Human sample collection

This study was approved by the Medical Research Ethics Committee of the Affiliated Changshu Hospital of Nantong University, Changshu, China (ethics approval number: 2018037). Written informed consent was obtained from all subjects. Human endometrial samples and peripheral blood samples were collected from patients with clinically normal pregnancies terminated for nonmedical reasons (n = 20) and unexplained spontaneous abortions (n = 20) at the Affiliated Changshu Hospital of Nantong University, Changshu, China. Criteria for a normal pregnancy include no history of spontaneous abortion or abnormal birth, absence of abdominal pain, vaginal bleeding, fever, or any other evident discomfort after conception. Preoperative examination revealed no pathogen infection and ultrasound examination confirmed the presence of original cardiac tube pulsation without significant uterine effusion. The criteria for unexplained spontaneous abortion include the absence of fetal heart tube pulsation during two consecutive vaginal ultrasound examinations conducted one week apart in early pregnancy. Other potential causes such as trauma, fever, genital malformation, infection, or chromosome abnormality were ruled out. Clinical characteristics of patients with unexplained spontaneous abortions and clinically normal pregnancy are presented in supplementary Table 2.

Animals

C57BL/6 mice were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (CAS, Shanghai, China). The animals were housed in a specific pathogen-free (SPF) facility at Shanghai Institutes for Biological Sciences, CAS. All animal experiments were conducted following the guidelines of the Institutional Animal Care and Use Committee of Shanghai Institutes for Biological Sciences, CAS (ethics approval number: 20200306-028), and complied with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.

Mouse model

Adult C57BL/6 mice (7–10 weeks old) were mated to establish pregnancy. The presence of a vaginal plug was considered day 0 of gestation. To block UCHL1 in-vivo, a working solution was prepared by adding 50 μL of LDN57444 (dissolved in DMSO at 4 mg/mL) or DMSO to 950 μL of corn oil. Subsequently, the mice were injected i.p. daily with the identical volume of working solution containing DMSO (control group) or 0.4 mg/kg LDN57444 (UCHL1 blockade group; S7135, Selleckchem, USA) from gestational day (GD) 5 to 7 (n = 6 per group). The mice were sacrificed on GD8 and the uterine tissues were collected.

The establishment of artificially induced decidualization has been previously described [27]. Briefly, C57BL/6 female mice at 7–8 weeks of age were ovariectomized (n = 6 per group). Two weeks after ovariectomy, the mice were injected with 100 ng of E2 daily for three days. After a two-day interval, the mice received daily injections of 1 mg of P4 and 6.7 ng of E2. Six hours after the third E2 and P4 injection, the right uterine horn was stimulated by 50 µL of sesame oil, while the contralateral horn remained untreated as a control. Daily injection of E2 and P4 was continued for 5 days to induce decidualization of the endometrium. The mice were sacrificed on Day 6 for tissue collection.

Isolation of DSCs and DICs

Decidual tissues were washed twice with PBS plus penicillin–streptomycin solution (P/S), and DSCs were isolated as soon as possible according to the previously described method [28]. Briefly, decidual tissues were cut into pieces and digested with 0.1% collagenase type IV (Roche, Vienna, Austria) and 0.002% DNase I (Sigma-Aldrich, Darmstadt, Germany) in DMEM/F-12 medium for 30 to 60 min at 37 °C. After digestion, the tissue pieces were filtered through sterile gauze pads (200 mesh) to remove cellular debris. The cell suspension was then centrifuged at 1500 rpm for 8 min, and the supernatant was discarded. Collected cells were resuspended in DMEM/F-12 medium, layered over different concentrations (60%, 40% and 20% from bottom top) of Percoll (GE Healthcare, Chicago, United States) and centrifuged at 2000 rpm for 20 min. The cells from the 20%/40% interface mainly consisted of DSCs, while those from the 40%/60% interface mainly consisted of decidual immunocytes (DICs). Finally, isolated DSCs were cultured in dishes with DMEM/F-12 supplemented with 10% FBS and incubated in a humidified incubator with 5% CO2 at 37 °C. Isolated DICs were cultured in RPMI-1640 medium (HyClone, Utah, United States) containing 10% FBS (Gibco, Massachusetts, United States) and 50 µg/mL penicillin/streptomycin.

pNK cell isolation

Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples collected from healthy pregnant women in the first trimester by Ficoll-Hypaque (Sigma-Aldrich) density gradient centrifugation at 800×g for 20 min. According to the manufacturer’s instructions (MiltenyiBiotec, Bergisch Gladbach, Germany), peripheral NK cells (pNKs) were obtained through negative selection using the human NK cell isolation kit. pNKs were cultured in RPMI-1640 medium (HyClone) supplemented with 10% FBS (Gibco) and 50 µg/mL penicillin/streptomycin. The purity of CD45+CD3−CD56+ pNKs was > 90%, as determined by flow cytometry assays (data not shown).

Cell coculture assay

For the transwell assay, DSCs isolated from different samples were cultured in medium as described above and treated with LDN57444 or identical volume of DMSO as indicated. The culture medium was then collected in 24-well plates. pNKs (1 * 105 cells/well) were placed in the upper compartment of the Transwell chamber inserts (5 μm aperture, Corning, Painted Post, NY, USA). After 48 h of coculture, the number of dNKs in the lower compartments was counted.

For the coculture assay, isolated DSCs or decidualized HESCs (described below) were seeded in 24-well plates and treated LDN57444 or identical volume of DMSO as control for 24 h. Then, the culture medium was discarded and pNKs were seeded into the plate to coculture with these cells for another 48 h. The suspended cells were collected for flow cytometric analysis.

Flow cytometry assays

pNKs cocultured with DSCs or decidual HESCs were collected, and the expression of CD45, CD3, CD56 and CD16 was analyzed by flow cytometry assays. Briefly, the collected pNKs were washed twice with PBS and stained with PerCP anti-human CD45 antibody (Biolegend, San Diego, CA, USA), FITC anti-human CD3 antibody (Biolegend), BV421 anti-human CD56 antibody (Biolegend) and APC-Cy7 anti-human CD16 antibody (Biolegend) diluted in PBS with 2% FBS at 4 °C for 30 min. pNKs were then washed twice and analyzed by flow cytometry assays.

DICs isolated from decidual tissue of mice treated with DMSO or LDN57444 were collected, and the expression of CD45, CD3 and NK1.1 was analyzed by flow cytometry assays. The procedure was described above, and the antibodies used were APC-Cy7 anti-mouse CD45 antibody (Biolegend), FITC anti-mouse CD3 antibody (Biolegend) and BV421 anti-mouse NK1.1 antibody (Biolegend).

Quantitative real-time PCR

Total RNA was extracted from cells or tissues using TRIzol Reagent (Sigma-Aldrich) and subsequently reverse-transcribed into cDNA using the PrimeScript RT Master kit (TaKaRa, Shiga, Japan). Quantitative real-time PCR was performed using FastStart Universal SYBR Green Master Kits (Roche) on a ViiA7 Real-time PCR System (Applied Biosystems) according to the manufacturer’s instructions. The sequences of the primers are listed in Supplementary Table 1.

Western blot

Total proteins were extracted using lysis buffer (150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris (pH 7.4), 5 mM EDTA) containing a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Sigma-Aldrich). Then, protein lysates were electrophoresed via SDS‒PAGE and analyzed by western blotting with antibodies against UCHL1 (Abcam CAT# ab8189), IGFBP1 (Cell Signaling Technology, CST CAT# 31025), GAPDH (CST CAT# 2118, RRID: AB_561053), β-actin (CST CAT# 4970, RRID:AB_2223172), P-ERK1/2 (CST CAT# 4370, RRID:AB_2315112), ERK1/2 (CST CAT# 4695, RRID:AB_390779), FOXO1 (CST CAT# 2880, RRID:AB_2106495), JAK2 (CST CAT# 3230), P-STAT3 (CST CAT# 9145), and STAT3 (CST CAT# 9139).

Immunohistochemistry

Human and mouse tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin after dehydration, cut at 5 µm thickness and mounted on slides. The paraffin slides were then deparaffinized and rehydrated using a graded alcohol series before H&E staining or immunohistochemistry (IHC). For IHC, the slides were boiled in 10 mM sodium citrate buffer (pH 6.0) for 10 min and then naturally cooled to room temperature. After blocking with 5% bovine serum albumin in PBS (pH 7.5), the samples were incubated overnight at 4 °C with anti-UCHL1 (Abcam CAT# ab8189), followed by incubation with an HRP-conjugated secondary antibody. Immunoreactivity was detected using a DAB kit (Gene Tech, South San Francisco, United States).

Immunofluorescence

Frozen samples of human decidual tissue, embedded in optimum cutting temperature compound (OCT, Sakura Finetek), were sectioned at 7 μm thickness for immunofluorescence. After washing with PBS, the sections were fixed with a mixture of MeOH/acetone (1:1) at − 20 °C for 5 min and blocked with 1% BSA for 1 h at 37 °C. Mouse anti-human UCHL1 (Abcam CAT# ab8189) and rabbit anti-human IGFBP1 (Abcam CAT# ab111203) were applied to slides overnight at 4 °C, followed by incubation with goat anti-mouse Alexa Fluor 488-IgG or goat anti-rabbit Alexa Fluor 555-IgG (eBioscience, California, United States) for 1 h at 37 °C in the dark. Finally, DAPI staining was performed.

Cell culture and in vitro decidualization

The immortalized human endometrial stromal cell line T-HESCs (ATCC CAT# CRL-4003TM, RRID: CVCL_C464) was a gift from Professor Haibin Wang from the Institute of Zoology, CAS (Beijing, China)31. HESCs were cultured in DMEM/F-12 medium containing 10% FBS and 50 µg/mL penicillin/streptomycin with 1 mM sodium pyruvate, 1% insulin-transferrin-selenium (ITS), 3.1 g/L glucose, 1.5 g/L sodium bicarbonate and 500 ng/mL puromycin (all from Thermo Fisher Scientific). The culture medium was replenished every three days. In vitro decidualization of HESCs was induced as previously reported3. In brief, HESCs were treated with DMEM/F-12 containing 2% FBS, 10 µM medroxy progesterone (MPA, Selleck Chemicals, Texas, United States) and 0.5 mM 8-Br-cAMP (Selleck Chemical) for 7 days, and the medium was replenished every 2 days. DMSO (equal volume to LDN57444), LDN57444 (dissolved in DMSO at 10mM) (indicated concentrations) or C188-2 (indicated concentrations) were added during the induction of decidualization in certain experiments.

ELISA

In the in vitro decidualization assay, HESCs were treated with either DMSO or LDN57444. Then, the culture supernatant was collected at the indicated time points and centrifuged to eliminate cellular debris. The concentrations of PRL, CXCL12, IL15 and TGF-β were then analyzed according to standard protocols.

Plasmid construct and cell transfection

To construct the UCHL1 knockdown shRNA plasmid, we cloned shRNA-encoding oligonucleotides into the lentiviral pLKO.1 puro vector, a gift from Bob Weinberg (Addgene plasmid # 8453; http://n2t.net/addgene:8453; RRID: Addgene_8453) [29], to target UCHL1 mRNA. The shRNA sequences were as follows: shUCHL1-1, 5ʹ-CGGGTAGATGACAAGGTGAAT-3ʹ, shUCHL1-2, 5ʹ-GTGTGAGCTTCAGA TGGTGAA-3ʹ, shUCHL1-3′ 5ʹ-CCAGCATGAGAACTTCAGGAA-3ʹ and scramble control 5ʹ-CCTAAGGTTAAGTCGCCCTCG-3ʹ, synthesized by Sangon Biotech.

For generation of the overexpression plasmid pLVX/UCHL1, the cDNA encoding the human UCHL1 gene was amplified from the cDNA of HESCs and inserted between the XhoI and XhaI sites of pLVX-IRES-ZsGreen1 (Clontech) and pCMV-Tag2B (Stratagene). The C90S mutation of UCHL1 used PCR-based site-directed mutagenesis based on pLVX/UCHL1 with the following primers: forward, 5ʹ-AATTCCTCTGGCACAATCGGACTTATTC-3ʹ, reverse 5ʹ-CGATTGTGCCAG AGGAATTCCCAATGG-3ʹ.

To establish UCHL1 knockdown or UCHL1-overexpressing HESCs, we performed lentivirus packaging and production according to the manufacturer’s protocol (http://www.addgene.org/tools/protocols/plko/). Briefly, lentiviral plasmids harboring the desired sequences were transfected into 293T cells, together with the packing plasmids pSPAX2 and pMD2G using Lipofectamine 2000 according to the reagent protocol. The supernatants were collected 48 h after transfection, filtered through a 0.45 µm filter and used to infect HESCs in a 6-well plate supplemented with 10 µg/mL polybrene (Thermo Fisher).

Statistics

All data were shown as the mean ± SEM and were obtained from at least three independent experiments. Significant differences were analyzed by the Mann–Whitney U test, one-way ANOVA and two-way ANOVA with GraphPad Prism (version 8.0, GraphPad Software) and Statistical Package for Social Science software (version 23.0, SPSS).