Insight of Autophagy in Spontaneous Miscarriage.

Spontaneous miscarriage (SM) refers to natural pregnancy loss before 20 weeks of gestation, though the exact definition remains controversial. It has drawn great social concern in light of increasing morbidity during latest decades 1 . Main risk factors for SM include fetal chromosomal abnormality 2 , maternal infections, uterine structural defects, endocrine disorders, and immune dysregulation 3 , 4 . However, in a certain part of cases, especially for recurrent spontaneous miscarriage (RSM), the underlying mechanisms still remain undefined, which urgently deserves more research on etiology, pathogenesis and effective therapeutic methods. Macroautophagy/autophagy is a self-degradative cellular process in eukaryotic cells, warranting survival under stress circumstances like energy or nutrition shortage, inflammatory events, oxidative stress and endoplasmic reticulum stress 5 , 6 . Besides keeping cellular homeostasis, autophagy has been flagged the functionality in physiological and pathological process, for instance, immune response, malignancy, and neurodegeneration 7 - 9 . Likewise, in reproductive system, autophagy has been reported to exist in physiological and pathological endometrium, involving in regulating menstrual cycle, development of endometriosis, adenomyosis and endometrial carcinoma 10 - 12 . Additional studies have brought further supporting evidence to relationship between autophagy with embryogenesis, implantation, decidualization and pregnancy maintenance 13 - 15 . In mechanism, autophagy contains five basic steps: nucleation, elongation, maturation, fusion and degradation (Figure 1 A) . First, autophagy is initiated by isolation membrane namely phagophore which later expands to engulf intracellular cargo and forms autophagosome. After that, autophagosome becomes mature via fusion with lysosome and the cargo is degraded for further recycle finally. In the aspect of molecule, autophagy is activated by the completion of unc-51-like kinase 1 (ULK1) complex, which consists of focal adhesion kinase family interacting protein of 200 kDa (FIP200), Atg13 and ULK1. ULK1 complex is negatively regulated by mammalian target of rapamycin complex 1 (mTORC1) and positively regulated by 5' adenosine monophosphate-activated protein kinase (AMPK) via directly suppressing and enhancing the activation of ULK, respectively 16 . Followed with phagophore formation, Atg5-Atg12 conjugation interacts with Atg16L to form Atg16L complex. Meanwhile, LC3 precursor convert to LC3-II with the assistance of Atg4B and phosphatidylethanolamine successively 17 . LC3-II locates and inserts into the extending phagophore membrane, typically serving as a critical marker of autophagy activity. Moreover, differing from non-selective autophagy, selective autophagy is regulated by the p62/SQSTM1 paradigm 18 . Considering autophagy is a continuous cellular process, for proper detection, the concept of autophagic flux must be mentioned. To date, both bafilomycin A1, which inhibits lysosomal degradation and chloroquine, which blocks autophagosome-lysosome fusion, are used to evaluate the functional status of autophagic flux 19 . There are several significant events to maintain successful pregnancy: fertilization and embryonic development, trophoblasts invasion, placental development, decidualization, enrichment and infiltration of decidual immune cells (Figure 1 B) . Recent studies imply that autophagy may associated with SM through regulating decidualization, trophoblast cells and immune cells at maternal-fetal interface 13 , 20 , 21 . Herein, we discuss the importance of autophagy during early pregnancy and offer a new insight into spontaneous miscarriage and relevant treatment.

Herein, we review the role of autophagy in regulating critical events during gestation, including fertilization and embryonic development, bio-functions of trophoblasts, placental development, decidualization, infiltration and residence of decidual NK, macrophage, and T cells. However, studies about autophagy in decidual macrophage and T cells are few, which needs more investigations. In addition, underlying mechanisms of RM mediated by aberrant autophagy are included, and thus we mention relevant potential therapeutic methods for SM by targeting autophagy. Particularly, rapamycin and vitamin D have been hotpots in recent decade for their multiple roles in regulating trophoblast survival, decidualization, or immunological milieu. More importantly, autophagy is a dynamic process and is involved in both maintaining homeostasis and causing pathological damage under stress. As a result, autophagy could act as a compensation in certain conditions which might lead to misinterpretations for results and mechanisms. Autophagy-related molecules might be new strategies for predicting SM, though the source of sampling and its power in scanning tests are still challenging problems. For example, Atg5 is not only a key factor in promoting trophoblast survival but also of great importance in regulating decidualization, monocyte-macrophage differentiation, mediating interaction between TGF-β and decidual T cells, possibly. Further well-designed studies are required to verify the role of autophagy in SM pathogenesis and the network of related regulatory mechanisms in order to make potential treatment methods into practice.

Around 15% pregnancies end up with SM, which is characterized by pregnancy loss before the 20 th week. In particular, RSM refers to occurrence of 3 or more consecutive miscarriages, and over half of cases show no identifiable causes 196 . Accumulating evidence indicates the important role of autophagy because of aberrant expression of autophagy-related markers in SM/RSM ( Table 2 ). LC3, LC3II/I, Atg5 and Beclin-1 are higher in RSM placentae 81 , 197 along with inhibited Shh signaling pathways 20 . Avagliano et al. have also shown that STBs in SM exhibit a higher density of LC3 staining with more autophagosomes than those of normal controls. HIF-1α increases in SM villi and decidua, while the ratio of Bax to Bcl-2 and cleaved caspase 3 are elevated in decidua of SM, without statistical significance, though. Thus, autophagy might be a protector from hypoxia-induced apoptosis 35 . Interestingly, as compared to women with their first conceptions, autophagy of peripheral blood mononuclear cells increases in women with prior spontaneous pregnancy losses but no deliveries 198 . However, here exists some discrepancies. Yang et al. have shown that plasmacytoma variant translocation 1 (PVT1) is decreased in villi from RSM patients. In vitro, PVT1 knockdown elicits impaired autophagy with increased mTOR, p62 and decreased Beclin-1, LC3- II/I, ULK1 in HTR-8/SVneo cell line 199 . Our previous studies display that autophagy is impaired in RSM. Smaller number of autophagosomes with aberrant distribution are exhibited in the villi of RSM patients 148 . Insufficient autophagy with lower Atg5, LC3B and higher p62 is observed in DSCs derived from SM patients. Besides, downstream molecules of autophagy mediating NK residence are also reduced 21 . Wei et al. have confirmed these results in rat model induced by antiphospholipid antibodies. Elevated p62 and mTOR as well as downregulated Beclin-1 and LC3-II are presented in placentae and the impaired autophagy could be rescued by rapamycin 200 . Except for different ways to evaluate of autophagy, cell types and sampling, various pathogenic mechanisms involved in could still be to blame for the inconsistence. Of note, autophagy is a dynamic process and might act as a compensation for cellular stress in SM. Furthermore, the exact causal relationship between altered autophagy and SM remains to be established based on larger scale of subjects and well-designed studies. In normal pregnancy, Yin Yang 1 (YY1) can interact with plasmacytoma variant translocation 1 (PVT1) promoter to upregulate its transcription, which further favors trophoblasts invasion and adhesion by autophagy via mTOR signaling. Effects followed with decreased PVT1 in villi samples might be involved in RSM pathogenesis, rendering PVT1 as a therapeutic target 199 . PLAC8, a newly-explored factor in tumorigenesis, has been regarded as a target of treating oral squamous cell carcinoma for its regulatory roles in ERK 201 , Wnt/β-catenin and PI3K/Akt/GSK3β pathways 202 , respectively. In trophoblasts, PLAC8 co-localizes with p53 and mediates its degradation, facilitating the viability, proliferation and differentiation via enhanced autophagy 106 . Punicalagin enhances autophagic flux to protect primary human STBs from apoptosis induced by stress conditions 203 . Hyperoside activates autophagy and combats inflammatory response in rat model with decreased IL-1β, IL-8, ICAM-1, VCAM-1 and complement C3, thereby rescues pregnancy losses via downregulating mTOR/S6K and TLR4/MyD88/NF-kB 200 . Studies above offer us insights into treatment of RM targeting autophagy. Rapamycin, a mTOR inhibitor to induce autophagy, could also reverse adverse pregnancy outcomes and elevates LC3 stanning in junctional and labyrinth zones in placentae of rats 200 . It effectively induces autophagy to reduce NK cytotoxicity in trophoblasts 148 and promote DSC autophagy and NK residence in decidua 21 . Antiphospholipid antibodies-induced NLRP3-mediated IL-1β secretion in human trophoblasts could also be inhibited by rapamycin 204 . In folate-deficient models, rapamycin alleviates impaired autophagy and defective decidualization 127 , 128 . However, rapamycin has also been reported for its potential in treating endometriosis 151 and endometrium-carcinoma 205 via enhancing NK cytotoxicity, reminding us that dose and side effects of rapamycin should be considered carefully when it comes to SM ( Table 3 ). To date, vitamin D has drawn great interests in reproductive system for its deficiency correlated with endocrine diseases and adverse pregnancy outcomes 206 . Especially for pregnancy complications presented with poor placentation, decreased vitamin D could be a risk factor 207 . A prospective cohort study conducted by Mumford et al. reports that sufficient preconception, instead of early pregnancy, concentrations of serum vitamin D are associated with better pregnancy outcomes among women who have suffered from one or two prior pregnancy losses before 208 . Recent research indicates autophagy may participate in interactions between vitamin D and placental development, but whether it's mediated by vitamin D receptor (VDR) still remains controversial. Reduction of maternal serum vitamin D and intracellular VDR may lead to impaired vitality (decreased LC3B/Beclin-1) and reduced ratio of VTs in placenta 209 . Additionally, the VDR protein is upregulated after supplementation without significant increase in its RNA and target gene level, indicating the importance of VDR but this effect is mediated by nongenomic response 210 . However, according to Wilson et al. , though genetic deficiency of VDR caused aberrant autophagy with greater expression of Atg4b and activated mTOR, no statistically significant differences are observed in fetal growth, overall morphological parameters in VDR null placentae in comparison to controls 211 . Vitamin D can rescue the impaired viability and invasion of trophoblasts induced by ROS-treated HTR-8 cell line 212 . Fetal mortality, organ dysfunction and metabolic disorders could be alleviated by vitamin D supplementation in ischemia placenta via activating autophagy and attenuating apoptosis 210 . During implantation and decidualization, vitamin D suppresses inflammation and positively regulates HOXA10 at maternal-fetal interface 213 . Importantly, vitamin D decreases Th17 cells as well as the ratio of Th17 to Treg in peripheral blood of SM patients 214 . Whether these effects are mediated by autophagy needs further confirmations. To be more specific, crosstalk among placenta, decidua and maternal serum vitamin D levels could play a critical role in pathogenesis of SM. Anyway, vitamin D should be a potential therapeutic method for SM in regulating immunological milieu and placentation ( Table 3 ).