [41].

The study of autophagy markers in preeclampsia demonstrated that there are different patterns during normal pregnancy and preeclampsia, in part, because of the environmental factors, like hypoxia. Cells exposed to hypoxic conditions demonstrated higher levels of LC3, beclin-1, and the autophagosome formation when compared to normal placentas. Saito and Nakashima [24] reported that poor placentation is induced by decreased infiltration of trophoblasts due to abnormal processing for autophagy, which is activated by soluble endoglin (sENG). Inactivation of autophagy represses trophoblast infiltration and vascular remodeling due to excessive hypoxia, causing poor placentation, as observed in preeclampsia [26]. Hung and collaborators showed that autophagy decreased with advancing of gestational age in placentas of normotensive women, through analyses of changes in LC3-II and p62 according to gestational weeks [42].

The determination of p62 levels also has a few reports and in addition with the analysis of LC3-II [2, 14, 43] may be important to determine autophagic status in the villous tissue.

This important finding has been the basis of several investigations on oxidative stress, an alteration identified in placental related disorders, and whether this oxidative stress is able to induce autophagy activation on placental explants. We developed an in vitro study with the objective of evaluating the effect of oxidative stress induced by hydrogen peroxide (H2O2) on the occurrence of autophagy activation in placental explants of pregnant women at term (39–40 weeks), without clinical or obstetric disorders identified and undergone to elective cesarean section. In the methodology, we intended to reproduce experimentally the pathophysiology of obstetric complications related to placental dysfunction caused by uterine circulatory alterations. These alterations are responsible for the establishment of the hypoxia-reoxygenation phenomenon also called as ischemia-reperfusion injury and consequent production of (ROS).

The results showed the higher gene expression of LC3-II, beclin-1, and p62 detected in cultures exposed to different concentrations of H2O2 and demonstrated that the oxidative stress generated was able to induce autophagy in placental explants (**Figure 2**). Material and methods of this experiment are shown as supplementary material to this chapter.

Gene expression of LC3-II (**Figure 2A**) was increased in tissues exposed to the concentration of 1000 μM of H2O2 compared to the non-exposed cultures, which means that autophagy was more activated in this concentration, in response to the oxidative effect. Gene expression of beclin-1 and p62 (**Figure 2B** and **C**) increased in front of increasing concentrations of H2O2 with the maximum value in 1000 μM of H2O2, showing statistical difference (p < 0.05) when compared to controls.

In the present study, the higher gene expression of LC3-II, beclin-1, and p62 detected in cultures exposed to H2O2 demonstrated that the oxidative stress generated was able to induce autophagy in placental explants.

The high expressions of mRNA for LC3-II and TNF-α (**Figure 3A** and **B**) demonstrated in our study corroborate with other reports, showing that placental

**81**

**Figure 3.**

**Figure 2.**

*Autophagy in Preeclampsia*

*DOI: http://dx.doi.org/10.5772/intechopen.85592*

*Activation of autophagy in cultures exposed to different concentrations of hydrogen peroxide. mRNA expression of LC3-II (A), beclin-1 (B), and p62 (C) in placental explants. Results expressed as mean ± SD. \* (p < 0.05) vs.* 

*Pro-inflammatory cytokine profile in cultures exposed to different concentrations of hydrogen peroxide. Gene and protein expression of pro-inflammatory cytokines TNF-α(A/B) and IL-1β (C/D) in placental explants.* 

*Results expressed as mean ± SD. \* (p < 0.05) vs. 0 (ANOVA). Source: own authorship.*

*0; # (p < 0.05) vs. 0, 10, 100 μM (ANOVA). Source: own authorship.*

*Autophagy in Preeclampsia DOI: http://dx.doi.org/10.5772/intechopen.85592*

**Figure 2.**

*Prediction of Maternal and Fetal Syndrome of Preeclampsia*

in LC3-II and p62 according to gestational weeks [42].

development [41].

the villous tissue.

consequent production of (ROS).

mentary material to this chapter.

ated was able to induce autophagy in placental explants.

of trophoblasts in pregnant women by LC3-II and beclin-1, markers which are analyzed and compared between term placentas, and in their first trimester of gestation [39]. Autophagy is essential for placental development and for maintaining pregnancy, and the disruption of autophagy in extravillous trophoblast (EVT) contributes to hypoplastic placentation. Placentas of patients with preeclampsia present high levels of autophagy, with lower LC3 activation, and higher apoptosis than normal pregnancies. When induced by external factors such as hypoxia, autophagy directly affects trophoblast infiltration during normal placental

The study of autophagy markers in preeclampsia demonstrated that there are different patterns during normal pregnancy and preeclampsia, in part, because of the environmental factors, like hypoxia. Cells exposed to hypoxic conditions demonstrated higher levels of LC3, beclin-1, and the autophagosome formation when compared to normal placentas. Saito and Nakashima [24] reported that poor placentation is induced by decreased infiltration of trophoblasts due to abnormal processing for autophagy, which is activated by soluble endoglin (sENG). Inactivation of autophagy represses trophoblast infiltration and vascular remodeling due to excessive hypoxia, causing poor placentation, as observed in preeclampsia [26]. Hung and collaborators showed that autophagy decreased with advancing of gestational age in placentas of normotensive women, through analyses of changes

The determination of p62 levels also has a few reports and in addition with the analysis of LC3-II [2, 14, 43] may be important to determine autophagic status in

This important finding has been the basis of several investigations on oxidative stress, an alteration identified in placental related disorders, and whether this oxidative stress is able to induce autophagy activation on placental explants. We developed an in vitro study with the objective of evaluating the effect of oxidative stress induced by hydrogen peroxide (H2O2) on the occurrence of autophagy activation in placental explants of pregnant women at term (39–40 weeks), without clinical or obstetric disorders identified and undergone to elective cesarean section. In the methodology, we intended to reproduce experimentally the pathophysiology of obstetric complications related to placental dysfunction caused by uterine circulatory alterations. These alterations are responsible for the establishment of the hypoxia-reoxygenation phenomenon also called as ischemia-reperfusion injury and

The results showed the higher gene expression of LC3-II, beclin-1, and p62 detected in cultures exposed to different concentrations of H2O2 and demonstrated that the oxidative stress generated was able to induce autophagy in placental explants (**Figure 2**). Material and methods of this experiment are shown as supple-

Gene expression of LC3-II (**Figure 2A**) was increased in tissues exposed to the concentration of 1000 μM of H2O2 compared to the non-exposed cultures, which means that autophagy was more activated in this concentration, in response to the oxidative effect. Gene expression of beclin-1 and p62 (**Figure 2B** and **C**) increased in front of increasing concentrations of H2O2 with the maximum value in 1000 μM of H2O2, showing statistical difference (p < 0.05) when compared to controls. In the present study, the higher gene expression of LC3-II, beclin-1, and p62 detected in cultures exposed to H2O2 demonstrated that the oxidative stress gener-

The high expressions of mRNA for LC3-II and TNF-α (**Figure 3A** and **B**) demonstrated in our study corroborate with other reports, showing that placental

**80**

*Activation of autophagy in cultures exposed to different concentrations of hydrogen peroxide. mRNA expression of LC3-II (A), beclin-1 (B), and p62 (C) in placental explants. Results expressed as mean ± SD. \* (p < 0.05) vs. 0; # (p < 0.05) vs. 0, 10, 100 μM (ANOVA). Source: own authorship.*

#### **Figure 3.**

*Pro-inflammatory cytokine profile in cultures exposed to different concentrations of hydrogen peroxide. Gene and protein expression of pro-inflammatory cytokines TNF-α(A/B) and IL-1β (C/D) in placental explants. Results expressed as mean ± SD. \* (p < 0.05) vs. 0 (ANOVA). Source: own authorship.*

hypoxic environment and the presence of TNF-α caused a positive regulation in the autophagy-related protein LC3-II in a trophoblastic cell line [26].

Gene expressions of TNF-α and IL-1β were higher in explants cultured with 1000 μM of H2O2 with significant difference (p < 0.05) to culture without H2O2 (**Figure 3A** and **C**). The protein expressions of these cytokines (**Figure 3B** and **D**) were higher in the supernatants from concentrations of 100 to 1000 μM of H2O2, with difference (p < 0.05) to non-exposed and cultures with 10 μM of H2O2. The protein expression of IL-1 β was higher in 1000 μM of H2O2 (p < 0.05) than in the culture of 100 μM of H2O2 (**Figure 3D**).

The similar patterns of p62, beclin-1, and LC3-II gene expressions suggest the production of mRNA for the p62 protein transcription, which will be degraded during autophagy activation. This protein plays a significant role on the regulation of oxidative stress, degenerative diseases, and carcinogenesis [44].

### **3. Autophagy and inflammasomes**

Inflammasome is a molecular platform composed of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) proteins. It is required for the activation of caspase-1 and subsequent maturation of the pro-inflammatory cytokines interleukin-1β and IL-18 [45]. NLRs respond to different endogenous or exogenous stimuli and activate caspase-1. During inflammation, these pro-inflammatory molecules are maturated in inflammasomes, which are in part regulated by the autophagy mechanism.

Actually, autophagy can control endogenous inflammasome activators, such as pro-IL-1β, which regulates the secretion of IL-1β and IL-18, thereby preventing an exaggerated inflammation [9, 45, 46].The activation of the inflammasome NLRP3 occurs via two signals: the first signal is provided by NF-kB activators and is a prerequisite for inflammasome activation via NLRP3 expression in macrophages [47]; the second signal activates NLRP3 inflammasome directly and involves host-derived adenosine triphosphate (ATP), uric acid crystals, bacterial toxins, or particulate matter (**Figure 4**) [48].

Some studies have demonstrated a mutual relationship between autophagy and inflammasomes. Autophagy negatively regulates inflammasome activation. Autophagy induction is dependent on the presence of specific inflammasome sensors, inflammasomes are ultimately degraded by autophagosomes via the selective autophagic receptor p62, and autophagy plays a role in the biogenesis and secretion of the pro-inflammatory cytokine IL-1β [45, 9, 49].

The role of autophagy in inflammasome regulation may depend on the context of danger signal. In the absence of a danger signal, autophagy can act removing IL-1β and inflammasome components while maintaining cellular homeostasis. In the presence of a danger signal, autophagy may act initially as a secretory pattern to diffuse inflammation while preventing cell death and pyroptosis. Recent studies showed that macrophages may activate autophagy in response to inflammasome activation, as a way to delay the onset of pyroptosis. According to the authors, the inhibition of autophagy resulted in increased activation of pyroptosis and the impact of these types of cell death regulation by autophagy need to be more studied on inflammatory process [50]. When genes of autophagy regulator Atg 16L1 or Atg7 are deleted or a chemical inhibitor of autophagy is applied, LPS-dependent inflammasome activation occurs suggesting that autophagy controls inflammasome activation and can limit production of cytokines IL-1β and IL-18 [51]. Induced autophagy to inhibit the inflammasome and excessive inflammation or marking directly specific NLRs (NOD-like

**83**

**Figure 5.**

*Autophagy in Preeclampsia*

tory process [52].

**Figure 4.**

(**Figure 3C** and **D**).

inflammasome activation [53].

*NLRP3 inflammasome. Source: own authorship.*

*DOI: http://dx.doi.org/10.5772/intechopen.85592*

receptors) to reduce its activity may be a promising strategy to reverse inflamma-

In a previous study published by our research group, we observed that some markers of oxidative stress, such as superoxide dismutase (SOD) and catalase are altered at high concentrations of hydrogen peroxide confirming that H2O2 induces oxidative stress on placental explants and demonstrated that this stress involves

We demonstrated that gene expressions of NLRP3 and caspase-1 have similar

patterns, with greater expression in cultures exposed to the concentration of 1000 μM of H2O2, which means that this concentration was able to activate the NLRP3 inflammasome (**Figure 5**). The activation of this complex may occur as a

*Inflammasome activation in placental explants in cultures exposed to different concentrations of hydrogen peroxide. mRNA expression of NLRP3 (A) and caspase-1 (B) in explants of placental explants. Results* 

*expressed as mean ± SD. \* (p < 0.05) vs. 0, 10 μM (ANOVA). Source: own authorship.*

In the present study, the oxidative stress induced by H2O2 on placental explants contributed to the inflammatory profile generated by activating the NLRP3 inflammasome, caspase-1 and inducing the release of IL-1β

*Activation of NLRP3 inflammasome. This process requires two signals: the first is dependent of NF-kB activators and the second demands ATP, uric acid crystals, bacterial toxins or particulate matter to activate* 

#### **Figure 4.**

*Prediction of Maternal and Fetal Syndrome of Preeclampsia*

culture of 100 μM of H2O2 (**Figure 3D**).

**3. Autophagy and inflammasomes**

autophagy mechanism.

particulate matter (**Figure 4**) [48].

of the pro-inflammatory cytokine IL-1β [45, 9, 49].

autophagy-related protein LC3-II in a trophoblastic cell line [26].

of oxidative stress, degenerative diseases, and carcinogenesis [44].

hypoxic environment and the presence of TNF-α caused a positive regulation in the

Gene expressions of TNF-α and IL-1β were higher in explants cultured with 1000 μM of H2O2 with significant difference (p < 0.05) to culture without H2O2 (**Figure 3A** and **C**). The protein expressions of these cytokines (**Figure 3B** and **D**) were higher in the supernatants from concentrations of 100 to 1000 μM of H2O2, with difference (p < 0.05) to non-exposed and cultures with 10 μM of H2O2. The protein expression of IL-1 β was higher in 1000 μM of H2O2 (p < 0.05) than in the

The similar patterns of p62, beclin-1, and LC3-II gene expressions suggest the production of mRNA for the p62 protein transcription, which will be degraded during autophagy activation. This protein plays a significant role on the regulation

Inflammasome is a molecular platform composed of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) proteins. It is required for the activation of caspase-1 and subsequent maturation of the pro-inflammatory cytokines interleukin-1β and IL-18 [45]. NLRs respond to different endogenous or exogenous stimuli and activate caspase-1. During inflammation, these pro-inflammatory molecules are maturated in inflammasomes, which are in part regulated by the

Actually, autophagy can control endogenous inflammasome activators, such as pro-IL-1β, which regulates the secretion of IL-1β and IL-18, thereby preventing an exaggerated inflammation [9, 45, 46].The activation of the inflammasome NLRP3 occurs via two signals: the first signal is provided by NF-kB activators and is a prerequisite for inflammasome activation via NLRP3 expression in macrophages [47]; the second signal activates NLRP3 inflammasome directly and involves host-derived adenosine triphosphate (ATP), uric acid crystals, bacterial toxins, or

Some studies have demonstrated a mutual relationship between autophagy and inflammasomes. Autophagy negatively regulates inflammasome activation. Autophagy induction is dependent on the presence of specific inflammasome sensors, inflammasomes are ultimately degraded by autophagosomes via the selective autophagic receptor p62, and autophagy plays a role in the biogenesis and secretion

The role of autophagy in inflammasome regulation may depend on the context of danger signal. In the absence of a danger signal, autophagy can act removing IL-1β and inflammasome components while maintaining cellular homeostasis. In the presence of a danger signal, autophagy may act initially as a secretory pattern to diffuse inflammation while preventing cell death and pyroptosis. Recent studies showed that macrophages may activate autophagy in response to inflammasome activation, as a way to delay the onset of pyroptosis. According to the authors, the inhibition of autophagy resulted in increased activation of pyroptosis and the impact of these types of cell death regulation by autophagy need to be more studied on inflammatory process [50]. When genes of autophagy regulator Atg 16L1 or Atg7 are deleted or a chemical inhibitor of autophagy is applied, LPS-dependent inflammasome activation occurs suggesting that autophagy controls inflammasome activation and can limit production of cytokines IL-1β and IL-18 [51]. Induced autophagy to inhibit the inflammasome and excessive inflammation or marking directly specific NLRs (NOD-like

**82**

*Activation of NLRP3 inflammasome. This process requires two signals: the first is dependent of NF-kB activators and the second demands ATP, uric acid crystals, bacterial toxins or particulate matter to activate NLRP3 inflammasome. Source: own authorship.*

receptors) to reduce its activity may be a promising strategy to reverse inflammatory process [52].

In the present study, the oxidative stress induced by H2O2 on placental explants contributed to the inflammatory profile generated by activating the NLRP3 inflammasome, caspase-1 and inducing the release of IL-1β (**Figure 3C** and **D**).

In a previous study published by our research group, we observed that some markers of oxidative stress, such as superoxide dismutase (SOD) and catalase are altered at high concentrations of hydrogen peroxide confirming that H2O2 induces oxidative stress on placental explants and demonstrated that this stress involves inflammasome activation [53].

We demonstrated that gene expressions of NLRP3 and caspase-1 have similar patterns, with greater expression in cultures exposed to the concentration of 1000 μM of H2O2, which means that this concentration was able to activate the NLRP3 inflammasome (**Figure 5**). The activation of this complex may occur as a

#### **Figure 5.**

*Inflammasome activation in placental explants in cultures exposed to different concentrations of hydrogen peroxide. mRNA expression of NLRP3 (A) and caspase-1 (B) in explants of placental explants. Results expressed as mean ± SD. \* (p < 0.05) vs. 0, 10 μM (ANOVA). Source: own authorship.*

consequence of a common form of cellular stress initiated by different stimuli, such as the release of ROS [54, 55].

The relationship between occurrence of inflammasome and autophagy activation may be explained by the elevation in gene expression of p62 under conditions of oxidative stress. Inflammasome can be degraded by autophagosomes through this protein [56].

## **4. Conclusion**

Taken together, the results of the present study confirm that H2O2 induces oxidative stress in placental explants, demonstrated by activation of NLRP3 inflammasome, which in turn induce the autophagy activation in order to control the inflammatory state. Activation of inflammasome and autophagy are essential elements of the innate immune system, and disorders in these processes have been implicated in various inflammatory and infectious diseases [57]. Oxidative stress may also contribute to placental tissue senescence and to the pathophysiology of some placental-related disorders of pregnancy, such as preeclampsia and fetal growth restriction [56]. Thus, initiatives to reduce stress on trophoblastic tissue should be considered for future researches.

Many studies have observed the effects of supplementation to prevent the effects of oxidative stress and autophagy in preeclampsia, such as the use of antioxidants, vitamins C and E, calcium, resveratrol and some natural products [58–61].

The use of natural products and hormones such as Vitamin D may be a new model to reduce inflammation by regulating autophagy, since there is a direct correlation between vitamin D levels and cell survival in pathologies associated with gestation. Vitamin D and its components such as vitamin D receptor (VDR) are molecules that are highly related to the autophagic process [62]. In this sense, the use of products with antioxidant and anti-inflammatory effects still need to be evaluated in order to reduce oxidative stress, induce autophagy, and decrease the activation of inflammasome in placental tissue.

#### **Acknowledgements**

Results showed in this chapter were supported by the Fundação de Amparo a Pesquisa do Estado de São Paulo, FAPESP (Grant No 2014/25611-5).

### **Conflict of interest**

The authors declare that they have no conflict of interest.

### **Supplementary material**

#### **1. Material and methods**

#### **1.1 Study population and ethics statement**

This study consisted of 15 healthy pregnant women with normal evolution of the pregnancy, with no personal history of hypertensive disorders in pregnancy. These pregnant women were admitted to the Obstetrics Unit of Botucatu Medical School,

**85**

*Autophagy in Preeclampsia*

*DOI: http://dx.doi.org/10.5772/intechopen.85592*

**1.2 Collection of placental tissue**

**1.4 Cell viability assay**

to the manufacturer's instructions.

Sao Paulo State University, Botucatu, SP, Brazil between November 2015 and May 2016. Gestational age was calculated from the last menstrual period and confirmed by ultrasound dating. Exclusion criteria included chronic hypertension, multiple gestation, prior preeclampsia, illicit drug use, and preexisting medical conditions such as diabetes, cancer, acute infectious disease, cardiovascular, autoimmune, renal and hepatic diseases. The study was approved by the Ethics Committee of the Botucatu Medical School, and written informed consent was obtained from all women involved in the study (CAAE Protocol number: 37160614500005411).

All placentas from normotensive pregnant women were delivered by cesarean section, without labor and were examined macroscopically and processed within 10 min of delivery. Fragments of approximately 5 × 5 cm were immediately removed from the central region of the placenta, constituting samples of the villous cytotrophoblast and the syncytiotrophoblast region in contact with the maternal side (basal plate). After collection, the trophoblastic tissue was washed in buffered saline (PBS) and separated from the decidual layer that is normally adhered to the basal plate. The terminal portions of the villi were evidenced in PBS (the villi were seen floating

The amount of villi used was 11 mg of placental tissue that was cultured in each

After the culture periods, the explants were removed and submitted to RNA extraction for further analysis of the expression of genes related to inflammation (cytokines), autophagy, and inflammasome. Culture supernatants were obtained, centrifuged at 2,000 g for 10 min and stored at −80°C for determination of cytokines.

The cell viability assay was conducted through the activity of the enzyme lactate dehydrogenase (LDH) in supernatants of placental explant after 24, 48, 72, and 96 h of culture and was determined by commercial kit (Sigma-Aldrich) according

well of 24-well plates (SPL Life Sciences, Korea) for 24 h for stabilization [63]. Cultures were performed in vitro in the absence of hydrogen peroxide or in the presence of 10, 100, and 1,000 μM of H2O2 for 4 h and 24 h in RPMI 1640 culture medium supplemented with 2 mM L-glutamine (Sigma-Aldrich, St Louis, MO, USA), 40 mg/ml antibiotic/antimycotic (Sigma-Aldrich), and 10% fetal bovine serum (Gibco BRL Life

Technologies, The Netherlands) inactivated (complete RPMI medium).

**1.5 Evaluation of the expression of transcripts related to inflammation**

The placental explants were evaluated for the expression of the genes encoding IL-1β, TNF-α, LC3-II, beclin-1, and p62 proteins at the transcriptional level. In addition, the gene expression of the inflammasome was evaluated through the NLRP3 and caspase-1 genes. Total RNA was extracted from the placentas using the Total RNA Purification Kit (Norgen Biotek Corp., Thorold, Canada) according to the manufacturer's protocol, and the Reverse Transcription-coupled polymerase chain reaction (RT-qPCR) was performed as described previously [64]. Briefly, isolated RNA was DNAse I Amp Grade (Invitrogen) treated. Subsequently, the synthesis of complementary DNA (cDNA) was conducted using ImProm-IITM Reverse Transcription System,

freely in the liquid) and dissected in small sections to constitute explants.

**1.3 Culture of placental explants with hydrogen peroxide**

#### *Autophagy in Preeclampsia DOI: http://dx.doi.org/10.5772/intechopen.85592*

*Prediction of Maternal and Fetal Syndrome of Preeclampsia*

should be considered for future researches.

activation of inflammasome in placental tissue.

**Acknowledgements**

**Conflict of interest**

**Supplementary material**

**1. Material and methods**

**1.1 Study population and ethics statement**

as the release of ROS [54, 55].

protein [56].

**4. Conclusion**

consequence of a common form of cellular stress initiated by different stimuli, such

The relationship between occurrence of inflammasome and autophagy activation may be explained by the elevation in gene expression of p62 under conditions of oxidative stress. Inflammasome can be degraded by autophagosomes through this

Taken together, the results of the present study confirm that H2O2 induces oxidative stress in placental explants, demonstrated by activation of NLRP3 inflammasome, which in turn induce the autophagy activation in order to control the inflammatory state. Activation of inflammasome and autophagy are essential elements of the innate immune system, and disorders in these processes have been implicated in various inflammatory and infectious diseases [57]. Oxidative stress may also contribute to placental tissue senescence and to the pathophysiology of some placental-related disorders of pregnancy, such as preeclampsia and fetal growth restriction [56]. Thus, initiatives to reduce stress on trophoblastic tissue

Many studies have observed the effects of supplementation to prevent the effects of oxidative stress and autophagy in preeclampsia, such as the use of antioxidants,

vitamins C and E, calcium, resveratrol and some natural products [58–61].

The use of natural products and hormones such as Vitamin D may be a new model to reduce inflammation by regulating autophagy, since there is a direct correlation between vitamin D levels and cell survival in pathologies associated with gestation. Vitamin D and its components such as vitamin D receptor (VDR) are molecules that are highly related to the autophagic process [62]. In this sense, the use of products with antioxidant and anti-inflammatory effects still need to be evaluated in order to reduce oxidative stress, induce autophagy, and decrease the

Results showed in this chapter were supported by the Fundação de Amparo a

This study consisted of 15 healthy pregnant women with normal evolution of the pregnancy, with no personal history of hypertensive disorders in pregnancy. These pregnant women were admitted to the Obstetrics Unit of Botucatu Medical School,

Pesquisa do Estado de São Paulo, FAPESP (Grant No 2014/25611-5).

The authors declare that they have no conflict of interest.

**84**

Sao Paulo State University, Botucatu, SP, Brazil between November 2015 and May 2016. Gestational age was calculated from the last menstrual period and confirmed by ultrasound dating. Exclusion criteria included chronic hypertension, multiple gestation, prior preeclampsia, illicit drug use, and preexisting medical conditions such as diabetes, cancer, acute infectious disease, cardiovascular, autoimmune, renal and hepatic diseases. The study was approved by the Ethics Committee of the Botucatu Medical School, and written informed consent was obtained from all women involved in the study (CAAE Protocol number: 37160614500005411).
