Melatonin Ameliorates Ovarian Hyperstimulation Syndrome (OHSS) through SESN2 Regulated Antiapoptosis

Background Ovarian hyperstimulation syndrome (OHSS) is one of the most severe complications after ovarian stimulation during assisted reproductive technology (ART). However, its pathogenesis still remains unclear. Melatonin is an important antioxidant factor in female reproduction and Sestrin-2 (SESN2) is reported to be involved in cellular response to different stress conditions. Whether or not melatonin and SESN2 are involved in OHSS is still a question to us clinicians. Methods and Results We collected the granulosa cells of OHSS patients and focused on the role of SESN2 in OHSS. We also studied the role and mechanism of melatonin plays in OHSS patients. We found that the expression of SESN2 was increased in the granulosa cells of OHSS patients (n = 24) than those in controls (n = 15). Incubation with angiotensin II (1 μM, 2 μM) in HUVECs and H2O2 (0.1 mM, 0.2 mM) in KGNs increased the generation of ROS concurrent with the increased expression of SESN2, while melatonin treatment partly restored SESN2 levels. The mechanism study demonstrated that SESN2 was deeply involved in the regulation of AMPK and mTOR, whereas melatonin partially restored angiotensin II or H2O2 induced the activation of AMPK phosphorylation and the inhibition of mTOR, 4EBP1 and S6K1 phosphorylation, all of which could trigger cell apoptosis. Conclusions These findings indicated that melatonin attenuated ROS-induced apoptosis through SESN2-AMPK-mTOR in OHSS. Thus, melatonin is likely to be a potential and important therapeutic agent for treating and preventing OHSS.


Introduction
Infertility is defned as the inability to be pregnant within one year of unprotected intercourse and its incidence has reached to 10-15% in recent years [1].It has become not only a medical concern but also a social issue with increasing prevalence in both developed and developing countries.In vitro fertilization (IVF) is wildly accepted over the past 35 years as an efective treatment for infertility during which controlled ovarian stimulation (COH) is almost always employed to retrieve more oocytes.Although COH might improve IVF outcome, it also increases the risk of an iatrogenic complication: ovarian hyperstimulation syndrome (OHSS).OHSS is not uncommon.Studies reported that the presence of its moderate and severe form was up to 10% of all IVF cycles [2].Te true incidence of OHSS is probably much underestimated since the symptoms of mild OHSS are easy to be ignored [3].Te symptoms of OHSS in its mild form can be untypical such as nausea and vomiting; however, moderate and severe OHSS may result in oliguria, hydrothorax, ascites, hepatorenal failure, acute respiratory distress syndrome, hemorrhage from ovarian rupture, thromboembolism, and ultimately, even death [4].Although OHSS increases the physical, psychological, and economic burden of the patients and their families, its pathogenesis is not completely understood and no specifc therapy is available for this syndrome [5,6].Terefore, prevention of OHSS becomes a crucial issue since its treatment is largely tamping down symptoms, rather than addressing causes.So far, several strategies including cycle cancellation, coasting, in vitro oocyte maturation have been used in practice to prevent OHSS.Ismet H et al. have shown that oxytocin as well as cabergoline alleviates OHSS in OHSS rat models [7].More studies should be focus on the prevention of OHSS.
Te precise cause of OHSS remains currently the subject of controversy.Nevertheless, high estradiol levels in the presence of human chorionic gonadotropin (hCG) increase the vascular endothelium permeability, leading to a massive shift of intravascular fuid into the third space.Tere are also evidences that during the pathogenesis of this iatrogenic complication, large amounts of angiotensin II, vascular endothelial growth factor (VEGF), interleukins (ILs), nitric oxide (NO), tumor necrosis factor-a (TNF-a), and other molecules are excessively produced, causing the overproduction of reactive oxygen species (ROS) which results in oxidant-antioxidant imbalance [8].Te vascular endothelium is then deteriorated by these imbalanced free radicals that cannot be antagonized by free radical scavengers; consequently, high vascular permeability occurs and fnally results in the aggravation of OHSS [9].
Melatonin is mainly secreted by pineal glands in human beings and is regulated by circadian rhythms.However, higher levels of melatonin are found in human follicular fuid than in plasma because melatonin is not only derived from the general circulation but also synthesized in the ovary (mainly by the granulosa cells) [10].Melatonin has a signifcant impact on female reproduction.It is considered essential for folliculogenesis, steroid production.Tere are also evidences that melatonin takes part in the control of pubertal onset, ovulation, sexual maturation and pregnancy protection [11].Melatonin as well as its metabolites has been proved to be a powerful radical scavenger.It reduces ROS levels in the ovary through receptor dependent and independent pathways [12,13].Recently, more and more attention has been paid to the importance of melatonin in female reproduction [14].
Sestrins are highly conserved and stress-inducible metabolic regulators which are ubiquitously expressed at diferent levels in all adult tissues [15,16].Te physiological functions of sestrins have not been fully elucidated yet.Te critical roles of sestrins in mammalian metabolism have been revealed by the deletion of these proteins which is incompatible with the survival of mice [17].Previous studies have also suggested that sestrins have a close relationship with age and oxidative stress associated diseases such as Alzheimer's disease, Parkinson's disease, and diabetes and have a favorable profle as potential therapeutic targets for these diseases [18,19].Sestrin-2 (SESN2) belongs to the sestrins family and functions as a suppressor of ROS accumulation as well as a neuroprotector [20].Te overexpression of SESN2 reduces ROS levels whereas SESN2 knockdown in cultured cells or mice increases ROS content [21,22].Moreover, any condition that leads to ROS accumulation induces SESN2 expression [19].Terefore, the increased ROS levels in OHSS may increase the expression of SESN2.Most previous studies on SESN2 have been focused on the nervous system and cardiovascular system; these studies have shown that SESN2 played an important role in preventing ROS damage, repairing mitochondria deterioration and maintaining the stability of inner environment [23,24].However, there are very few studies on the expression and the function of SESN2 in human reproduction.Since OHSS is closely associated with the excessive production of ROS and melatonin is supposed to be a powerful radical scavenger, the objective of this study was to investigate whether SESN2 are induced in OHSS and whether melatonin can alleviate oxidative stress in OHSS as well as the potential role of SESN2 in OHSS.

Clinical Sample Collection.
Te diagnosis of OHSS is based on the guideline of OHSS by Practice Committee of the American Society for Reproductive in 2016 [25].Patients present with abdominal distention, nausea, vomiting and relevant results of laboratory investigations or ultrasound scan are diagnosed as OHSS during our ART procedures after being excluded of other diseases.Te severe symptoms of OHSS include oliguria, anuria, ascites, hydrothorax and thromboembolism, acute respiratory distress syndrome.Clinical parameters including age, BMI, AMH, basal FSH, basal E 2 , E 2 on hCG administration day, number of oocytes retrieved were collected.Patients received hCG when the diameter of their follicles was >18 mm.Oocytes were collected 36 h after hCG injection by transvaginal ultrasoundguided puncture and aspiration of the follicles with a diameter of 18 to 20 mm.Te granulosa cells from patients on oocyte retrieval day were collected and purifed with Ficoll-Pague ™ PLUS (GE-HealthCareBio-Science, Sweden).Our research was approved by the Reproductive Ethics Committee of Ren Ji Hospital (No. 2015030308).Te informed consent was obtained from each patient before oocyte retrieval.

Cell
Culture.Human umbilical vein endothelial cells (HUVECs) and human granulosa cell line (KGNs) were kindly provided by Shandong University.HUVECs represent endothelial cells and KGNs are used to represent ovarian granulosa cells in previous studies [26].

Te Evaluation of Intracellular ROS of Cultured Cells.
Intracellular ROS levels were measured using fuoroprobe CM-H 2 DCFDA (Sigma-Aldrich, Saint Louis, Missouri, USA).Cells cultured in six-well plates were incubated in DMED/F12 with 10 mM CM-H 2 DCFDA for 20 min at 37 °C in a dark, humidifed chamber.CM-H 2 DCFDA fuorescence was measured using a fuorescence microscope (Zeiss, Germany) with a digital imaging system at an excitation wavelength ranging from 430 to 480 nm and the intensity of the fuorescence were analyzed using IMAGE J 1.34 s (National Institutes of Health, USA).
2.5.Western Blot.30 μg proteins were applied to an SDS gel for electrophoresis and were then transferred to polyvinylidene fuoride membranes.Nonspecifc binding sites were blocked using 5% non-fat milk for 90 min at room temperature.After blocking, the membrane was incubated overnight at 4 °C with the following primary antibodies: anti-SESN2 antibody ( 2.6.Statistical Analysis.All data were analyzed using SPSS 22.0 software (IBM, Armonk, New York, USA).Te differences between two groups were analyzed using Student's t-test.Te diferences among three groups or above were analyzed using one-way ANOVA analysis followed by the Newman-Keuls multiple comparison test.Te data are represented as mean ± standard deviation (SD) and all experiments were repeated at least three times.P < 0.05 was regarded as statistically signifcant.To further confrm the results observed, we also used KGNs cells to mimic ovarian granulosa cells.Te same phenomenon was observed in KGNs cells.Te ROS generation and SESN2 levels in cultured KGNs cells were signifcantly increased after incubation with H 2 O 2 (0.1 mM, 0.2 mM) (Figures 2(A) and 2(C), 2(E), 2(G)).

Melatonin Signifcantly Restored SESN2 Levels in Cultured
HUVECs and KGNs.Since ROS and SESN2 levels are increased in OHSS model and melatonin is a powerful radical scavenger, we explored the role of melatonin in treating OHSS.Te treatment with melatonin (10 μM) for 24 h signifcantly decreased SESN2 level induced by angiotensin II (P � 0.037) (Figures 3(a) and 3(b)), similarly, the treatment with melatonin (1 mM) for 12 h signifcantly decreased SESN2 level in KGNs cells induced by H 2 O 2 (Figures 3(c) and 3(d)).

Melatonin Inhibited Apoptosis through SESN2 Regulated
Signaling Pathway in HUVECs and KGNs.Previous studies have demonstrated that melatonin plays an antioxidant role and regulates antiapoptotic pathway [31][32][33][34].To better investigate the role and mechanism melatonin plays in OHSS, we determined the classic apoptosis related molecule levels in angiotensin II induced HUVECs treated with melatonin.Te results demonstrated that incubation with angiotensin II (1 μM) signifcantly increased the levels of p17 caspase 3 and decreased BCL2 level, which were partly restored by the treatment with melatonin (10 μM)(P < 0.05) (Figures 4(a) 4(b)).SESN2 has previous been demonstrated to be involved in the AMPK-mTOR signaling pathway which is related to apoptosis.Terefore, we studied this signal pathway and found that incubation with angiotensin II (1 μM) in cultured HUVECs signifcantly increased the levels of p-AMPK and decreased the levels of p-mTOR and its downstream molecules (p-S6K1, p-4EBP1), which were partly restored by the treatment with melatonin (10 μM) (P < 0.05) (Figures 4(a Obstetrics and Gynecology International

Discussion
In this study, we demonstrated that SESN2 level was increased in the granulosa cells of OHSS patients and was involved in the oxidative stress of OHSS by regulating the apoptosis of endothelium cells.Incubation with angiotensin II in cultured HUVECs and H 2 O 2 in cultured KGNs induced augmented ROS generation and increased SESN2 expression, both of which were restored by the treatment of melatonin.Tese benefcial efects of melatonin could be explained partly by regulating antiapoptosis through SESN2-AMPK-mTOR.OHSS is associated with more morbidity and mortality than other complications during IVF process.Despite strides to reduce the incidence of this potentially fatal and completely iatrogenic complication, it remains a serious health concern for a signifcant percentage of patients undergoing IVF.Te prediction and prevention of OHSS are crucial for clinicians.In our research, OHSS patients exhibit signifcantly higher E 2 levels on HCG day compared to non-OHSS patients, while basal E 2 levels remain unchanged.FSH secreted by the pituitary gland stimulates the granulosa cells in antral follicles to produce and release E 2 during the early follicular stage.E 2 , in turn, regulates the FSH level through negative feedback.In the early stages of decreased ovarian function, with a reduced number of antral follicles, the pituitary gland must secrete more FSH to maintain similar basal E 2 levels.As a result, basal FSH levels increase while basal E 2 levels remain the same.Most OHSS cases occur in women with better ovarian reserves, which explains why OHSS patients have lower basal FSH levels compared to non-OHSS women but higher AMH levels, while their basal E 2 levels do not difer signifcantly, as demonstrated in our study.OHSS patients typically have better ovarian reserves and a greater number of ovarian follicles.During controlled ovarian stimulation with exogenous GnRH, their E 2 levels on HCG day are higher than those of non-OHSS patients.It was previously thought that elevated plasma E 2 concentrations or rapid increases in E 2 levels during HCG ovulation induction indicated a higher sensitivity to HCG.However, it is now understood that E 2 levels alone are not reliable predictors of OHSS [35,36].Our study corroborates previous research, demonstrating that it is not the basal E 2 levels but rather the E 2 levels on HCG day that can serve as predictors of OHSS [37,38].

Obstetrics and Gynecology International
Te development of OHSS is always accompanied by elevated E 2 levels during the process of ART, and E 2 has been implicated as a potential etiologic factor, one of the possible reasons is that elevated E 2 increases capillary permeability so VEGF and other chemical mediators or precursors which augment fuid extravasation increase and thus OHSS developed.Te overabundance of ROS released by VEGF and infammatory factors causes the dilatation of endothelium, which leads to a massive shift of the body fuids from the vessels into the third space and increases the severity of infammation and tissues injuries during the process of OHSS.Higher levels of melatonin are found in human follicular fuid than in plasma because melatonin is not only derived from the general circulation but also synthesized in the ovary [10].Melatonin and its metabolites are free radical scavengers.Terefore, we speculate that the role of melatonin in the follicular fuid is to protect the oocytes and granulosa cells from being deteriorated by the oxidative stress and free radicals.Te stimulation and deterioration of vascular endothelial cells caused by ROS are the critical factors of OHSS.We found out that SESN2 level was closely associated with ROS, while melatonin could ameliorate SESN2 level induced by angiotensin II or H 2 O 2 .SESN2 is a cytoplasm stressassociated protein that accumulates in cells exposed to hypoxia, stress and DNA damage.Here, we demonstrated signifcantly higher level of SESN2 was expressed in high risk OHSS patients compared to that in controls, which may be attributed to an increased oxidative stress in OHSS patients.Tese results are constant with previous studies which showed that any condition which leads to ROS accumulation may induce SESN2 expression [19].Oxidative stress drives endothelium deterioration, which contributes to the development and progression of OHSS.Anti-infammatory and antioxidant agents prevent endothelium dilatation and reduce the oxidative imbalance in OHSS.Melatonin, as one of the most powerful free radical scavengers prevents granulosa cells in ovaries and endothelium from the deterioration of these free radicals.
Te mechanisms by which melatonin provide antioxidant protection in OHSS are not fully understood.Previous studies have demonstrated that melatonin regulates antiapoptotic pathway [31][32][33][34].BCL2 family and caspase family are important for regulating the atresia of antral follicles.Deletion of BCL2 increases the apoptosis of preantral follicle while the overexpression of caspases results in increased number of apoptotic follicles [10].Our study demonstrated that the p17 caspase 3 level was increased while BCL2 level was decreased OHSS oxidative models, which testifed that the apoptosis signaling pathway was activated in OHSS oxidative models.Melatonin, could partly reverse p17 caspase 3 and BCL2 levels in OHSS oxidative model, which revealed the antioxidant efect of melatonin in OHSS by preventing oxidative stress-mediated apoptosis.
In recent years, more and more studies have shown that AMPK-mTOR molecules were also closely associated with apoptosis.Chen et al. have shown that H 2 O 2 could induce apoptosis of neurons through activating AMPK and inhibiting mTOR [20].Arsikin et al. have also demonstrated that 6hydroxydopamine induced apoptosis through AMPK in SH-SY5Y neuroblastoma cells [39].Our study demonstrated that ROS could active SESN2-AMPK-mTOR which resulted in the apoptosis in OHSS oxidative models.Melatonin prevents degenerative diseases in the nervous system by inhibiting apoptosis through regulation of the mTOR-mediated pathway.
Te study has a few limitations.First, we selected KGNs as our human granulosa cell line to construct our OHSS oxidative models.However, it would be more accurate to validate our fndings using other granulosa cell lines, such as SVOGs.Second, our results cannot be immediately applied to humans without the conduction of large-scale clinical trials.Further research is necessary to determine the precise dosage of melatonin in OHSS patients.

Conclusion
Te results described herein help us understand the benefcial efects of melatonin in OHSS patients.Te antioxidative and antiapoptotic properties of melatonin seem to produce positive efects on OHSS.Considering the safety of exogenous melatonin has been testifed in many studies [40][41][42], our present fndings will provide us the potential for clinical application of melatonin to prevent OHSS and defne the most appropriate timing when the administration of melatonin should be efectively carried.Tis is the very initial step of our study and our results may not be adapted to OHSS patients without large-scale clinical studies.In our further study, we should focus on the exact mechanisms of the protective role of melatonin to support our study and large-scale clinical studied should be conducted to explore the long-term efects, way of administration, and appropriate drug dosage of melatonin in OHSS patients.

Figure 1 :
Figure 1: Te expression of SESN2 in granulosa cells of OHSS patients and controls.(a) Te expression of SESN2 mRNA in granulosa cells of OHSS (n � 24) patients and controls (n � 15) (t � −3.231, P � 0.003); (b) the representative pictures of Western blotting of SESN2 and GAPDH in the granulosa cells of OHSS patients and controls; (c) the comparison of SESN2/GAPDH protein ratio in the granulosa cells of OHSS patients (n � 24) and controls (n � 15).* P < 0.05; * * P < 0.01.

Figure 2 :
Figure 2: Te oxidative stress and SESN2 levels in HUVECs and KGNs induced by diferent concentrations of angiotensin II or H 2 O 2 .(A) (a-c) the representative pictures of controls, 1 μM, 2 μM angiotensin II treated HUVECs dyed with DCFH-DA, respectively; (d-f ) the representative pictures of controls, 0.1 mM, 0.2 mM H 2 O 2 treated KGNs dyed with DCFH-DA, respectively; (B) the comparison of mean oxidative fuorescence intensity in HUVECs.Te mean of the results in controls was assigned an arbitrary value of 1.0 and the results (mean ± SD) of other cells are expressed as the relative intensity; (C) the comparison of mean oxidative fuorescence intensity in KGNs.Te mean of the results in controls was assigned an arbitrary value of 1.0 and the results (mean ± SD) of other cells are expressed as the relative intensity.Bar: 50 μm; (D) the representative picture of Western blotting of SESN2 and GAPDH in controls, 1 μM, 2 μM angiotensin II treated HUVECs; (E) the representative picture of Western blotting of SESN2 and GAPDH in controls, 0.1 mM, 0.2 mM H 2 O 2 treated KGNs; (F) the comparison of SESN2/GAPDH ratio after being incubated with angiotensin II for 24 h; (G) the comparison of SESN2/ GAPDH ratio after incubation with H 2 O 2 for 12 h.Te results are expressed as mean ± SD. * P < 0.05; * * P < 0.01; * * * P < 0.001.Ctrl: control.
Showed an Increased Expression of SESN2 in Granulosa Cells.Te clinical characteristics of the patients are presented in Table 1.Te OHSS patients had younger age, lower BMI, lower base FSH levels than the women in non-OHSS group and higher AMH levels, higher E 2 levels on hCG day; moreover, OHSS patients retrieved more oocytes than non-OHSS group (P < 0.05).Furthermore, the suppressor of ROS accumulation, SESN2, both its mRNA (OHSS n � 24, control n � 15) (P � 0.003) and protein levels (OHSS n � 24, control n � 15) (P � 0.038) were signifcantly increased in the granulosa cells of OHSS patients (Figures1(a) and 1(b)).

Table 1 :
Clinical characteristics of the study population.
* P < 0.05 compared with control group; * * P < 0.01 compared with control group.