Spatial activation of autophagy in human placenta-related tissue during labor: a possible mechanism for labor onset

Introduction: The study aimed to explore the mechanism of labor onset via investigation on autophagy in placental tissues of normal pregnant women. Methods: Placentas at the �rst, second, and third months of gestation and membrane samples from uniparous women without signs of uterine contractions (gestation group) were collected as well as placentas and membranes from uniparous women (parturition group) who underwent vaginal delivery (parturition-initiation group; PI group) or cesarean sections (parturition-non-initiation group; PNI group) without any medical complications. Autophagosomes were observed by transmission electron microscopy (TEM). Immuno�uorescence and western blotting (WB) were used to quantify the protein levels of autophagy markers LC3A and LC3B in placenta and fetal membrane. TEM, immunohistochemistry (IHC) and WB were used to compare the autophagy in different parts of placenta and fetal membrane in the PI and PNI groups. Results: TEM and IHC results showed that the number of autophagosomes and autophagolysosomes increased in the gestation group, and WB results suggested increased LC3B/A ratio between the placenta and the fetal membrane in gestation group. Autophagy in the maternal side of placenta in PI group was signi�cantly increased, and the autophagy level in the placenta and fetal membrane near rupture became higher in both PI and PNI groups. Discussion: Autophagy


Introduction
Labor is the process in which the fetus is separated from its mother.A series of physiological processes are ful lled under the coordination and cooperation of the nervous system, endocrine system and immune system from gestation to parturition [1].However, the mechanisms of labor initiation in humans are unclear, and accumulated evidence has suggested that labor initiation consists of independent and synergistic physiological events.In ammatory signals, decidua activation, endocrine coordination between fetus and mother, cervical maturation and remodeling [2], uterine muscle contraction and cervical dilation, rupture of membranes and placenta separation [3][4][5][6][7] and other factors and regulatory pathways are involved.Abnormal delivery such as overdue delivery and premature delivery is one of the major causes of neonatal death.Therefore, it is necessary to understand the mechanism of labor initiation which is also the prerequisite of normal delivery.
Autophagy is a highly conserved, intracellular degradation process widely found in various biological cells during evolution, helping to remove misfolded or aggregated proteins and digest damaged organelles through the lysosomal pathway.During pregnancy, autophagy has been reported to affect fertilization [8,9], embryo development [10], endometrial exudation and maternal-fetal interface crosstalk [11].Abnormal autophagy is associated with a variety of pregnancy complications, such as preeclampsia [12][13][14][15], fetal growth restriction [16] and preterm birth (PTB) [17,18].Studies have shown that hyperin ammation is associated with autophagy in the uterus and placenta [19,20].In addition, our study based on an oxidative stress-induced model showed that compared to maternal cells, fetal cells are more capable of activating in ammatory responses through the p38MAPK and p53 pathways and promoting cellular senescence.
Microtubule-associated protein 1 light chain 3 (LC3) is one of the marker proteins of autophagosome formation in mammalian cells, which helps form a stable structure on the inner or outer membranes at all stages of autophagy-related structures by combining with phosphatidylethanolamine (PE).Normally, LC3 exists in the cytoplasm in the form of LC3A (cytosolic form) and is modi ed to the active LC3B (membrane bound form) by posttranslational modi cation when autophagy is activated [21].Therefore, the content and ratio of LC3B and LC3A can re ect cell autophagy activity.
In this study, we attempted to explore the mechanism of labor initiation by detecting autophagy in the maternal-fetal interface tissues (placenta and fetal membrane) of normal pregnant women at different gestational ages in PI group the PNI group to provide theoretical basis for pregnancy physiology.

Study population and sample collection
Placenta samples among uniparous and nulliparous women in gestation group without signs of uterine contraction were collected at rst, second and third month of gestation.
Placenta and fetal membrane were collected from term and uniparous as well as nulliparous women undergoing vaginal delivery (PI group) and caesarean section (PNI group) indicated as maternal request without any medical complications, respectively.Exclusion criteria: 1) Medical complications including hypertension, pre-eclampsia, diabetes, cholestasis or any other medical/surgical disease; 2) Abnormal placental conditions including abnormal placental adherent, placenta previa or abruption; 3) Fetal malformation, fetal growth restriction, macrosomia or distress; 4) Abnormal labor progress including prolonged labor or uterine atony; 5) Premature rupture of membrane, chorioamnionitis.
This study have obtained written informed consent from all patients providing samples under approval by the Ethics Committee of Nanfang Hospital, Southern Medical University.

Autophagosome Observation by Transmission Electron Microscopy(TEM)
Placenta tissues were cut into 1mm pieces immediately after collection and placed into EM xative (2% glutaraldehyde, 0.1 M sodium cacodylate) at 4℃.The specimens were then washed with double distilled water for 30 minutes and were subsequently dehydrated in 50%, 100% and pure propylene oxide.After embedding of placenta tissue at room temperature for 12 hours, the resin was polymerized at oven at 55℃ for 24 hours.Afterwards, an 80-nm section were made for coating with lead citrate and uranyl acetate.A transmission electron microscope (TEM) (JEM-1400, JEOL, Tokyo, Japan) were used for examination.Autophagosomes in each specimen were quanti ed by two independent observers who were blinded to which group was examined.

Statistics
All statistical analyses were processed blind using Prism 7.0 software (GraphPad, La Jolla, CA, USA).Data were expressed as Mean + SD in bar charts.The statistical signi cance of results was analyzed by Student's t-test, P < 0.05.

Autophagy occurs in placenta at different gestational weeks
The autophagosome in placentas from rst, second and third month pregnant women were investigated by TEM.In Fig. 1, autophagolysosome were observed in placenta at different gestational weeks.The electron microscopic analysis also showed appearance of vacuolar mitochondria and autophagolysosome.There were signi cantly more autophagosomes quanti ed in placentas at the 3rd month than at the previous two months.

Autophagy in the placenta and fetal membrane was higher in PI group than in PNI group
The autophagy activity of different tissues in PI group and PNI group was detected.TEM was used to observe the autophagosomes in the placenta and fetal membrane of the two groups.Results showed more autophagosomes and autophagolysosomes in gestation group than in parturition group (Fig. 2a).WB was used to detect autophagy marker protein levels.The results showed that the LC3B/A ratio of placenta to fetal membrane in the gestation group was higher than that in the parturition group (P < 0.001, Fig. 2b).

The longitudinal distribution of placental autophagy was different
We further compared the differences of autophagy expression in different parts of the same placental tissue in PI group.TEM was used to compare the number of autophagy-associated structures in different sections of the placenta, and the results demonstrated more autophagy-associated structures in the maternal side of the placenta (Fig. 3a).WB was used to detect the expression levels of autophagy-related proteins in different sections of the placenta in PI and PNI groups.Results showed higher autophagy levels in the maternal side of the placenta in PI group, while there was no difference in PNI group (Fig. 3b).

The transverse distribution of autophagy in placenta and fetal membrane was different
Different groups of autophagy expression were also detected through a variety of methods.From different parts of the tissue in PI and PNI group, TEM results showed more structures of autophagy in the placenta and fetal membrane in the former group.The level of autophagy in the placenta tissue near the insertion of the umbilical cord and the fetal membrane near the natural rupture was also higher (Fig. 4a).LC3B protein IHC analysis showed that PI group has tan grain in the placenta and fetal membrane tissues, which is nearer to the break than in PNI group (Fig. 4b-c).IHC results showed that LC3B protein in fetal membrane tissue was different in the near and far rupture of PNI group compared with PI group (P < 0.05, Fig. 4d).LC3B protein in placental tissue was signi cantly different in the near and far laceration sites of PI group compared with that of PNI group (P < 0.01, Fig. 4d).

Discussion
Autophagy is detected at placenta and related membrane in our study, where the number of autophagosome increases with gestational weeks and signi cantly rises at labor stage.The autophagy activity in the placenta at labor exhibits spatial distribution and is more obvious at the maternal site of placenta and proximal part of fetal membrane adjacent to the cord insertion.
Previously, activation of autophagy has been reported in early placenta and speci cally located in extravillous cytotrophoblasts (EVT), is supposed to participate in the trophoblast migration, invasion and remodeling of spiral artery for normal placentation [22].By using TEM and immuno uorescence, placental autophagy were demonstrated at both early and late gestation in Huang et al.'s study [11].Moreover, Kanninen et al. [23] revealed increased autophagy activity in peripheral blood mononuclear cells throughout gestation.These ndings are consistent with our data, which showed autophagy activity in placentas at all gestation but signi cantly higher in the later stage.It has been widely accepted that the placentas undergo a processive aging during the short duration of pregnancy [24].As an essential system to maintain cellular homeostasis, autophagy has been revealed to be related to the aging of multiple organs, including hearing [25], integumentary [26] or hematopoietic system [27].
Hence, the presence of autophagy in placenta through gestation might also play an important role in placenta aging.Furthermore, we demonstrated an increased autophagy activity in placental tissue or membrane after labor onset.Similarly, Wang L et al. [28] detected the autophagy-speci c genes in myometrium during labor and speculated the participation of autophagy in labor initiation.After the onset of labor, a physiological episode of hypoxia and reoxygenation occurs due to uterine contraction.More autophagy activity tends to degrade metabolic products in all related tissues, including myometrium, placenta and fetal membrane to preserve energy and metabolic homeostasis.Interestingly, autophagy was found more active at the maternal side of placenta after labor onset.Placenta is a fetal-maternal organ where the fetal portion is closer to villous chorion while the maternal site is adjacent to decidua basalis [29].Hence, we speculate that the autophagy signals associated with labor onset might arise from the basal decidua.Moreover, the autophagy activity is higher around the rupture part of membrane.
The study found the heterogeneity in autophagy in placenta and fetal membrane at different sites.In the placenta, autophagy is more active in the maternal site.Therefore, maternal autophagy activity is higher during delivery, suggesting the activation of autophagy signals.According to the anatomy, the placenta can be divided into the site near the start of umbilical cord, the central placenta and the site away from the edge of the start of the umbilical cord.Increase in autophagosome and cell cavitation at the central placenta suggests the higher autophagy level in the central than the edge of the placenta.Considering central placenta as the possible main functional area, cells differentiate and thrive.Meanwhile, it may also be the site of early changes after activation of autophagy pathway which plays a potential role in labor initiation.There were signi cant differences in the level of autophagy in the maternal placenta between PI and PNI groups, suggesting that the birth initiation during parturition may be related to the increase of autophagy, and the activation of autophagy pathway may come from the maternal site.
According to the anatomical structure, the fetal membrane is divided into the place near the natural rupture of the endocervical opening and the place far from the endocervical opening.The results showed that the autophagy level of fetal membrane tissue at the endocervical opening was higher, indicating the possible cause of rupture by autophagy.Rupture of membranes signals the initiation of labor, which in turn proves that autophagy is involved in the initiation of labor.
Labor initiation requires signal transmission, but due to the lack of vascular tissue in the fetal membrane, other small biological molecules may be responsible.There is evidence that exosomes of fetal origin can be transported from the fetal side to the maternal side [30], indicating their potential role in paracrine communication between fetal and maternal tissues.Salomon et al. [31] found that exosomes can be released by placenta and affect pregnancy.Therefore, we speculated that labor initiation signals may be transmitted by exosomes and other small biological molecules, but how autophagy plays a role in labor initiation through exosomes is still unclear and further studies are needed to elaborate the mechanisms.

Conclusion
Autophagy can occur in the placenta and fetal membranes, with its activity particularly high in the start phase of delivery.We assume that autophagy may be activated by some factors in the normal late pregnancy condition, while the excessive autophagy levels in the placental and fetal membranes may reduce the number of cells and degrade the function of placental and fetal membranes.Eventually, the tissues fail to provide the necessary conditions for pregnancy, thus leading to the initiation of labor.