*5.3. Preterm Labor*

Atg16L1 is essential for forming autophagosomes with the Atg5-Atg12 complex, which is associated with Crohn's disease [75]. Atg16L1-knockout macrophages produced high levels of inflammatory cytokines such as IL-1β and IL-18 via the TIR-domain containing adaptor-inducing interferon-β (TRIF)-dependent signaling pathway, indicating that autophagy suppresses intestinal inflammation [76]. Atg16L1 knockout mice gave birth prematurely in response to lipopolysaccharide (LPS) [32]. Thus, autophagy is involved in resistance to infection by removing inflammasomes to regulate inflammation. Turnover of organelles mediated by selective autophagy would be an important mechanism by which autophagy prevents inflammation [77]. If it does not work properly, accumulation of damaged organelles induces activation of NLRP3 inflammasomes. Uric acid, which increases in preeclampsia, activates inflammasomes via activation of NLRP3 inflammasomes in monocytes [78]. Paradoxically, a treatment of either LPS, a Toll-like receptor (TLR) 4 ligand, or peptidoglycan with poly(I:C), TLR2 and TLR3 ligands, inhibited autophagy via decrease of Atg4c and Atg7 proteins in placentas using inflammation-induced preterm labor models [79]. This might be a consequence of excessive autophagy activation; in other words, autophagic capacity might be exhausted with continuous infection. As for the other type of premature delivery model, in which p53 knockout induced senescence in uterine decidual cells with activation of mTOR signaling, rapamycin treatment, which activates autophagy, reduced preterm birth as well as the incidence of neonatal death [55,80]. Thus, autophagy restoration has a positive effect on premature delivery; mTOR-mediated autophagy inhibition is related with premature delivery. As for spontaneous deliveries at term, little evidence is provided for the role of autophagy in humans. One important caution is given for the study; we have to consider at least the mode of delivery, in which labor pain might affect autophagy status in the placentas, when comparing autophagy status in human placentas [81].

### **6. Caution When Interpreting Autophagy-Related Experiments**

The importance of estimating autophagy is to precisely calculate the velocity of autophagy flow. Estimating autophagy using a single method is impossible, and is more di fficult in vivo—and in humans—than in vitro, or in animal models [82,83]. In western blot analyses of cell cultures, the increase in the MAP1LC3-II/actin ratio, sometimes replaced with the MAP1LC3-II/MAP1LC3-I ratio, indicated autophagy activation in response to lysosomal inhibitors, such as bafilomycin A1 or chloroquine, compared with cell cultures without inhibitors. Thus, the dynamics of autophagy flux are comparable to autophagy inhibitors in living cells. Autophagy cannot be precisely estimated from "human" fixed tissues. Some studies, however, reported increases of MAP1LC3 mRNA and protein as an indicator of autophagic activity in placental tissues, but the increase does not imply activation of autophagy in other tissues [82,83]. Though numbers of MAP1LC3 puncta in immunofluorescence analysis are equal to the number of autophagosomes, the increase of MAP1LC3 puncta could be the result of the fusion of an autophagosome and lysosome being blocked, as well as autophagy activation. To precisely estimate autophagy in an organ or tissue, Kaizuka et al. developed a new method using MAP1LC3 fluorescent probes, in which one is degradative and the other is not [84]. However, autophagy activation still remains di fficult to measure in human tissues. In fixed tissues, the number of autophagosomes and autolysosomes should be evaluated. A step in the formation of an autolysosome, co-localization of MAP1LC3 dots, and lysosomal-associated membrane protein 1 (LAMP1), which are composed of autophagosomes and lysosomes, can be useful in confirming the formation of the autolysosome. The ratio of autolysosomes to autophagosomes could be used to estimate autophagy in fixed tissues. A marked accumulation of p62, which was seen in liver-specific autophagy-deficient mice, would be useful as well [85]. This is because the accumulation of p62 in cytoplasmic inclusion bodies impair cellular viability [86]. Accumulation of p62 was seen in some trophoblast cell lines, in which autophagy was suppressed by an Atg4BC74A mutation, and EVTs in biopsies taken from the placental bed of women with preeclampsia [23]. This would be a marker of autophagy inhibition in placental tissues. Rubicon inhibited autophagic flux, which led to the accumulation of p62 in a mouse model. Increased expression of Rubicon in nonalcoholic fatty liver disease would increase autophagy inhibition and lead to complications [87]. Taken together; the ratio of autolysosomes to autophagosomes, p62 accumulation, and Rubicon may allow an estimation of autophagy in placental tissues.
