bqae049-html.html

© The Author(s)  2024. Published  by Oxford University  Press on behalf of the Endocrine  Society.
All rights reserved.  For commercial  re-use, please contact reprints@oup.com  for reprints  and
translation  rights  for reprints.  All other  permissions  can be obtained  through  our RightsLink
service via the Permissions  link on the article page on our site

—

for further information please

contact journals.permissions@oup.com. This article is published and distributed under the
terms of the Oxford University Press, Standard Journals Publication Model
(https://academic.oup.com/pages/standard-publication-reuse-rights)

BMP4 in human endometrial stromal cells can affect

decidualization by regulating FOXO1 expression

Yanjie Huang

, Fangfang Dai

, Liping Chen, Zhidian Li, Hua Liu

1, *

, Yanxiang

Cheng

1,*

Yanjie Huang, Department of Obstetrics and Gynecology, Renmin Hospital of Wuhan

University, 99 Zhang Zhidong Road, Wuhan, Hubei 430060, People’s Republic of

China. E-mail:

2021283020234@whu.edu.cn

. ORCID ID: 0009-0006-9886-3796

Department of Obstetrics and Gynecology, Renmin Hospital of Wuhan University,

Wuhan, Hubei 430060, China

#These authors have contributed equally to this work and share the first authorship

* Corresponding authors

：

Professor Yanxiang Cheng, Department of Obstetrics and Gynecology, Renmin

Hospital of Wuhan University, 99 Zhang Zhidong Road, Wuhan, Hubei 430060,

People’s Republic of China. E-mail: yanxiangCheng@whu.edu.cn. ORCID ID: 0000-

0002-9218-5246

Professor Hua Liu, Department of Obstetrics and Gynecology, Renmin Hospital of

Wuhan University, 99 Zhang Zhidong Road, Wuhan, Hubei 430060, People’s

Republic of China. E-mail:

liuhua008@tom.com.

ORCID ID: 0009-0009-5591-9325

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

DISCLOSURE SUMMARY: The authors have nothing to disclose.

Keywords:

Recurrent spontaneous abortion, decidualization, BMP4, FOXO1,

maternal-fetal interface.

Abstract

Background

: Recurrent spontaneous abortion (RSA) is defined as the loss of two

or more consecutive intrauterine pregnancies with the same sexual partner in the first

trimester. Despite its significance, the etiology and underlying mechanisms of RSA

remain elusive. Defective decidualization is proposed as one of the potential causes of

RSA, with abnormal decidualization leading to disturbances in trophoblast invasion

function.

Methods

: Decidual samples were collected from both RSA patients and healthy

controls to assess BMP4 expression. In vitro cell experiments utilized the hESC cell

line to investigate the impact of BMP4 on decidualization and associated aging, as

well as its role in the maternal-fetal interface communication. Subsequently, a

spontaneous abortion mouse model was established to evaluate embryo resorption

rates and BMP4 expression levels.

Results

: Our study identified a significant downregulation of BMP4 expression

in the decidua of RSA patients compared to the normal control group. In vitro

experiments demonstrated that BMP4 knockdown resulted in inadequate

decidualization and inhibited associated aging processes. Mechanistically, BMP4 was

implicated in the regulation of FOXO1 expression, thereby influencing

decidualization and aging. Furthermore, loss of BMP4 hindered trophoblast migration

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

and invasion via FOXO1 modulation. Additionally, BMP4 downregulation was

observed in RSA mice.

Conclusions

: Our findings highlighted the downregulation of BMP4 in both RSA

patients and mice. BMP4 in human endometrial stromal cells was shown to modulate

decidualization by regulating FOXO1 expression. Loss of BMP4 may contribute to

the pathogenesis of RSA, suggesting potential avenues for abortion prevention

strategies.

Abbreviation:

RSA: Recurrent spontaneous abortion;

hESCs: human

endometrial stromal cells; qRT

‐

PCR: quantitative real

‐

time polymerase chain

reaction; PRL: prolactin; IGFBP1: insulin-like growth factor binding protein-1;

DSCs: decidua stromal cells; BMP4: Bone morphogenetic protein 4; TGF-β:

Transforming growth factor β; FOXOs: Forkhead transcription factors; HC: Healthy

control.

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

Introduction

Recurrent spontaneous abortion (RSA) is defined as two or more consecutive

spontaneous abortions with the same sexual partner before 20-24 weeks of

gestation

(1)

Globally, the incidence of RSA in women of reproductive age is

approximately 5%

(2)

. RSA can have adverse effects on the physical health, mental

well-being, and family dynamics of affected couples. While chromosomal

abnormalities, endocrine factors, and anatomical factors are recognized causes of

RSA, there remain unknown factors contributing to the condition in some patients

(1)

Therefore, it is imperative to elucidate the pathogenesis of RSA and explore effective

treatment options for affected individuals.

Decidualization refers to the transformation of human endometrial stromal cells

(hESCs) into decidua stromal cells (DSCs) under progesterone stimulation during the

secretory phase. Achieving mature decidualization is crucial for successful embryo

implantation. The transient presence of senescent cells has been shown to facilitate

decidualization, aiding in the normal implantation of embryos

(3)

. Conversely, the

absence of senescent decidual cells can result in implantation failure or early

pregnancy loss

(4)

. Abnormal decidualization is associated with pathological pregnancy

such as abortion

(5-7)

, preeclampsia

(8)

and recurrent implantation failure

(9)

. However,

the underlying mechanisms remain largely elusive.

Bone morphogenetic protein 4 (BMP4), a member of the transforming growth

factor β (TGF-β) superfamily, regulates various biological processes, including cell

growth, apoptosis, and differentiation

(10)

While BMP4 has been implicated in early

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

embryonic development

(11-13)

its role in abortion has not been extensively studied. In

our investigation, we observed a significant decrease in BMP4 expression within the

decidua of RSA patients for the first time, suggesting a potential involvement of

BMP4 in RSA.

Forkhead transcription factors (FOXOs), named after the forkhead gene of

Drosophila melanogaster

(14)

encompass four FOXO subtypes (FOXO1, FOXO3,

FOXO4, and FOXO6) in mammalian cells. During decidualization, FOXO1 emerges

as a pivotal transcription factor initiating the expression of decidualization marker

genes (IGFBP1 and PRL). Additionally, FOXO1 is implicated in cell cycle arrest,

apoptosis, cell senescence, and other cellular behaviors crucial to decidualization

(15-

18)

Our study reveals a significant reduction in BMP4 expression within the decidua

of patients with RSA. Furthermore, our findings demonstrate that BMP4 depletion

correlates with impaired decidualization. It is plausible that BMP4 downregulation

diminishes FOXO1 expression, thereby inhibiting cellular aging processes and

resulting in insufficient maturation of DSCs, consequently impairing decidualization

and potentially culminating in miscarriage. Additionally, impaired decidualization

may compromise the migration and invasion abilities of trophoblast cells, disrupting

communication at the maternal-fetal interface. Collectively, our results underscore the

potential significance of abnormal BMP4 loss as a mechanism underlying impaired

decidualization in RSA patients.

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

Materials and methods

2.1 Decidual Tissue Collection

Decidua samples were collected at Wuhan University People's Hospital, and

decidua tissues from RSA patients and patients with elective termination of pregnancy

less than 20 weeks were collected with the informed consent of the subjects, all of

whom were younger than 35 years old. The inclusion criteria were as follows: (1)

Unexplained recurrent abortion; (2) Both blood and urine pregnancy tests were

positive; (3) Intrauterine pregnancy was confirmed by ultrasound. The exclusion

criteria are as follows

(19)

: (1) diagnosis of uterine abnormalities, such as congenital

anatomic abnormalities, fibroids, adenomyopathy, endometrial polyps, etc. (2)

diagnosis of chromosomal abnormalities or endocrine disorders; (3) Rule out infection

or immune disease

(20)

After the sample was collected, the blood on the tissue was

cleaned with PBS, partially fixed with paraformaldehyde, and partially quickly frozen

in liquid nitrogen. Human protocols were approved by the Ethics Committee of

Clinical Research, Renmin Hospital of Wuhan University (WDRY2020-K218).

2.2 Lentivirus Infection

Overexpression of lentivirus and small RNA interference (shRNA) were used in

this

study. Lentivirus was purchased from Shanghai Genechem Gene Medical

Technology Co., LTD. The reagent concentration followed the manufacturer's

instructions to determine the optimal infection concentration. The MOI=30 was

selected for the follow-up experiment. Cells transfected with lentivirus were cultured

in (DMEM)/F-12 supplemented with 10% FBS. After transfection for 72 h, the

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

infected cells with stable gene expression were selected with purinomycin (5 μg/mL).

Overexpression and knockdown efficiency were verified by quantitative real‐time

polymerase chain reaction (qRT‐PCR) or Western blotting.

2.3 Cell Culture and Treatment

The human choriocarcinoma cell line HTR-8/SVneo was donated by the

Reproductive Center of Sun Yat-sen University.

HTR-8/SVneo was cultured with 10%

fetal bovine serum (FBS) (Gibco, Life Technologies, Grand Island, NY) and 100

units/mL of penicillin and streptomycin (Invitrogen, Waltham, MA, United States) in

Dulbecco's modified Eagle's medium (DMEM)/F-12 (Meilunbio, China).

hESCs

(Xiamen Immocell Biotechnology Co., Ltd., China, Cat: #

IM-H442) was

supplemented

in the phenol red-free Dulbecco's modified Eagle's medium

(DMEM)/F-12 (Meilunbio, China) plus 10% FBS at 37°C in 5% CO

atmosphere.

hESCs were treated with 1 mol/L medroxyprogesterone-17-acetate (MPA)

(MedChemExpress, USA, Cat: # HY-B0469) and 0.5 mmol/L N6,20-

Odibutyryladenosine cAMP sodium salt (db-cAMP) (MedChemExpress, USA, Cat:

#HY-B0764) to induce decidualization in vitro. T

he medium was changed every 2 d.

The concentration of exogenous FOXO1 inhibitors (AS1842856, MCE, United States,

Cat: HY-100596) was 100 nM.

2.4 qRT‐PCR

Total RNA was extracted from cells or tissues with TRIzol reagent (Invitrogen)

according to the manufacturer's instructions. The spectrophotometer (NanoDrop

2000c; Thermo Fisher Scientific, Waltham, MA, United States) was used to detect

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

RNA concentration and purity. Total RNA (1 μg) was reverse-transcribed into cDNA

using a reverse transcription kit (Yeasen Biotechnology Co., Ltd. Cat#11198ES08,

China), the qPCR was performed according to the manufacturer's specification of

SYBR GREEN PCR Master Mix (Shanghai Yisheng Co., Ltd.) in the Bio-Rad PCR

system's Applied Biosystems CFX96TM Real-Time PCR system, and the experiment

was triplicate. In cell experiments, PCR products were detected using the 2

−ΔΔCt

technique relative to glyceraldehyde-phosphate dehydrogenase (

GAPDH

) for standard

gene expression levels, and in human and animal tissue experiments, the 2

−ΔCt

technique relative to

GAPDH

was used to detect the PCR products

(21)

Primer sequences used were as follows: h-

GAPDH

sense 5’-GTA TCG TGG

AAG GAC TCA TGA C-3’,

GAPDH

antisense 5’-ACC ACC TTC TTG ATG TCA

TCA T-3’, h-

BMP4

sense 5’-GAT GTG GGC TGG AAT GAC-3’, h-

BMP4

antisense

5’-GGT TGG TTG AGT TGA GGT G-3’, h-

FOXO1

sense 5’-TCT ACG AGT GGA

TGG TCA AGA-3’, h-

FOXO1

antisense 5’-ATG AAC TTG CTG TGT AGG GAC-

3’, h-

PRL

sense 5’-CTA CAT CCA TAA CCT CTC CTC A-3’, h-

PRL

antisense 5’-

GGG CTT GCT CCT TGT CTT C-3’, h-

IGFBP1

sense 5’-CTA TGA TGG CTC

GAA GGC TC-3’, h-

IGFBP1

antisense 5’-TTC TTG TTG CAG TTT GGC AG-3’,

GAPDH

sense 5’-TCG CTC CTG GAA GAT GGT GAT-3’, m-

GAPDH

antisense

5’-CAG TGG CAA AGT GGA GAT TGT TG-3’ and m-

BMP4

sense 5’-TTG ATA

CCT GAG ACC GGG AAG-3’, m-

BMP4

antisense 5’-ACA TCT GTA GAA GTG

TCG CCT C-3’.

The amplification efficiency of all primers was 90-110% (107.90%

for h-

GAPDH

, 94.77% for h-

BMP4

, 102.74% for h-

PRL

, 98.35% for h-

IGFBP1

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

102.56% for h-

FOXO1

, 104.35% for m-

GAPDH

, and 101.52% for m-

BMP4

2.5 Western blot

RIPA buffer including PMSF protease inhibitors (Beyotime Biotechnology,

China) and a phosphatase inhibitor (Beyotime Biotechnology, China) was used to

extract total protein from homogenized tissue or cultured cells. The Enhanced BCA

Protein Assay Kit (Beyotime Biotechnology, China, Cat: #

KR0008) was used to

extract the protein. SDS-PAGE was applied to isolate equal quantities of protein (50

μg protein per hole) and then transfer the protein to 0.45 mm/0.22 mm polyvinylidene

difluoride (Merck Millipore, Billerica, MA). After being blocked with 5% skim milk

powder at room temperature for 1 h, anti-GAPDH (ABclonal Cat# AC001,

RRID:

AB_2619673

), BMP4 (ABclonal Cat# A11315,

RRID: AB_2758503

), PRL (ABclonal

Cat# A1618,

RRID: AB_2763630

), IGFBP1 (Cell Signaling Technology Cat# 31025,

RRID: AB_2798998

), FOXO1 (Proteintech Cat# 66457-1-Ig,

RRID: AB_2881826

p53 (ABclonal Cat# A0263,

RRID: AB_2757076

), p21 (ABclonal Cat# A1483,

RRID: AB_2761709

), and p16 (ABclonal Cat# A0262,

RRID: AB_2757075

) were

used as primary antibodies overnight in a shaking bed at 4°C. Then, the secondary

antibodies were used HRP Goat Anti-Rabbit IgG (H+L) (ABclonal Cat# AS014,

RRID: AB_2769854

) or HRP Goat Anti-Mouse IgG (H+L) (ABclonal Cat# AS003,

RRID: AB_2769851

) to incubate the membranes for 1 h at room temperature. Adding

the ECL Enhanced Plus Kits (RM0021P, ABclonal, China), the chemiluminescent

detection system (Bio-Rad, United States) was used to detect protein expression.

2.5 Senescence β-Galactosidase (SA-β-Gal) Staining

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

Senescence β-Galactosidase Staining Kit (Beyotime Biotechnology, China, Cat:

#C0602) was applied to stain cells. According to the manufacturer's instructions, after

being washed with PBS once, the hESC cells were firstly fixed at room temperature

for 15 min with β-galactosidase staining fixing solution. After the fixing solution was

removed, the cells were cleaned with PBS three times for 3 min. After PBS was

removed, a certain proportion of staining working solution (10 μL β-galactosidase

solution A, 10 μL β-galactosidase solution B, 930 μL β-galactosidase solution C, 50

μL X-Gal solution) was added according to the instructions. Incubated overnight at

37°C without CO

. Three areas were randomly selected with a fluorescence

microscope at ×200 magnification to observe the proportion of the number of stained

cells in the whole visual field.

2.6 Immunohistochemistry

The newly collected endometrial tissues were fixed with 4% paraformaldehyde,

embedded with paraffin wax, and routinely dewaxed, rehydrated, and sectionalization.

Then high-pressure thermal repair was used, citrate buffer of pH 6.0 was used to

infiltrate all slices, and after boiling for 5 min in a pressure cooker, the slices were

soaked for natural cooling, and then soaked in 3% H

for 15 min away from light.

After blocking endogenous peroxidase and blocking serum, they were incubated

overnight with BMP4 primary antibody (1:100) at 4°C, and then the secondary

antibody was incubated in a humidor with HRP Goat Anti-Rabbit IgG (H+L) at 37

℃

for 30 min. Finally, it was stained with diaminobenzidine and reversed stained with

hematoxylin. Protein expression was analyzed with ImageJ Pro Plus version 6.0

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

software (Media Cybernetics Inc.).

2.7 Immunofluorescence

Reactive oxygen species were detected with dihydroethidium (superoxide anion

fluorescent probe) kit (Yeasen Biotechnology Co., Ltd., China, Cat: #50102ES02).

According to the instructions, the mother liquor was diluted to the desired working

concentration with a buffer solution (PBS). The cells were cleaned with PBS 3 times,

and the basic medium and working solution were mixed at a ratio of 1:1 and added to

the culture plate, and incubated for 1 h at room temperature or 37°C without light.

The fluorescence signal was examined under a fluorescence microscope with a

magnification of 200

2.8 Transwell Assays

Transwell inserts with 8.0 μm diameter chamber were pretreated with Matrigel

(40



L, dilution 1: 8, sigma, St Louis, MO). The invasion ability of trophoblast cells

was assessed by counting the number of cells that penetrated Matrigel and reached the

lower surface of the inserts, and the migration ability of trophoblast cells was

estimated by transwell inserts without Matrigel. hESCs transfected with ov-NC, ov-

BMP4, sh-NC, and sh-BMP4 lentivirus were uniformly implanted in the lower

chamber, and decidualization was induced by MPA and db-cAMP. Other drugs were

added 48 h after induction. After 4 d, 1

cells/mL HTR-8/SVneo trophoblast cells

were implanted in transwell inserts pretreated with 1:8 diluted 40 μL Matrigel and

cultured with basal medium. 2

cells/mL hESCs were cultured in the lower

chamber with 20% FBS in the medium after induction. The Co-cultured times for

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

migration and invasion were 24 h and 48 h, respectively. Then the transwell chamber

was cleaned 3 times with PBS at room temperature, the cells on the surface of

Matrigel were wiped with a cotton swab, the chamber was placed in methanol for 15

min, stained with crystalline iodine for 10 min, and cleaned 3 times with PBS. Under

a fluorescence microscope (Olympus, Japan), cells penetrating to the lower surface

can be observed with a magnification of 200×. Three regions were randomly selected

and the number of cells was calculated. The experiment was repeated three times

independently.

2.9 Spontaneous Abortion Animal Model Construction

Twenty C57 female mice and ten C57 male mice were purchased from Beijing

Vital River Laboratory Animal Technology Co. Ltd., China. The number of male and

female mice was evenly divided and mated, ten C57 female mice were used to

construct a normal pregnancy mouse model, and others were generally used to

construct a spontaneous abortion mouse model. The first day of seeing a vaginal plug

was 0.5 d of gestation, at the 7.5 d of gestation, 0.25 mg/kg LPS was injected

intraperitoneally to induce abortion at the 7.5 d of gestation. All female mice were

killed at 13.5 d of gestation, and the embryo absorption rate (number of embryos

absorbed/number of total embryos *100%) was measured and calculated. All animal

studies were approved by the ethics committee for laboratory animal welfare

(IACUC) of Renmin Hospital of Wuhan University [No. WDRM animal (f) No.

20201207].

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

2.10 Statistical Analysis

Each experiment was conducted in triplicate and repeated three times. All data

were analyzed with GraphPad Prism statistical software (

version

8.0). The difference

between the two groups was analyzed by student t-test. Three independent replicate

experiments were conducted in the study. All data were presented as the mean ± SEM.

< 0.05, the difference was considered statistically significant.

Results

3.1 The expression of BMP4 was significantly decreased in the decidual tissue of

the RSA patient

To determine whether BMP4 had a certain relationship with the pathogenesis of

RSA, we first collected the decidua tissues of RSA patients and

the

healthy control

(HC) group.

It indicated that the mRNA and protein levels of BMP4 in decidua tissue

of RSA patients were significantly lower than that in the HC group (

Figure 1A-C

The results of IHC showed that BMP4 staining in the HC group was strongly positive

and strongly expressed in both cytoplasm and nucleus, while only a small amount of

positive staining was found in the RSA group (

Figure 1D

). These results suggested

that BMP4 may play a role in the decidualization of endometrial stromal cells.

3.2 BMP4 facilitated the decidualization of endometrium

To further investigate the specific role of BMP4 in the pathogenesis of RSA, we

first induced decidualization of hESC in vitro. After induced decidualization with db-

cAMP and MPA for 6 d, the DSC cells were enlarged and rounded, which was

consistent with the characteristics of cell morphological reprogramming during

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

decidualization (

Figure 1H

). Compared with hESC, the mRNA level of

BMP4

DSC increased by about 20 times, and the protein level increased by about 1.6 times

(

Figure 1E-G

). This revealed that BMP4 was associated with decidualization.

To investigate the effect of BMP4 on endometrial decidualization, hESC was

transfected with sh-BMP4 and ov-BMP4. After being transfected with lentivirus, the

cells still had long spindle interstitial cell morphology. qPCR and Western blot were

used to verify the effectivity of knockdown and overexpression of BMP4

(Figure 2A-

). Then, we induced decidualization of hESC transfected with sh-BMP4 and ov-

BMP4. The results showed that the mRNA and protein levels of decidualizing

biomarkers (IGFBP1 and PRL) were significantly decreased in the sh-BMP4 group,

and highly higher in the ov-BMP4 group (

Figure 2D-F

). In addition, the cell

morphology in the sh-BMP4 group was more similar to that of fibroblasts. The cell

morphology of the control group and the ov-BMP4 group was more similar to that of

epithelial cells (

Figure 2G

3.3 BMP4 affected the cellular aging associated with decidualization

Well-differentiated decidualization is accompanied by a certain degree of cell

senescence, and the lack of cell maturity will affect the normal function of

decidualization

(4, 22, 23)

. To investigate how BMP4

regulated decidualization, db-cAMP

and MPA were used to induce hESC with knockdown and overexpressed BMP4 4 d.

The results showed that the knockdown of BMP4 inhibited the protein levels of p53,

p21, and p16, while overexpression of BMP4 enhanced the expression of these genes

(

Figure 3A-D

). Using Senescence Associated β-Galactosidase (SA-β-Gal) Staining to

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

stain DSC after 4 d of induction, it was observed that the number of stained cells

decreased significantly in the sh-BMP4 group compared with the control group. The

number of stained cells increased highly in the ov-BMP4 group (

Figure 3E, F

). Since

aging is accompanied by the accumulation of ROS in cells, we further observed the

fluorescence intensity of cells in each group with a fluorescence microscope. The

results showed that the trend of ROS was consistent with the above experimental

results

(Figure 3G, H)

. Overall, BMP4 regulated the cellular aging associated with

decidualization.

3.4 BMP4 regulated the expression of transcription factor FOXO1 to influence

decidualization and cell aging

Many

studies have shown that FOXO1 initiates the decidualization of endometrial

stromal cells, and can also mediate cell aging, apoptosis, and cell cycle arrest

(22, 24)

We first investigated whether BMP4 can regulate FOXO1 expression. The results

showed that the expression level of FOXO1 was decreased after BMP4 was knocked

down, overexpression of BMP4 can increase the expression level of FOXO1 (

Figure

4A, B

). To determine whether FOXO1 was involved in BMP4-mediated

decidualization, we added AS1842856 (FOXO1-specific inhibitor) into the medium of

the ov-BMP4 group with decidualization induction, and we continued to culture the

cells for two days. It can be seen that compared with the ov-BMP4 group, the protein

levels of decidualization markers (IGFBP1 and PRL) in the ov-BMP4 group with

AS1842856 were highly decreased, and the expression of aging-related factors (p53,

p21, and p16) was also reduced. However, the expression of BMP4 was not affected

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

(

Figure 4C, D

). Compared with the ov-BMP4 group, the proportion of β-

galactosidase positive cells also decreased effectively in the ov-BMP4 group with

AS1842856,

only 28% of the former group (

Figure 4E, F

), and the fluorescence

intensity of ROS was also inhibited

(Figure 4G, H)

In summary, BMP4 regulated the decidualization of endometrium by regulating

the expression of FOXO1 downstream, as well as the associated cell aging.

3.5 BMP4 affected

migration and invasion ability of trophoblast cells in

maternal-fetal interface crosstalk

The pathogenesis of abortion is multi-factorial and complicated. The normal

interaction between decidua and villi is necessary to maintain the stability of the

maternal-fetal interface

(25)

. We hypothesized that low expression of BMP4 in decidua

would inhibit villi migration and invasion. To verify the hypothesis, hESC transfected

with lentivirus was inoculated in 24-well plates to induce decidualization for 4 d, and

HTR-8/SVneo cells were planted in transwell chambers and co-cultured with DSC for

24 h and 48 h (

Figure 5A

). Compared with the control group, the migration and

invasion ability of HTR-8/SVneo cells co-cultured with DSC were significantly

reduced in the sh-BMP4 group. The migration and invasion ability of HTR-8/SVneo

co-cultured with the ov-BMP4 group was significantly enhanced (

Figure 5B-E

). To

investigate whether BMP4 also affected the migration and invasion ability of

trophoblast cells through FOXO1, AS1842856 was selectively added to hESC with

decidualization induced for 2 d

(Figure 5F

). The results showed that when

AS1842856 was added to the medium, the migration and invasion ability of HTR-

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

8/SVneo cells was decreased compared with that in the ov-BMP4 group

(Figure 5G-

. The above experiments indicated that the high expression of BMP4 during

decidualization could affect the migration and invasion ability of trophoblast cells

through FOXO1.

3.6 The expression of BMP4 was reduced in the miscarriage mice model

To verify the results of the above tissue and cell experiments, we constructed a

miscarriage mouse model (

Figure 6A

) to explore the function of BMP4 on the

abortion of mice. As shown in

Fig 6B, C

, the embryo absorption rate of aborted mice

was significantly higher than that of the control group. More importantly, it was found

that the mRNA expression level of

BMP4

was decreased in decidual tissues of aborted

mice (

Figure 6D

). The above results demonstrated that the expression level of BMP4

was also reduced in the decidua of miscarriage mice. This indicated that BMP4 was

indeed involved in the process of miscarriage in vivo.

Discussion

The etiology of RSA is multifaceted. Apart from well-established physiological,

pathological, and anatomical factors, there is growing evidence indicating that

abnormal decidualization underlies RSA

(26)

. In our study, we illustrated that BMP4

deficiency in human decidual tissue triggers impaired decidualization, consequently

resulting in adverse pregnancy outcomes such as RSA. While previous research has

predominantly explored BMP4's roles in tumorigenesis

(27)

, bone homeostasis

(28)

, and

tissue regeneration

(29)

, and other areas, there remains a scarcity of studies on its

relevance to pregnancy and its outcomes. Our research represents a pioneering effort

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

in demonstrating BMP4's significant role in the decidualization process of

endometrial stromal cells. Following successful decidualization, BMP4 expression,

initially low, markedly increases. Suppression of BMP4 expression impedes

decidualization progression and the natural aging and apoptosis associated with this

process, adversely affecting blastocyst implantation, placental development, and fetal

growth and development

(30)

Aging cells exhibit increased activity of aging-related β-galactosidase (SA-β-

gal), expression of Senescence-Associated Secretory Phenotype (SASP), and

corresponding aging protein markers

(31)

. Evidence suggests an association between

decidualization and cellular aging

(32)

. While premature aging damage to decidua cells

can lead to abnormal decidua and uncontrollable inflammation

(3)

, in a normal decidua

process, acute decidual senescence and transient SASP post-ovulation support

pregnancy by maintaining an appropriate balance of cell differentiation and

senescence. Our study indicates that BMP4 deficiency may result in inadequate aging

of decidual cells, contributing to abnormal decidualization and abortion.

During the decidualization process, FOXO1 is commonly recognized as a

transcription factor that initiates the transcription of decidualization marker genes. It

directly binds promoters, regulating the transcription of IGFBP1 and PRL, thereby

promoting the occurrence of decidualization

(33)

. Moreover, FOXO1 may also play a

role in the aging of IL-8-dependent intimal stromal cells

(22, 34)

contributing to a more

favorable environment for blastocyst implantation and placenta development. In our

study, we observed that the knockout of BMP4 resulted in decreased expression of

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

FOXO1, leading to the inhibition of both decidualization and cell senescence to a

certain extent. Upon the addition of a FOXO1-specific inhibitor (AS1842856), the

expression of decidualization marker genes and senescence-related molecules

decreased, along with a reduction in senescent cells and reactive oxygen species

(ROS). Notably, the expression of BMP4 remained unaffected. This finding

substantiates that FOXO1 acts as the downstream molecule of BMP4 in the regulation

of decidualization and senescence. Other studies suggest that this regulatory process is

likely mediated through the classical PI3K/AKT pathway

(15, 35, 36)

Furthermore, the trophoblast and decidua exert reciprocal influences on each

other, with a myriad of cytokines produced by both compartments capable of

interacting and influencing their respective functions. Within the co-culture system

utilized in this study, we observed that BMP4 secreted by decidual cells also impacts

the migration and invasion capabilities of trophoblast cells. Specifically, the migration

and invasion capacities of HTR-8/SVneo cells treated with the culture medium

supernatant of decidual cells were markedly diminished in the sh-BMP4 group. In a

rescue experiment, the addition of exogenous AS1842856 to the ov-BMP4 group led

to a decrease in the migration and invasion abilities of HTR-8/SVneo cells. This

suggests that BMP4 can influence trophoblast invasion through FOXO1, potentially

modulating the epithelial-mesenchymal transition process. However, the precise

underlying mechanism warrants further exploration. Finally, in a successful RSA mice

model, we demonstrated a significant reduction in BMP4 expression within the

decidua tissue of aborted mice compared to normal pregnant mice. This finding

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

further underscores the pivotal role of BMP4 in the pathogenesis of RSA.

Furthermore, both BMP2 and BMP4 are members of the BMP family. Previous

studies have highlighted BMP2's impact on the decidualization of decidua stromal

cells by modulating FOXO1 expression through the WNT4 signaling pathway

(37, 38)

BMP2 is also known to promote trophoblast proliferation, steroid synthesis,

angiogenesis, and placental function through the autocrine/paracrine pathway

(39)

Despite BMP4 being recognized as a pivotal molecule in regulating embryonic stem

cell differentiation and mesodermal differentiation

(40)

, its role in abortion has been

relatively understudied. Building on prior research, our innovative exploration sought

to determine whether BMP4 could similarly regulate FOXO1 expression and

influence trophoblast cell function. Our experiments substantiated that BMP4 indeed

shares comparable biological functions with BMP2. It not only affects decidualization

by modulating FOXO1 expression but also enhances the migration and invasion

abilities of trophoblasts. However, further experimental research is required to

elucidate the extent of the similarities in biological functions between BMP4 and

BMP2.

Conclusion

In summary, we observed a reduction in BMP4 expression in the decidual tissue

of RSA patients (

Figure 7

). In our in vitro cell experiments, we established that

BMP4 plays a crucial regulatory role in the process of endometrial decidualization.

The loss of BMP4 resulted in the downregulation of FOXO1 expression, impeding

normal decidualization and contributing to cell senescence and apoptosis associated

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

with decidualization. Additionally, BMP4 disrupted the homeostasis of the maternal-

fetal interface and inhibited the invasion function of the trophoblastic layer. Animal

experiments further confirmed the decreased expression of BMP4 in the decidual

tissue of RSA mice. These findings illuminate the role of BMP4 in the pathogenesis

of RSA. Furthermore, more research is needed to assess whether a potential

therapeutic strategy based on BMP4 could be proposed for the diagnosis.

Declarations

6.1 Data Availability Statement

The original contributions presented in the study are included in the

article/Supplementary Material, further inquiries can be directed to the corresponding

authors.

6.2 Ethics approval and consent to participate

The studies involving human participants were reviewed and approved by the

Renmin Hospital Research and the form and use of decidua tissue were approved by

the Ethics Committee (WDRY2020-K218). All patients/participants provided their

written informed consent to participate in this research. The animal research was

reviewed and approved. All animal studies were approved by the ethics committee for

laboratory animal welfare (IACUC) of Renmin Hospital of Wuhan University [No.

WDRM animal (f) No. 20201207].

6.3 Consent for Publication

All authors agree to publication.

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

6.4 Competing interests

The authors declare no conflict of interest.

6.5 Funding

This work was supported by the National Natural Science Foundation of China

(grant number 82071655); Natural Science Foundation of Hubei Province (grant

number 2022CFB252); cross-innovation talent project in Renmin Hospital of Wuhan

University (grant number JCRCZN-2022-016); Undergraduate education quality

construction comprehensive reform project (grant number 2022ZG282).

6.6 Authors' contributions

YJH, YXC: designed this research. FFD: analyzed the data. YJH, LPC, ZDL:

interpreted the study. YJH: drafted the article. FFD, HL: revised this study. All authors

approved of the version to be published.

6.7 Acknowledgements

The authors would like to express their sincere gratitude to all RSA patients who

participated in this study.

7. References

Ford HB, Schust DJ. Recurrent pregnancy loss: etiology, diagnosis, and therapy. Rev

Obstet Gynecol. 2009;2(2):76-83.

Krieg SA, Fan X, Hong Y, Sang QX, Giaccia A, Westphal LM, et al. Global alteration in

gene expression profiles of deciduas from women with idiopathic recurrent pregnancy loss. Mol

Hum Reprod. 2012;18(9):442-50.

Deryabin PI, Borodkina AV. Stromal cell senescence contributes to impaired endometrial

decidualization and defective interaction with trophoblast cells. Hum Reprod. 2022;37(7):1505 -

24.

Rawlings TM, Makwana K, Taylor DM, Mole MA, Fishwick KJ, Tryfonos M, et al. Modelling

the impact of decidual senescence on embryo implantation in human endometrial assembloids.

Elife. 2021;10.

Zhou Q, Yan G, Ding L, Liu J, Yu X, Kong S, et al. EHD1 impairs decidualization by

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

regulating the Wnt4/beta-catenin signaling pathway in recurrent implantation failure.

EBioMedicine. 2019;50:343-54.

Ticconi C, Pietropolli A, Di Simone N, Piccione E, Fazleabas A. Endometrial Immune

Dysfunction in Recurrent Pregnancy Loss. Int J Mol Sci. 2019;20(21).

Robertson SA, Care AS, Moldenhauer LM. Regulatory T cells in embryo implantation and

the immune response to pregnancy. J Clin Invest. 2018;128(10):4224-35.

Garrido-Gomez T, Castillo-Marco N, Cordero T, Simon C. Decidualization resistance in

the origin of preeclampsia. Am J Obstet Gynecol. 2022;226(2S):S886-S94.

Peters CS, Hernandez L, Sheffield N, Chittom-Swiatlo AL, Kocka FE. Cost containment of

formalin-preserved stool specimens for ova and parasites from outpatients. J Clin Microbiol.

1988;26(8):1584-5.

10.

Roberts RM, Ezashi T, Temple J, Owen JR, Soncin F, Parast MM. The role of BMP4

signaling in trophoblast emergence from pluripotency. Cell Mol Life Sci. 2022;79(8):447.

11.

Yang M, Rito T, Metzger J, Naftaly J, Soman R, Hu J, et al. Depletion of aneuploid cells in

human embryos and gastruloids. Nat Cell Biol. 2021;23(4):314-21.

12.

Wei Y, Wang T, Ma L, Zhang Y, Zhao Y, Lye K, et al. Efficient derivation of human

trophoblast stem cells from primed pluripotent stem cells. Sci Adv. 2021;7(33).

13.

Yabe S, Alexenko AP, Amita M, Yang Y, Schust DJ, Sadovsky Y, et al. Comparison of

syncytiotrophoblast generated from human embryonic stem cells and from term placentas. Proc

Natl Acad Sci U S A. 2016;113(19):E2598-607.

14.

Weigel D, Jurgens G, Kuttner F, Seifert E, Jackle H. The homeotic gene fork head

encodes a nuclear protein and is expressed in the terminal regions of the Drosophila embryo.

Cell. 1989;57(4):645-58.

15.

Adiguzel D, Celik-Ozenci C. FoxO1 is a cell-specific core transcription factor for

endometrial remodeling and homeostasis during menstrual cycle and early pregnancy. Hum

Reprod Update. 2021;27(3):570-83.

16.

Gellersen B, Brosens JJ. Cyclic decidualization of the human endometrium in

reproductive health and failure. Endocr Rev. 2014;35(6):851-905.

17.

Wang Z, Liu Y, Liu J, Kong N, Jiang Y, Jiang R, et al. ATF3 deficiency impairs the

proliferative-secretory phase transition and decidualization in RIF patients. Cell Death Dis.

2021;12(4):387.

18.

Nadkarni S, Smith J, Sferruzzi-Perri AN, Ledwozyw A, Kishore M, Haas R, et al.

Neutrophils induce proangiogenic T cells with a regulatory phenotype in pregnancy. Proc Natl

Acad Sci U S A. 2016;113(52):E8415-E24.

19.

Carp H. Immunotherapy for recurrent pregnancy loss. Best Pract Res Clin Obstet

Gynaecol. 2019;60:77-86.

20.

Schreiber K, Sciascia S, de Groot PG, Devreese K, Jacobsen S, Ruiz-Irastorza G, et al.

Antiphospholipid syndrome. Nat Rev Dis Primers. 2018;4:17103.

21.

Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method.

Nat Protoc. 2008;3(6):1101-8.

22.

Brighton PJ, Maruyama Y, Fishwick K, Vrljicak P, Tewary S, Fujihara R, et al. Clearance of

senescent decidual cells by uterine natural killer cells in cycling human endometrium. Elife.

2017;6.

23.

Hirota Y, Daikoku T, Tranguch S, Xie H, Bradshaw HB, Dey SK. Uterine -specific p53

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

deficiency confers premature uterine senescence and promotes preterm birth in mice. J Clin

Invest. 2010;120(3):803-15.

24.

Tsuru A, Yoshie M, Kojima J, Yonekawa R, Azumi M, Kusama K, et al. PGRMC1 Regulates

Cellular Senescence via Modulating FOXO1 Expression in Decidualizing Endometrial Stromal

Cells. Biomolecules. 2022;12(8).

25.

Tan HX, Yang SL, Li MQ, Wang HY. Autophagy suppression of trophoblast cells induces

pregnancy loss by activating decidual NK cytotoxicity and inhibiting trophoblast invasion. Cell

Commun Signal. 2020;18(1):73.

26.

Tang L, Xu XH, Xu S, Liu Z, He Q, Li W, et al. Dysregulated Gln-Glu-alpha-ketoglutarate

axis impairs maternal decidualization and increases the risk of recurrent spontaneous

miscarriage. Cell Rep Med. 2023;4(5):101026.

27.

Martinez VG, Rubio C, Martinez-Fernandez M, Segovia C, Lopez-Calderon F, Garin MI,

et al. BMP4 Induces M2 Macrophage Polarization and Favors Tumor Progression in Bladder

Cancer. Clin Cancer Res. 2017;23(23):7388-99.

28.

Zuo H, Yang D, Wan Y. Fam20C Regulates Bone Resorption and Breast Cancer Bone

Metastasis through Osteopontin and BMP4. Cancer Res. 2021;81(20):5242-54.

29.

Wertheimer T, Velardi E, Tsai J, Cooper K, Xiao S, Kloss CC, et al. Production of BMP4 by

endothelial cells is crucial for endogenous thymic regeneration. Sci Immunol. 2018;3(19).

30.

Kelleher AM, Setlem R, Dantzer F, DeMayo FJ, Lydon JP, Kraus WL. Deficiency of PARP-1

and PARP-2 in the mouse uterus results in decidualization failure and pregnancy loss. Proc Natl

Acad Sci U S A. 2021;118(40).

31.

Hernandez-Segura A, Nehme J, Demaria M. Hallmarks of Cellular Senescence. Trends

Cell Biol. 2018;28(6):436-53.

32.

Tong M, Kayani T, Jones DM, Salmon JE, Whirledge S, Chamley LW, et al.

Antiphospholipid Antibodies Increase Endometrial Stromal Cell Decidualization, Senescence, and

Inflammation via Toll-like Receptor 4, Reactive Oxygen Species, and p38 MAPK Signaling.

Arthritis Rheumatol. 2022;74(6):1001-12.

33.

Christian M, Zhang X, Schneider-Merck T, Unterman TG, Gellersen B, White JO, et al.

Cyclic AMP-induced forkhead transcription factor, FKHR, cooperates with CCAAT/enhancer-

binding protein beta in differentiating human endometrial stromal cells. J Biol Chem.

2002;277(23):20825-32.

34.

Sun X, Shi B, Zheng H, Min L, Yang J, Li X, et al. Senescence -associated secretory factors

induced by cisplatin in melanoma cells promote non-senescent melanoma cell growth through

activation of the ERK1/2-RSK1 pathway. Cell Death Dis. 2018;9(3):260.

35.

Chattopadhyay T, Singh RR, Gupta S, Surolia A. Bone morphogenetic protein-7 (BMP-7)

augments insulin sensitivity in mice with type II diabetes mellitus by potentiating PI3K/AKT

pathway. Biofactors. 2017;43(2):195-209.

36.

Xu S, Wang J, Zhong J, Shao M, Jiang J, Song J, et al. CD73 alleviates GSDMD -mediated

microglia pyroptosis in spinal cord injury through PI3K/AKT/Foxo1 signaling. Clin Transl Med.

2021;11(1):e269.

37.

Deng J, Zhao HJ, Zhong Y, Hu C, Meng J, Wang C, et al. H3K27me3 -modulated

Hofbauer cell BMP2 signalling enhancement compensates for shallow trophoblast invasion in

preeclampsia. EBioMedicine. 2023;93:104664.

38.

Liu M, Deng W, Tang L, Liu M, Bao H, Guo C, et al. Menin directs regionalized decidual

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

transformation through epigenetically setting PTX3 to balance FGF and BMP signaling. Nat

Commun. 2022;13(1):1006.

39.

Kumar S, Lakshmi Devi H, Singh Jalmeria N, Punetha M, Pandey Y, Samad HA, et al.

Expression and functional role of bone morphogenetic proteins (BMPs) in placenta during

different stages of pregnancy in water buffalo (Bubalus bubalis). Gen Comp Endocrinol.

2020;285:113249.

40.

Hayaei Tehrani RS, Sayahpour FA, Esfandiari F. A comparison between BMP4 and SB4 in

inducing germ line gene expression pattern during embryonic stem cells differentiation.

Differentiation. 2022;123:9-17.

Figure Legends

Figure 1

The expression of BMP4 in decidual tissues and its changes during decidualization.

(A) qPCR was used to detect the mRNA level of

BMP4

in decidual tissues of RSA

patients and HC (n=12 for the HC group, n=13 for the RSA group). The relative

mRNA level relative to

GAPDH

was calculated by the 2

-ΔCt

method, (B, C) the protein

level of BMP4 relative to GAPDH was detected by Western blot (n=3 for each group),

and (D) the intracellular localization and expression of BMP4 protein were detected

by immunohistochemical staining. Brown represents the expression of the target gene.

The black arrow refers to the stained decidual stromal cells. Scale bar=100 μm, (E, F)

hESC cell line was used for decidualization in vitro, and the relative expression level

of BMP4 protein before and after decidualization was detected by Western blot, (G)

the relative mRNA level of

BMP4

before and after decidualization was detected by

qPCR, (H) the morphological changes of hESC before and after decidualization.

Scale

bar=100μm. Each experiment was independently performed three times. **

< 0.01

and ***

< 0.001

. control. HC, healthy control pregnancy; RSA, recurrent

spontaneous abortion.

Figure 2

Effect of BMP4 on decidualization. (A) Fluorescent expression of hESC

transfected with lentivirus. Scale bar=50 μm, (B, C) qPCR and Western blot were

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

used to test the knockdown and overexpression efficiency of BMP4 after transfection

with lentivirus, (D-F) Western blot assay and qPCR were used to detect the effects of

knockdown and overexpression of BMP4 on decidualization, (G) the effects of

knockdown and overexpression of BMP4 on DSC morphology. Scale bar=50 μm.

Data represent mean ± SEM from three separate experiments and were shown as the

mean ± SEM. *

< 0.05, **

< 0.01, and ***

< 0.001 vs. control.

Figure 3

The effect of BMP4 on aging during decidualization. (A-D) Western blot was

used to detect the degree of decidualization and senescence in DSC after BMP4

knockdown and overexpression, (E, F) Senescence β-Galactosidase Staining Kit was

used to stain DSC, and the proportion of aging cells was counted under an inverted

microscope. Scale bar=100 μm, (G, H) fluorescence signal of intracellular ROS was

observed under fluorescence microscope. Scale bar=100 μm.

Data represent mean ±

SEM from three separate experiments and were shown as the mean ± SEM. *

< 0.05,

< 0.01, and ***

< 0.001 vs. control.

Figure 4

The regulation of FOXO1 by BMP4 and the effect of FOXO1 on decidualization

and cell senescence. (A, B) The regulation of FOXO1 by BMP4 was detected by

Western blot and qPCR, (C) schematic diagram of the dosing time of FOXO1-specific

inhibitor (AS1842856) and the rescue experiment, (D) the relative protein expression

levels of BMP4, decidualization marker genes (IGFBP1 and PRL) and aging-related

genes (p52, p21, and p16) were detected by Western blot after AS1842856 was added,

(E, F) the Senescence β-Galactosidase Staining Kit was used to stain three DSC

groups, and the proportion of senescent cells in the total cell number was counted

under inverted microscope,

respectively. Scale bar=100μm, (G, H) ROS fluorescence

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024

signals in each group were observed under an inverted microscope, and the relative

optical density was calculated. Scale bar=100 μm.

Each experiment was

independently performed three times. ns

≥ 0.05, *

< 0.05, **

< 0.01, ***

0.001, and ****

< 0.0001 vs. control.

Figure 5

Effects of BMP4 on maternal-fetal interface crosstalk. (A) Diagram of the co-

culture experiment process, and (B) Transwell migration of HTR-8/SVneo cells co-

cultured with lentivirus transfected hESC. Scale bar=100μm, (C) quantification of cell

migration, and (D) Transwell invasion of HTR-8/SVneo cells co-cultured with

lentivirus-transfected hESC. Scale bar=100 μm, (E) quantification of cell invasion,

(F) schematic diagram of the dosing time of AS1842856 and the rescue experiment,

(G, H) Transwell migration of HTR-8/SVneo cells co-cultured with lentivirus-

transfected hESC after addition of AS1842856 and quantification of the results. Scale

bar=100 μm, (I, J) Transwell invasion of HTR-8/SVneo cells co-cultured with

lentiviral hESC after addition of AS1842856 and quantification of the results. Scale

bar=100μm.

Each experiment was independently performed three times. *

< 0.05,

< 0.01, ***

< 0.001, and ****

< 0.0001 vs. control.

Figure 6

The expression of BMP4 in abortion mice. (A) Schematic diagram of the construction

of the aborted mouse model, (B, C) The successful construction of the aborted mouse

model was proved through data analysis of the mouse embryo absorption rate, (D)

qPCR was used to detect the mRNA level of

BMP4

in RSA mice and control mice.

n=3 for each group. *

< 0.05 and **

< 0.01 vs. control.

Figure 7

Schematic diagram of the role of BMP4 during decidualization and in crosstalk at

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/endo/advance-article/doi/10.1210/endocr/bqae049/7659434 by UCSD-Philosophy user on 02 May 2024