Original Article

Autophagy in Normal and Abnormal EarlyHuman Pregnancies

Laura Avagliano, MD, PhD1, Laura Terraneo, PhD1,Eleonora Virgili, BD1, Carla Martinelli PhD2, Patrizia Doi, MS1,Michele Samaja, PhD1, Gaetano Pietro Bulfamante, MD1,and Anna Maria Marconi, MD1

AbstractAutophagy is an inducible catabolic process by which cells degrade and recycle materials to survive stress, starvation, and hypoxia.The aim of this study was to evaluate autophagy at the fetal–maternal interface, to assess autophagy involvement during the earlyphase of human gestation, and to explore autophagic modification in case of early abnormal pregnancy outcome. Specimens werecollected from first-trimester normal gestations undergoing legal termination of pregnancy and first-trimester sporadic sponta-neous miscarriages. Autophagy was studied in villous and decidual samples by transmission electron microscopy, immunohisto-chemistry, immunofluorescence, and Western blotting. Autophagy markers were found in cytotrophoblast, syncytiotrophoblast,extravillous trophoblast, and decidual stromal cells. Autophagy is physiologically involved in early normal gestation. Comparedwith normal pregnancy, spontaneous miscarriage presents an increase in autophagy expression in villous specimens due to anincrement in concentration of autophagic vacuole in syncytiotrophoblast, suggesting a cytoprotective mechanism of the cellsto respond to microenvironmental challenge.

Keywordsautophagy, apoptosis, pregnancy, trophoblast, miscarriage

Introduction

Autophagy is an evolutionary conserved, intracellular inducible

catabolic process by which injured organelles, damaged proteins,

and pathogens are sequestered into autophagosomes, double

membrane-bound vesicles that fuse with lysosomes for proteoly-

tic degradation and material recycling.1 Microtubule-associated

protein light chain 3 (LC3), the mammalian homologue of yeast

autophagy-related gene 8, intervenes in the late stages of autop-

hagosome formation. Among LC3 isoforms, LC3-II, the phos-

phatidylethanolamine conjugated product of LC3-I obtained

after LC3 activation,1,2 is currently used as a specific marker

of autophagy for its role during autophagosome genesis.3

Previous studies have investigated the occurrence of autop-

hagy during the early stages of mammalian pregnancy in the

preimplantation period for oocyte-to-embryo transition4-6 but

no extensive in vivo assessment of autophagy at the fetomater-

nal interface after implantation has been performed in humans.

At the site of implantation, apoptosis permits placenta develop-

ment,7 and a relatively hypoxic environment warrants an ade-

quate embryonic and placental growth.8 Although many

cellular and molecular events are expected to regulate the var-

ious steps of fetomaternal interactions, the mechanisms that

regulate the early stage of pregnancy are not yet fully

understood. The aim of the present study was to evaluate the

occurrence of autophagy during the first trimester of pregnancy

to verify whether it is involved in the early phase of gestation

and whether autophagy expression differs between normal

pregnancy (NP) and spontaneous miscarriage (SM).

Materials and Methods

Sample Collection

Normal pregnancies. Tissues were obtained from 10 healthy,

nonsmokers women (age 28.5 + 8.1 years) undergoing legal

termination of pregnancy within 12 weeks of amenorrhea

(mean gestational age 9.4 + 1.1 weeks; range 8-11 weeks);

1 Department of Health Sciences, San Paolo Hospital Medical School,

Universita degli Studi di Milano, Milan, Italy2 Department of Biomedical Sciences for Health, Universita degli Studi di

Milano, Milan, Italy

Corresponding Author:

Laura Avagliano, Unit of Obstetrics and Gynecology, Department of Health

Sciences, San Paolo Hospital Medical School. University of Milano, Via A. di

Rudinı 8. 20142 Milano, Italy.

Email: laura.avagliano@unimi.it

Reproductive Sciences2015, Vol. 22(7) 838-844ª The Author(s) 2014Reprints and permission:sagepub.com/journalsPermissions.navDOI: 10.1177/1933719114565036rs.sagepub.com

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in all cases gestational age and fetal viability were confirmed

by ultrasound before suction curettage. All women gave their

consent to the study. All procedures were performed in general

anesthesia. At the time of suction curettage, the gestational tis-

sues were removed in sterile conditions and macroscopically

assessed, through several washes in sterile phosphate-

buffered saline solution. For each woman, samples of decidua

and villi were collected and stored separately: immediately

after the washes, tissues collected for immunohistochemical

and immunofluorescence analysis were fixed in 10% formalin

solution; samples for electron microscopy were fixed in 2.5%glutaraldehyde; samples for Western blotting were frozen in

liquid nitrogen and stored at �80�C for protein extraction.

Spontaneous miscarriages. Tissues were obtained from archive

specimens from our previous work as described.9 All cases

were sporadic miscarriage. Only embryonic pregnancies were

included and all cases had normal embryonic karyotype. Smo-

kers were excluded, therefore 10 cases were eligible for this

study. Spontaneous miscarriage and NPs were comparable for

gestational age (gestational age 9.2 + 0.6 weeks; P ¼ .76) and

maternal age (age 30.9 + 4.8 years; P ¼ .43).

Immunohistochemistry

Immunohistochemical studies were carried out in all samples

(n ¼ 20: 10 NPs and 10 SMs) on 4-mm thick tissue sections

as described previously.10-12 Primary antibodies were as fol-

lows: rabbit polyclonal LC3 (NB100-2220, Novus Biologicals,

Littleton, Colorado; dilution 1:1000), mouse monoclonal anti-

CK-7 (OV-TL 12/30, Dako, Glostrup, Denmark; dilution

1:100), mouse monoclonal anti-HLA-G (SC-21799, Santa Cruz

Biotechnology, Dallas, Texas; dilution 1:2000), and rabbit mono-

clonal cleaved caspase-3 (#9664, Cell Signaling Technology,

Danvers, Massachusetts; dilution 1:800).

Immunofluorescence

Immunofluorescence studies were conducted in the same

samples used for immunohistochemistry (n ¼ 20: 10 NPs

and 10 SMs), as described previously11 using rabbit antiLC3

(NB100-2220, Novus Biologicals, Littleton, Colorado; dilution

1:500). Fluorophore-conjugated secondary antibodies were

used and nuclei were subsequently counterstained with 40,6-

diamidino-2-phenylindole (Invitrogen, Carlsbad, California).

Fluorescence images were viewed and captured using Imager.Z1

microscope (Zeiss, Feldbach, Switzerland).

Transmission Electron Microscopy

Tissues from 8 cases (4 NPs and 4 SMs) were fixed in 2.5%glutaraldehyde in 0.13 mol/L phosphate buffer at pH 7.2-7.4;

specimens were postfixed in 1% OsO4, dehydrated in ethanol

plus propylene oxide, and embedded in epoxy resin. Ultrathin

sections of 50 to 60 nm were counterstained with uranyl acetate

and lead citrate. A Jeol JEM 1010 (Tokyo, Japan) electron

microscope was used for examination. Cases used for

transmission electron microscopy evaluation were a subgroup

of the total population due to tissue availability restrictions.

Autophagic vacuoles were identified, in villi and decidua, as

double membrane compartments containing material in various

stages of degradation. For each case, the area of autophagic

vacuoles was calculated by 2 observers (LA and CM) on

60000� printed micrographs (previously converted in digital

bitmap images by Image Processing and Analysis in Java-Ima-

geJ) in 3 randomly selected areas of villi, both in cytotropho-

blast and in syncytiotrophoblast. Quantification of autophagy

was measured as a ratio of autophagic vacuole area to the total

cytoplasmic area. The mean ratio from the 3 areas was consid-

ered as the final value for each case.

Western Blotting

Villous (n¼ 20: 10 NPs and 10 SMs) and decidual (n¼ 20: 10s

NP and 10 SMs) samples were homogenized in a glass potter in

a 1:5 ratio (weight:volume) with 50 mmol/L Tris-HCl pH 7.4,

5 mmol/L EDTA, 1 mmol/L DTT, 2% SDS, and Protease Inhi-

bitor Cocktail (Sigma Aldrich, St. Louis, Missouri) and centri-

fuged at 14 000g for 15 minutes. Protein concentration within

the supernatant was measured by the Coomassie Plus Protein

Assay reagent Kit (Pierce, Rockford, Illinois). An equal

amount of protein from each sample (30 mg) was subjected to

electrophoresis in a 10% polyacrylamide gel for LC3 and Bcl2

or in 8% polyacrylamide gel for hypoxia-inducible factor 1a(HIF-1a), caspase-3, Bax, and transferred onto nitrocellulose

membrane. Membranes were incubated in a blocking solution

(Tris-buffered saline [TBS] containing 5% powdered nonfat

milk and 0.01% Tween-20) at room temperature for 1 hour.

Membranes were incubated overnight at 4�C with the pri-

mary antibody, followed by incubation with horseradish

peroxidase-conjugated secondary antibody. The used primary

antibodies and dilutions were as follows: rabbit polyclonal

anti-HIF-1a (H-206X: sc-10790, Santa Cruz Biotechnology,

Dallas, Texas; 1:300 in TBS-Tween 1�), rabbit polyclonal

LC3 (NB100-2220, Novus Biologicals, Littleton, Colorado;

1:1000 in 5% bovine serum albumin [BSA]), mouse monoclo-

nal Bcl2 (C-2: sc-7382, Santa Cruz Biotechnology, Dallas,

Texas; 1:500 in TBS-Tween 1X), mouse monoclonal b-actin

(A5316, Clone AC-74, Sigma Aldrich, St Louis, Missouri,

1:5000 in 5% BSA), mouse monoclonal Bax (B-9: sc-7480,

Santa Cruz Biotechnology, Dallas, Texas; 1:500 in TBS-

Tween 1�), and rabbit monoclonal caspase-3 (#9665, Cell Sig-

naling, Danvers, Massachusetts; 1:1000 in 5% BSA). After

washes in TBS-Tween 1�, membranes were incubated with the

secondary antibodies at room temperature for 1 hour.

Secondary antibodies used were as follows: peroxidase-

conjugated goat antirabbit immunoglobulin (IgG; AffiniPure,

Jackson Immunoresearch, West Grove, Pennsylvania), diluted

1:10 000 in 5% milk for LC3, Beclin-1, and HIF-1a, or 1:10

000 in TBS-Tween 1� for caspase-3, and goat anti-mouse IgG

(AffiniPure, Jackson Immunoresearch, West Grove, Pennsyl-

vania), diluted 1:10 000 in 5% milk for b-actin or 1:10000 in

TBS-Tween 1X for Bax and Bcl2.

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Chemiluminescence was detected by incubating the mem-

brane with LiteAblot Chemiluminescent substrate (Lite Ablot,

EuroClone, Milano, Italy) followed by X-ray film exposure

(Kodak X-Omat Blue XB-1 Film, Eastman Kodak Company,

Rochester, New York).

Protein expression was quantified densitometrically (OD/

mm2) by Quantity One 4.2.1 image analysis software (Bio-

Rad, Segrate, Italy). To correlate the blot intensity with the

actual protein amount, calibration curves were run in blots

within the dynamic range of the instrument. After the back-

ground signal was subtracted, densitometry data were nor-

malized with respect to a single reference extract that was

inserted in every run, thereby representing the positive con-

trol to allow normalization and encompass intra- and interrun

variability. Protein levels are expressed as densitometry ratio

with b-actin. Western blotting analysis permits to distinguish

cytosolic and membrane-bound forms of LC3, LC3-I (18

kDa), and LC3-II (16 kDa), respectively.

Statistical Analysis

Analysis of immunoblots is reported as mean and standard

error of the mean. The Wilcoxon matched-pairs test and

Mann-Whitney U test were used, as appropriated, to compare

protein levels in villi and decidua and to compare autophago-

some content in cytotrophoblast and syncytiotrophoblast. The

P values �.05 were considered significant. Statistical tests

were performed using Instat 3, GraphPad software.

Results

Normal Pregnancies

Signs of autophagy were detected both in villous and decidual

specimens of first-trimester pregnancies (Figure 1C, E, and G):

LC3 staining was observed both in villous syncytiotrophoblast

and in cytotrophoblast by immunohistochemical method; LC3

immunostaining was also observed in trophoblast-anchoring

columns and extravillous trophoblast (EVT). Moreover, decid-

ual stromal cells displayed a positive immunoreactivity both in

basalis and in parietalis decidua.

We confirmed the localization of autophagy by elec-

tron microscopy in cytotrophoblast, syncytiotrophoblast, and

decidua by observing autophagosomes, a double-membrane

structures containing cytoplasmic material at various stage of

degradation (Figure 1I-L). To detect differences in autophagy

between villous and decidual samples, LC3-II protein expres-

sion was measured by Western blotting analysis. The expression

of LC3-II was comparable in decidual and villous specimens.

Spontaneous Miscarriages

Localization of LC3 staining in SM was comparable to NP, but

in syncytiotrophoblast an increased intensity of staining was

observed by immunofluorescence, finding an increase in punc-

tate dots (Figure 2A). We confirmed the increase of autophagy

in syncytiotrophoblast by semiquantitative electron micro-

scopy analysis, detecting a higher concentration of

autophagosome in syncytiotrophoblast of SMs compared to

NPs (Figure 2D and E). We detected an increased LC3-II

expression in villi from SMs than villi from NPs by Western

blotting analysis (Figure 2B and C).

Autophagy Expression and Expression of Markersof Hypoxia or Apoptosis

A proper oxygen level and cells turnover are necessary at the

implantation site to warrant an adequate evolution of preg-

nancy. Therefore, to explore the possible correlation of autop-

hagy with hypoxia, we measured the expression of HIF-1a, a

transcription factor stabilized in hypoxic conditions, by West-

ern blotting. The expression of HIF-1a was similarly low in vil-

lous and decidual samples of NP, whereas in cases of SM, the

expression of HIF-1a increased both in villi and in decidua

with the highest level in decidual samples (Figure 3A).

Moreover, to compare the expression of autophagy to apop-

tosis, we measured the expression of Bax (that promotes pro-

grammed cells death) and Bcl-2 (that inhibits programmed

cells death) in villous and decidual specimens by Western blot-

ting analysis, using for each case the same sample of tissue pre-

viously used to investigate autophagy. The ratio of Bax–Bcl-2

protein was used to indicate the susceptibility to apoptosis.

Comparable low level of apoptosis was observed both in villi

and in decidua of NP, whereas an increase in apoptosis was

observed in decidua of SM although without statistical signifi-

cance (Figure 3B). Cleaved caspase-3 expression was also

evaluated to detect apoptosis and it was observed in all samples

of decidua of miscarriage, according to the trend of Bax/Bcl-2

expression (Figure 3C) and in agreement with immunohisto-

chemical staining (Figure 3D).

Discussion

The present study assess autophagy expression during early

human pregnancy. Few previous studies have evaluated autop-

hagy in placental villi13,14 or decidua15 of the first trimester of

gestation. A novelty of our study is the analysis, at the same

time, of both the sides of blastocyst implantation, considering

the embryonic side by evaluating villi and the maternal side

by evaluating decidua.

Interestingly, the results of the present study show that

autophagy at the fetomaternal interface is constitutively

expressed in normal early human pregnancy. During normal

conditions, in villous specimens, we observed autophagy

expression in both the trophoblastic layers, with higher expres-

sion in cytotrophoblast than in syncytiotrophoblast. This obser-

vation is in agreement with a previous report,13 confirming the

physiological occurrence of autophagy in placental villi during

the first weeks of gestation.

The strength of our study is the novelty of the evaluation of

autophagy in SM. We observed an increased autophagy expres-

sion in villous samples due to an increment of autophagic

vacuoles concentration in syncytiotrophoblast. Syncytiotro-

phoblast is the outer trophoblastic layer, directly in contact with

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maternal blood. Since autophagy can intervene in placental

trophoblasts during stressor conditions as a cytoprotective

process,16 we suggest that the increase of autophagy in syncy-

tiotrophoblast could reflect a prosurvival tentative of the cells

to respond to the environmental changes.

Moreover, we detected autophagy markers in decidua speci-

mens, trophoblastic-anchoring columns, and EVT. It is known

that EVT migrates from the columns through the decidual

stroma, reaches the spiral arteries, penetrates the wall of the

vessels, and colonizes the lumen forming the trophoblastic

plugs and conducting to vascular remodeling.17 Deficiency in

EVT invasion and inadequate vascular remodeling can lead

to adverse pregnancy outcome such as miscarriage, preeclamp-

sia, and intrauterine growth restriction.18 Previous studies per-

formed on trophoblastic cell lines suggested that autophagy is

involved in trophoblast invasion,14 even if the results are con-

flicting: Some authors observed that autophagy induced by

hypoxia supports trophoblast invasion,15,19 whereas other

authors observed an increase in autophagy but a decrease in tro-

phoblast invasion in relation to the inhibition of HIF1-a expres-

sion.20 Differences in autophagy expression between decidua

from NP and SM were not detected in the present study, while

Figure 1. Localization of autophagy in normal pregnancy. A and B, negative controls. C-H, Localization of autophagy marker (LC3) by immu-nohistochemical staining. (C, E, and G), LC3 staining (autophagy marker); (D and F) cytokeratin 7 staining (CK7—marker of trophoblastic cells);(H) HLA-G staining (marker of endovascular trophoblast). Signs of autophagy are detectable in villous trophoblast (C and E) in decidual samples(E and G) and in endovascular trophoblast (G). Positivity are detectable not only in trophoblastic cells but also in some stromal cells both invillous and in decidual tissues. Original magnification 40� in (A), (B), (C), (D), (G), and (H); 20� in (E) and (F). I-L, Evaluation of autophagosomeby transmission electron microscopy. I, Villous sample: autophagosomes are clearly identifiable at higher magnification (inset) both in syncytio-trophoblast and cytotrophoblast. L, Decidual sample: double membrane autophagic compartment containing material in degradation is detect-able at higher magnification (inset). No Ab indicates no primary antibody control; C-, slice incubated with a matching concentration of nonspecificrabbit immunoglobulin; a, anchoring villous; ac, anchoring column; e.va.t, endovascular trophoblast; f, floating villous; gl, decidual gland; MMV,microvillous membrane; S, syncytiotrophoblast; C, cytotrophoblast; Fc, fetal capillary. LC3, microtubule-associated protein light chain 3.

Avagliano et al 841

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apoptosis and hypoxia were increased in SM. Our finding of

increased apoptosis in first-trimester decidua of SM is in agree-

ment with a previous report where apoptosis was responsible

for the pregnancy loss.21 In our samples of SM, we found an

increased expression of autophagy in villous specimens but a

low expression of apoptosis; on the contrary, in decidua speci-

mens we found low levels of autophagy but an increased sus-

ceptibility to apoptosis. It is possible that the opposite trend

of expression of autophagy compared to hypoxia and apoptosis

markers in villi and decidua of SM might reflect the role of

autophagy in antagonizing hypoxia-induced apoptosis, but fur-

ther investigation is needed to clarify this issue.

This study has some limitations. First, samples obtained

from legal interruption of pregnancies include viable tissues,

whereas samples from SMs include tissues from nonviable

pregnancies; it is therefore not possible to determine whether

autophagy has been induced in vivo before or after fetal

demise. Second, samples were stored in liquid nitrogen before

the analysis, whereas a previous study suggested a role of cold

stress in the induction of autophagy,22 therefore our results

might be influenced by the freezing of samples before the anal-

ysis. Third, we could not exclude that the intersample variabil-

ity in villi affected our results: Autophagy is a dynamic and

continuous process and LC3-II is just a marker of one stage

of it. Different stages of the evolution during the maturation

from the phagophore through the autolysosome in our samples

could affect the presence or the level of LC3-II.

In conclusion, our study demonstrates the occurrence of

autophagy in vivo at the fetomaternal interface during the first

trimester of normal human gestation. A low level of autophagy

Figure 2. Autophagy expression in normal pregnancy (NP) and spontaneous miscarriage (SM). A, immunofluorescence staining (LC3 in green)in villi (v) and decidua (d). Arrows indicate syncytiotrophoblast. Note the increase number of fluorescent punta dots in syncytiotrophoblasticlayer of spontaneous miscarriage, well identifiable in the insets. White scale bar: 25 mm, Yellow scale bar: 50 mm. B, Representative Westernblotting bands of autophagy markers LC3-I and II (18 and 16 kDa, respectively) and b-actin (42 kDa, housekeeping gene for normalization).Despite the high intersample variability in villous samples, the amount of LC3-II normalized onto b-actin in NP was always lower than LC3-IIin SP, as shown in (C). C, Western blotting analysis showed that the level of LC3-II expression was higher in villi obtained from spontaneousmiscarriage. *P ¼ .02 villi SM versus villi NP (Mann-Whitney test); **P ¼ .004 villi SM versus decidua SM (Wilcoxon test). D, Autophagosomecontent in placental villi, expressed as the ratio between the sum of the areas of autophagosomes and the total area, evaluated in cytotrophoblastand syncytiotrophoblast by transmission electron microscopy. * P¼ .05 syncytiotrophoblast SM versus syncytiotrophoblast NP (Mann-Whitneytest). E, Autophagosome detection by transmission electron microscopy in a case of spontaneous miscarriage. Arrow heads indicate autopha-gosome vacuoles in syncytiotrophoblast and cytotrophoblast. c indicates cytotrophoblast; s, syncytiotrophoblast; MMV, microvillous membrane;Fc, fetal capillary. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)

842 Reproductive Sciences 22(7)

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at this site may represent a physiological event necessary to the

normal evolution of pregnancy. For the first time to our knowl-

edge, our study demonstrates that SM specimens show higher

autophagy expression in villi. Further studies are needed to

clarify the role of autophagy in SM and to distinguish whether

the autophagy expression changes represent the cause or the

consequence of the early adverse pregnancy outcome.

Authors’ Note

Laura Avagliano had the idea, collected and analyzed the data, and

wrote the manuscript. Laurea Terraneo and Eleonora Virgili per-

formed the immunoblotting analysis and approved the last version

of the manuscript. Carla Martinelli performed the electron micro-

scopic study and approved the last version of the manuscript. Patrizia

Doi performed the immunohistochemical staining and approved the

last version of the manuscript. Michele Samaja discussed the data,

revised the manuscript, and approved its last version. Gaetano Bulfa-

mante analyzed and discussed the data, revised the manuscript, and

approved its last version. Anna Maria Marconi revised and approved

the last version of the manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to

the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, author-

ship, and/or publication of this article.

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