Women attending the Reproductive Center of Renmin Hospital of Wuhan University (Wuhan, China) were enrolled in the present study between January 2016 and January 2019. A total of 81 patients at between 7-9 weeks of gestation were enrolled, including 21 RM patients (RM group), 34 women undergoing electively induced abortion of a pregnancy (control group) and 26 women who underwent spontaneous abortion (abortion group). The inclusion criteria were an age of <35 years, a normal body mass index (BMI) and a regular menstrual cycle. Women with any endometrial or uterine pathology, endometriosis genetic abnormalities or anovulation were excluded. RM was defined as clinically spontaneous abortion occurring prior to 20 weeks of gestation on at least two occasions. Decidual and placental villous tissues were washed in PBS and collected soon following curettage under sterile conditions, and were divided into two aliquots. One aliquot was fixed in 10% formaldehyde for terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labelling (TUNEL), immunohistochemistry and immunofluorescence, and the other was stored at −80°C for western blot analysis. All samples were collected following the provision of informed consent from the patients, and all associated procedures were performed with the approval of the internal Review and Ethics Boards of Renmin Hospital of Wuhan University.

The human THP-1 peripheral blood acute mono-cytic leukemia cell line was obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The THP-1 cells were grown in RPMI-1640 medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 mg/ml streptomycin in an atmosphere containing 5% CO₂ at 37°C. The HTR-8/SVneo cell line, derived from human extravillous trophoblasts, was obtained from the China Center for Type Culture Collection (Wuhan, China). These cells were cultured in Dulbecco's modified Eagle's medium (DMEM; high glucose; Hyclone; GE Healthcare Life Sciences) supplemented with 10% FBS and antibiotics.

The THP-1 cells were differentiated into macrophages by incubation with 50 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 48 h. Macrophages were confirmed using an immunofluorescence assay with primary antibody against CD68 (Novus Biologicals, LLC, Littleton, CO, USA), and five fields (magnification, ×400) were selected for analysis using ImageJ software (version 1.46; National Institutes of Health, Bethesda, MD, USA). A trophoblast and macrophage co-culture model was established to determine the effect of macrophage-derived cytokines on the apoptosis of trophoblast-derived cells. In brief, 1.5×10⁵ HTR-8/SVneo cells in 2 ml DMEM containing 10% FBS were seeded into the lower chambers and 5×10⁵ differentiated macrophages (M0 in 1 ml RPMI-1640 with 10% FBS) were placed into the upper chambers of a Transwell plate (Corning Incorporated, Corning, NY, USA), and cultured for 24 h at 37°C for cell attachment. Subsequently, the upper chambers containing M0 macrophages were placed on top of the lower chambers containing HTR-8/SVneo, followed by incubation at 37°C for another 48 h.

Cell apoptosis was assessed using a fluorescein isothiocyanate (FITC)-Annexin V Apoptosis Detection kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and flow cytometry using FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA). In brief, the trophoblasts were collected and stained with propidium iodide/FITC-Annexin V for 15 min at 4°C in the dark, followed by the addition of binding buffer and analysis by flow cytometry within 10 min. The data were analyzed using the FlowJo VX10 software (Tree Star, Inc., Ashland, OR, USA). All experiments were performed in triplicate.

The cells and tissues were lysed with lysis buffer containing 1 mM phenyl methane sulfonyl fluoride (both from Beyotime Institute of Biotechnology, Haimen, China) and protease and phosphatase inhibitors (21). Total protein was extracted from the supernatants of the cell lysates following incubation in lysis buffer for 30 min on ice, followed by centrifugation at 4°C for 15 min at 13,000 g. The supernatant was collected and the protein concentrations were quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Equal quantities of protein (40-60 µg) were separated by 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (EMD Millipore, Bedford, MA, USA) at 200 mA. The membranes were blocked in 5% low-fat milk for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Rabbit anti-B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax; cat. no. 2774; 1:1,000 dilution), rabbit anti-Bcl-2 (cat. no. 2876; 1:1,000 dilution; both from Cell Signaling Technology, Inc., Danvers, MA, USA) and mouse anti-GAPDH (cat. no. 60004-1-Ig; 1:5,000 dilution; ProteinTech Group, Inc., Chicago, IL, USA). Following incubation, the membranes were washed with PBS with 0.1% Tween-20 (PBST) and incubated with horseradish peroxidase-conjugated secondary antibodies (cat. no. SA00001-1 and SA00001-2; 1:5,000 dilution; goat anti-mouse or goat anti-rabbit; ProteinTech Group, Inc.) for 1 h at 37°C. The proteins were detected using a Chemiluminescence Western Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and visualized using autoradiography. Densitometric analysis of band intensities was performed using ImageJ software (version 1.46).

Immunohistochemical staining was performed as previously described (22). In brief, formalin-fixed, paraffin-embedded decidual and placental villous tissues were cut into 4-µm sections, dewaxed in xylene and rehydrated with an ethanol series of descending grades followed by PBS. Subsequently, the samples were incubated in 3% hydrogen peroxide for 15 min at room temperature to block the endogenous peroxidase activity. Antigen retrieval was performed by heating in a microwave oven with sodium citrate buffer. The sections were then incubated in 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 20 min at room temperature to block any non-specific binding, followed by incubation with primary antibodies against CD68 (cat. no. NB100-683; 1:100 dilution; Novus Biologicals, LLC), CD86 (cat. no. ab53004; 1:100 dilution), CD163 (cat. no. ab87099; 1:50 dilution; both Abcam, Cambridge, MA, USA), Bax (1:400 dilution) and Bcl-2 (1:400 dilution; both Cell Signaling Technology, Inc.) at 37°C for 1 h. The sections were then washed and incubated with goat anti-mouse/rabbit secondary antibody (1:2,000 dilution; ProteinTech Group, Inc.) for 30 min at 37°C. Antibody binding was identified as a brown precipitate following staining with 3,3-diaminoben-zidine tetrahydrochloride (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA). Images were acquired using an optical microscope at a magnification of ×400. Five visual fields with tissue occupying >80% of the area were selected for analysis and the number of immuno-positive cells was calculated using ImageJ software (version 1.46).

Double immunofluorescence was performed to evaluate the expression of FasL on CD68⁺ and CD86⁺ macrophages and the distribution of CD68⁺ macrophages and CK7⁺ trophoblasts. In brief, the sections were incubated with primary antibodies against CD68 (1:200 dilution; Novus Biologicals, LLC) or CD86 (1:500 dilution; Abcam) overnight at 4°C, followed by incubation with cyanine-3 (Cy3)-conjugated goat anti-mouse antibody (cat. no. BA1031; 1:100 dilution; Boster Biological Technology, Wuhan, China) for 30 min at 37°C, followed by incubation with bovine serum albumin for 30 min at room temperature and then with primary antibodies against FasL (cat. no. sc-33716; 1:100 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or CK7 (cat. no. 17513-1-AP; 1:100 dilution; ProteinTech Group, Inc.) at 4°C overnight. The sections were then incubated with FITC-conjugated goat anti-rabbit antibody (cat. no. BA1105; 1:100 dilution; Boster Biological Technology) for 30 min at 37°C. The sections were washed with TBST and visualized under a fluorescence microscope at a magnification of ×400. Cy3 was detected as red and FITC as green fluorescence, and the blue fluorescence signal represented the nuclear DNA stained by 4′,6-diamidino-2-phenylindole (DAPI). Five random fields from stained sections were selected. The proportion of CD68⁺ and CD86⁺ macrophages with FasL was calculated as the number of CD68⁺FasL⁺ or CD86⁺FasL⁺ cells divided by the number of CD68⁺ or CD86⁺ cells in the field using ImageJ software (version 1.46).

Apoptosis in the decidual and placental villous tissues was detected using an in situ TUNEL detection kit (Promega Corporation, Madison, WI, USA). In brief, the paraffinized sections were dewaxed in xylene, rehydrated and washed in PBS, and then stained according to the manufacturer's protocol. The nuclei were visualized with 0.1 µg/ml DAPI (Beyotime Institute of Biotechnology) in PBS at 30°C for 30 min. Cells containing the characteristic fragmented nuclear chromatin and brown nuclear staining were considered to be apoptotic. The apoptotic index was calculated as follows: Apoptotic index = (number of TUNEL - positive nuclei)/(number of total nuclei) × 100%. Images were captured with a fluorescence microscope at a magnification of ×400 and analyzed using ImageJ software (version 1.46).

The THP-1 cells stimulated with PMA or PBS were stained with anti-FasL antibody to determine the expression of FasL on macrophages. In brief, THP-1 cells were collected following stimulation with PMA or PBS, centrifuged at 300 × g for 10 min at room temperature and resuspended in PBS. The cells (1×10⁶/tube) were stained with primary antibody against FasL (cat. no. sc-33716; 1:100 dilution; Santa Cruz Biotechnology, Inc.) overnight at 4°C, and then incubated with FITC-conjugated Affinipure goat anti-rabbit immunoglobulin G (H+L; cat. no. SA00003-2; 1:100 dilution; ProteinTech Group, Inc.) for 1 h at 37°C. Finally, the fluorescence of the cells was analyzed by flow cytometry (FACS Canto II; BD Biosciences).

Values were expressed as the mean ± standard error of the mean. Statistical analyses were performed using SPSS 19.0 (IBM Corp., Armonk, NY, USA) using Student's t-test or one-way analysis of variance with Bonferroni's post hoc test. The clinical characteristics were compared between the normal and RM patients using the Kruskal-Wallis test. The experiments were repeated at least three times for each group. P<0.05 was considered to indicate a statistically significant difference.

To examine the expression and distribution of macrophages in spontaneous abortion and in miscarriages in terms of recurrence, three groups were included in the present study. The clinical characteristics of the recruited patients are listed in Table I. No significant differences were observed among the three groups in terms of maternal age, BMI and gestational age. However, the number of miscarriages in the RM group and abortion group were significantly higher and the numbers of live births were significantly lower compared with those in the control group (P<0.01 for both).

The decidual tissues from the three groups were found to contain macrophages that were stained with the primary antibody against CD68. The majority of the CD68⁺ macrophages from subjects with RM were distributed in the cytoplasm of decidual stromal cells and certain macrophages dispersed around blood vessels and glands, and the localization of CD68⁺ macrophages in the decidua of the control subjects and abortion group was similar to that of the subjects from the RM group (Fig. 1A). Cells positive for CD163 (an M2 marker) and CD86 (an M1 marker) were apparent in the three groups, mainly in the cytoplasm of stromal cells (Fig. 1A). In the RM patients, the CD68⁺ macrophage population in the decidual tissues was significantly increased, accompanied by an increased CD86⁺ macrophage population (Fig. 1B and C) and a reduced CD163⁺ macrophage population (Fig. 1D), compared with the control group. In the abortion group, the CD68⁺ macrophage population did not differ significantly, and the expression levels of the CD86⁺ and CD163⁺ macrophage populations were similar to those in the RM group (Fig. 1C). These results indicated that increased macrophage infiltration and an aberrant macrophage population in the decidua may be associated with miscarriage.

In the present study, CK7 was selected as a trophoblast marker. As presented in Fig. 2, the immunofluorescence analysis indicated that the number of CD68⁺ cells was significantly higher in the RM patients compared with that in control subjects, which is in accordance with the results of the immunohistochemical analysis (Fig. 1A and B). Furthermore, CD68⁺ macrophages were located in the periphery of the trophoblast cells from the subjects with normal pregnancies, whereas in the decidual tissues of abortion and RM patients, significant infiltration of CD68⁺ macrophages into CK7⁺ trophoblast cells was observed (Fig. 2). These results demonstrated that the macrophage distribution was significantly altered in the women in the abortion and RM groups.

To investigate the association between apoptosis and fetal loss, TUNEL was used to detect cell apoptosis and western blot analysis was used to determine the expression of apoptosis-promoting protein Bax and the anti-apoptotic protein Bcl-2 in the decidual and placental villous tissues. The TUNEL assay revealed that the proportion of cells with brown nuclear staining (apoptotic cells) and the associated apoptotic index was significantly higher in the abortion and RM groups compared with that in the control group (Fig. 3A and B), indicating that apoptosis in the decidua from the abortion and RM patients was higher than that from the controls. Furthermore, the results of the western blot analysis revealed that the protein level of Bcl-2 was reduced and the protein level of Bax was increased compared with levels in the control group, which was further confirmed by the immunohistochemical and immunofluorescence staining (Fig. 3C-F). As expected, similar results were observed in the placental villous tissues (Fig. 4A-E). These results demonstrated that elevated apoptosis in the decidual and placental villous tissues is associated with fetal loss.

As FasL is able to induce apoptosis (18), the expression of FasL has been previously detected in macrophages, with patients with RM exhibiting elevated expression of FasL (15-17). The present study performed dual immunofluorescence analysis of CD68 and FasL to identify the expression of FasL on macrophages in the decidua. The results (Fig. 5A-D) indicated that CD68⁺ dMΦ expressed minimal FasL protein, with only a weak signal in normal women; by contrast, the decidua was rich in FasL⁺ macrophages in the RM patients (Fig. 5A and C). In addition, the expression of FasL on CD86⁺ macrophages was detected by dual immunofluorescence. The proportion of CD86⁺FasL⁺ macrophages in the abortion and RM groups were significantly higher than that in the control group (Fig. 5B and D). These results demonstrated that the abnormal expression of FasL on dMΦ may be associated with miscarriage, and even RM.

As significantly increased macrophage infiltration was observed in the abortion and RM patients (Fig. 1A and B), and as macrophages are able to induce apoptosis (23), a tropho-blast and macrophage co-culture model was used to determine whether macrophages induce apoptosis in trophoblasts. As presented in Fig. 6A, both the inactivated and activated THP-1 cells (M0) expressed CD68, although the expression of CD68 in the activated THP-1 cells was increased (Fig. 6A). In addition, the expression level of FasL was increased in the M0 macrophages (Fig. 6B and C), and the co-culture model demonstrated that M0 macrophages promoted the apoptosis of trophoblast-derived cells (Fig. 6D and E). In order to verify the role of FasL in macrophage-induced trophoblast apoptosis, an intervention experiment was performed, in which anti-FasL blocking antibody or isotype control antibody (R&D Systems, Inc., Minneapolis, MN, USA) was added to the co-culture model. Macrophages induced significant cell apoptosis, whereas the addition of anti-FasL blocking antibody significantly inhibited this effect (Fig. 6D and E). These results indicated that macrophages mediate the apoptosis of trophoblast cells through FasL-dependent pathways.