Bushen Huoxue recipe was provided by HuaRun SanJiu Medical and Pharmaceutical Co., Ltd. (Shenzhen, China). The six herbal components of BSHXR were presented (Table 1), which were mixed at a weight ratio of 7:6:6:7:6:6. The voucher specimens were deposited at the Research and Development Center of China Resources SanJiu Medical and Pharmaceutical Co., Ltd. (Shenzhen, China), and authenticated by Anhui University of Chinese Medicine (Anhui, China). The six herbs were crushed into crude grains and a solution was achieved by refluxing with boiling water. The solution was subsided by ethanol and granules were obtained through concentration and vacuum-drying. One gram dry BSHXR granule is equivalent to 10 g Astragali radix, 3.33 g Angelicae sinensis radix, 6 g Ligustici Chuanxiong Rhizoma, 20 g Cuscutae semen, 15 g Taxilli Herba, and 6.67 g Dipsaci Radix raw medical herbs. In the present study, granules were dissolved in boiling water and stirred evenly before use. Herbal granules were stored under dry condition in 4°C.

To ensure the quality and stability of BSHXR, we used high performance liquid chromatography (HPLC) to identify the active compounds in BSHXR, and three batches were examined. The batch numbers were as follows: Astragali radix (1503001W, 1503001S, and 1604001W), Angelicae sinensis radix (1412005W, 1412002S, and 1604007S), Ligustici Chuanxiong Rhizoma (1607002W, 1507002S, and 1608001S), Cuscutae semen (1505001S, 1507001S, and 1607001S), Taxilli Herba (1508001W, 1404001W, and 1607001W), and Dipsaci Radix (1608001W, 1508001S, and 1607001W). The substances used for the preparation of reference solution were purchased from China National Institutes for Food and Drug Control (Beijing, China). HPLC was performed on Waters 1525-2489 HPLC system (Milford, CT, United States), and Waters SunFire C₁₈ column (250 mm × 4.6 mm, 5 μm) was used for analysis. The mobile phase consisted of acetonitrile (A) and 0.04% phosphoric acid (B) in gradient elution mode as following: 0–65 min, 10%–28% A, 90%–72% B; 65–80 min, 28%–45% A, 72%–59% B. The flow velocity was 1.0 ml/min and the temperature of the column was maintained at 30°C. The injection volume was 20 μl.

The overall graphic scheme is summarized (Figure 1). Mice of the Kun-ming breed (8-week-old female mice and 10-week-old male mice) were purchased from the Laboratory Animal Center of Tongji Medical College, Huazhong University of Science and Technology. Approval of all experimental procedures was obtained from the Tongji Medical College Ethics Committee (Huazhong University of Science and Technology).

The mice were caged in a specific pathogen-free environment with 12 h light:12 h darkness rhythm and fed ad libitum. Female mice were randomly divided into four groups as follows: control group, model group, BSHXR1 (normal dose 11.4 g/kg) group, and BSHXR2 (low dose 5.7 g/kg) group. After acclimation for 1 week without any experimental intervention, female mice were inspected daily for vaginal smears. In the model group, the BSHXR1 group and the BSHXR2 group, COH was induced in female mice at diestrus with an intraperitoneal injection of 7.5 IU pregnant mare serum gonadotrophin (PMSG; Hangzhou Animal Medicine Factory, China) followed by 7.5 IU of human chorionic gonadotropin (hCG; Lizhu Pharmaceutical Factory, China) 48 h later. After superovulation, female mice were caged with male mice overnight for mating at a ratio of 1:1. In the control group, female mice were injected with normal saline at the appropriate times and mated with male mice during estrus at a ratio of 1:1. For all groups, a vaginal plug on the next morning was taken as evidence of successful mating and that day was considered to be day 1 of gestation (D1).

From D1, the vaginal plug-positive female mice of the control and model groups were given 0.3 ml normal saline daily by oral gavage. As for BSHXR1 and BSHXR2 groups, 11.4 g/kg (normal dose) and 5.7 g/kg (low dose) BSHXR was administered daily by oral gavage. The normal dose of BSHXR is 76 g/day for an adult weighing 60 kg in the clinic, thus we calculated the normal dose of BSHXR for mice according to the conventional method (Wei et al., 2010) The BSHXR granules were dissolved in boiling water and stirred evenly to room temperature before use.

On D5, D6, and D8, some of the vaginal plug-positive female mice were sacrificed, and pregnant mice were identified by the morphology of the uterus. The numbers of implantation sites on D5, D6, and D8 in the four treatment groups were recorded. Blood samples were collected and centrifuged at 3000 ×g for 15 min at 4°C. After centrifugation, serum was separated and stored at -80°C for measurement of hormones. Ovaries and uterus were removed for morphological analysis and quickly frozen for RNA and protein extraction.

To further study the effects of BSHXR on pregnancy outcome, some vaginal plug-positive female mice from each group was kept until spontaneous delivery and the overall condition of the fetuses were observed. We recorded the number of live/dead fetuses per pregnancy and the lengths of gestation period. Ultimately, the pregnancy rate and the rate of post-implantation pregnancy loss were calculated as previously described (Bindali and Kaliwal, 2002; Rudge et al., 2007).

For morphological analysis, the ovaries and uterus were collected and fixed in 4% paraformaldehyde (PFA) overnight, dehydrated and then embedded in paraffin. Hematoxylin and eosin staining (HE staining) was performed on ovary and uterus sections. Images were obtained from an Olympus DP73 upright microscope (Olympus, Tokyo, Japan) with the CellSens Standard acquisition software.

The serum levels of estrogen and progesterone were measured by radioimmunoassay (RIA) (Beijing North Institute of Biological Technology, Beijing, China) according to the manufacturer’s instructions.

After removing the embryos, total RNA was extracted from the homogenized decidual tissue using RNAiso Plus (TaKaRa) according to the manufacturer’s instructions. The concentration and purity of RNA was measured by a nucleic acid/protein analyzer (Thermo, Rockford, IL, United States). cDNA synthesis was conducted with a PrimeScript RT reagent kit (TaKaRa) in a reaction volume of 20 μl. Quantitative Real-Time PCR (qRT-PCR) analysis was carried out using SYBR Green qPCR kit (TaKaRa) and Applied Biosystems StepOne Real-Time PCR System (Applied Biosystems) according to the manufacturer’s instructions. The PCR conditions were as follows: initial denaturation for 30 s at 95°C, 40 cycles of 5 s at 95°C, annealing and elongation for 30 s at 60°C. Each sample was run in triplicate (n = 5–6 per group) and β-actin was used as the normalization reference. The primer sequences originated from NCBI GenBank database and had been previously verified. The specific primer sequences are listed in Table 2. Fold-change of gene expression was calculated using 2^-ΔΔC_T method (Livak and Schmittgen, 2001; Schmittgen and Livak, 2008).

Decidual tissues with the embryo removed were lysed in RIPA lysis buffer with protease inhibitors (Goodbio Technology Co., Wuhan, China). Then, lysates were centrifuged at 12,000 ×g for 10 min and the protein concentration of the supernatants were measured by BCA assay kit (Goodbio Technology Co., Wuhan, China). Protein samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. After transferring, the PVDF membranes were blocked with 5% non-fat dry milk in 0.5% TBS-Tween for 1 h at room temperature. Anti-ERα (sc-542; 1:1000; Santa Cruz Biotechnology), anti-PR (ab133526; 1:1000; Abcam), anti-VEGFA (AF-493-NA; 1:1000; R&D Systems), anti-ANGPT2 (ab155106; 1:1000; Abcam), anti-FGF2 (sc-79; 1:100; Santa Cruz Biotechnology), and anti-β-actin (GB13001-1; 1:1000; Goodbio Technology Co, Wuhan, China) antibodies were used for incubation overnight at 4°C. After the incubation of primary antibodies overnight, the membranes were washed with TBST three times for 10 min each, and incubated with fluorescent secondary antibody (P/N 926-32211, 1:5000; Odyssey) for 1 h at room temperature. Bands were visualized with Odyssey Infrared Image System (LI-COR, Lincoln, NE, United States), and ImageJ software (National Institutes of Health, United States) was used to quantify the relative protein expression. All assays were performed three times independently and the raw data of three independent experiments are shown in Supplementary Figure S1.

Paraffin sections of the uterus were de-paraffinized in xylene and rehydrated in declining concentrations of ethanol. Next, antigen retrieval was performed with citric acid buffer (pH = 6.0) at 95°C for 20 min. Then, sections were incubated with 3% H₂O₂ for 25 min at room temperature to inhibit endogenous peroxidase activity, and then blocked in 10% rabbit serum for 30 min at room temperature. Anti-CD31 (ab28364; 1:50; Abcam), Anti-ERα (sc-542; 1:50; Santa Cruz Biotechnology), anti-PR (sc-538; 1:50; Santa Cruz Biotechnology), anti-VEGFA (AF-493-NA; 1:100; R&D Systems), anti-ANGPT2 (sc-20718; 1:50; Santa Cruz Biotechnology), and anti-FGF2 (sc-79; 1:100; Santa Cruz Biotechnology) antibodies were used as primary antibodies for incubation overnight at 4°C, followed by washing 5 min three times in PBS. Tissue sections were then incubated with the corresponding secondary antibody (GB23204; 1:200; Goodbio Technology Co.) for 50 min at room temperature. After developing with 3,3′-diaminobenzidinetetrahydrochloride (DAB), cell nuclei were counterstained using Harris hematoxylin for 3 min. Finally, the sections underwent dehydration and were sealed with neutral balsam. Immunohistochemical pictures were taken using an Olympus DP73 upright microscope (Olympus, Tokyo, Japan) with the CellSens Standard acquisition software. Cell nuclei were stained blue and yellowish-brown staining was considered positive.

Data with a normal distribution were represented as mean ± SD, and data with a skewed distribution were represented as medians (first quartile, third quartile). All data were analyzed by SPSS 20.0 software (SPSS Inc., Chicago, IL, United States). One-way analysis of variance (ANOVA) was used to compare differences amongst the four groups, more specifically, a LSD or Dunnett’s T3 test was used depending on equal variances of data. Furthermore, Chi-square test was applied for comparison of rate. A p-value < 0.05 was considered statistically significant.

BSHXR consists of six herbs (composition summarized in Table 1). The qualitative determination of BSHXR was performed by HPLC. Five peaks were identified by comparing the retention times with the reference standard, and the main constituents of BSHXR were as follows: ferulic acid; calycosin-7-glucopyranoside; hyperoside; quercitrin; asperosaponin VI. Their retention times were 30.50 min, 31.75 min, 35.75 min, 62.75 min, and 76.50 min, respectively (Figures 2A,B). Three batches of BSHXR were tested by HPLC (Figure 2C).

Implantation and reproductive outcomes were observed in our study (Table 3). There were no significant differences in pregnancy rate, number of implantation sites on D5, D6, D8 and number of live fetus between the four groups. However, the number of live fetuses was highly variable in the model group, and post-implantation pregnancy loss was remarkably elevated in the model group compared to control (P < 0.05). BSHXR1 treatment reduced post-implantation pregnancy loss significantly (P < 0.05) while BSHXR2 treatment also reduced pregnancy loss, but not significantly. Moreover, compared with the increased number of dead fetuses in the model group, no dead fetuses were observed in the other three groups (P < 0.05). The gestational period of the model group was significantly longer than the control group (P < 0.001), and both BSHXR groups could shorten the gestation period to the normal length as that of the control group (P < 0.001).

To further confirm the effects of BSHXR on implantation, we also compared the morphology of the pregnant uterus on D6 and D8 when the implantation sites were visible for comparison (Figures 3A,B). Compared with the well-nourished implantation sites in the control group, it was evident that the implantation sites in the model group were relatively smaller and poorly developed. Both doses of BSHXR could improve the development of implantation sites, but the effects of BSHXR1 were better than that of BSHXR2.

Since the steroid hormones produced by the ovary are vital for implantation and early pregnancy, we analyzed the morphology and weight of the ovary, as well as the serum levels of estrogen and progesterone. In the HE section of the ovary, it was found that more corpora lutea were presented in the model group, and the volume and weight of the ovary was increased compared to the control group (P < 0.001 or P < 0.05) (Figures 4A,B), reflecting the hyperstimulated status of ovary due to COH. In contrast, BSHXR1 and BSHXR2 groups had less corpora lutea and decreased volume and weight of the ovary compared with those of the model group (P < 0.001 or P < 0.01 or P < 0.05). Furthermore, the serum level of estrogen was elevated in the model group on D8 when compared to the control group (P < 0.05) (Figure 4C), and both BSHXR1 and BSHXR2 treatments reduced the excessive increase in estrogen significantly (P < 0.01 or P < 0.05). Moreover, compared with the serum levels of progesterone in the control group, the model group exhibited a marked increase in progesterone at D5, D6, and D8 (P < 0.001 or P < 0.01 or P < 0.05) (Figure 4D), while BSHXR1 and BSHXR2 could reduce the secretion of progesterone to the normal serum levels (P < 0.001 or P < 0.01 or P < 0.05).

In order to observe the effects of BSHXR on implantation and subsequent decidualization, we performed morphological assessment of uterine tissue sections on D5, D6, and D8. On D5, the glandular cells were cuboidal in the model group compared to columnar in the control and BSHXR groups (Figure 5A). On D6 and D8, the cytoplasm of decidual cells in the primary decidual zone (PDZ) and second decidual zone was reduced in the model group compared to the control group, while BSHXR1 and BSHXR2 could increase the content of cytoplasm in decidual cells (Figures 5B,C). Apart from the alteration of cytoplasmic content in decidual cells on D6 and D8, it was also discovered that the blood vessels around the embryo were remarkably diminished in the model group on D8 compared with the control group. Both BSHXR1 and BSHXR2 could increase the blood vessels around the embryo, though the effect of BSHXR1 was better than that of BSHXR2 (Figure 5C).

To further explore the underlying mechanism of BSHXR’s pro-implantation effect, we measured the mRNA and protein expression of ERα and PR in the decidua and their localization in the pregnant uterus (Figures 6, 7). It was noticed that on D5 and D6, ERα was mainly expressed in stroma, luminal and glandular epithelium (Figures 6A,B). In the model group, the expression of ERα was reduced in the stroma, luminal and glandular epithelium on D5 and D6 compared to the control group, and this was prevented by BSHXR1 and BSHXR2 treatment. Again, the effect of BSHXR1 was better than that of BSHXR2.

On D8, ERα was mainly expressed in the stroma and luminal epithelium (Figure 6C). In the model group, the expression of ERα was reduced in the stroma on D8 compared to the control group, and this could be prevented by BSHXR1 and BSHXR2. In addition to the reduction of ERα protein expression, mRNA expression was also reduced in the model group (P < 0.01) when compared to the control group on D5, D6, and D8, and BSHXR1 could significantly increase ERα mRNA and protein expression on D5, D6, and D8 (P < 0.001 or P < 0.01 or P < 0.05). Although the mRNA expression of ERα was increased in the BSHXR2 group on D5, the protein level was not different compared to the model group. On D6 and D8, BSHXR2 significantly increased the mRNA and protein levels of ERα (P < 0.001 or P < 0.01 or P < 0.05) (Figures 6D–F).

Additionally, it was discovered that PR was mainly expressed in the stroma, and the luminal and glandular epithelium on D5 and D6 (Figures 7A,B). On D5, a slight reduction in the luminal epithelial PR expression in the model group was observed, but this was improved in the BSHXR groups (Figure 7A). On D6, there was no notable difference in PR expression in the stroma or the epithelium between the four groups (Figure 7B). On D8, PR expression in the stroma was reduced in the model group compared with the control group, and both BSHXR1 and BSHXR2 could increase stromal expression (Figure 7C). However, quantification of protein and transcript levels of PR showed that there was no difference in PR protein or mRNA expression between the four groups on D5 and D6 (Figures 7D–F), while on D8, PR mRNA and protein levels were reduced in the model group (P < 0.01) compared with the control group. Both BSHXR1 and BSHXR2 could significantly raise the mRNA and protein levels of PR on D8 (P < 0.01 or P < 0.05).

Since we found defective blood vessel formation around the embryo in the model group, we further investigated the effects of BSHXR on decidual angiogenesis in pregnant mice. As shown by HE staining, on D8, blood vessels were significantly reduced in the model group compared to the control group, and abnormalities in vascular density and morphology of the vascular sinus folding (VSF) were observed (Figure 8). In the decidua of control animals, the sprouting of blood vessels became active on D6, and the enlargement and elongation of the VSF was boosted on D8. Compared with the control group, a decrease in vascular density and defective enlargement and elongation of the VSF from D6 to D8 were observed in the model group, while BSHXR could increase vascular density and promote the development of blood vessels, though the effect of BSHXR1 was better than BSHXR2.

Representative immunohistochemical staining of VEGFA, ANG2, and FGF2 in the pregnant uterus is presented in Figures 9A, 10A, 11A, respectively. VEGFA was mainly distributed in the luminal and glandular epithelium on D5 and D6, with some expression in the stroma; while by D8, VEGFA has almost disappeared from the luminal epithelium and is present only in the stroma. At the mRNA level, VEGFA expression was significantly increased on D6 compared to D5, coinciding with the sprouting of blood vessels that also became evident at D6. This supports a role of VEGFA in decidual angiogenesis. There was no notable difference in VEGFA mRNA and protein expression from D5 to D8 between the four groups (Figures 9B–D).

Furthermore, ANG2 was mainly expressed in the mesometrial region and its expression continually increased from D5 to D8, and levels were especially high in the VSF region on D8. Low levels of ANG2 mRNA was observed on D5 and D6, but it was highly induced on D8. As the enlargement and elongation of the VSF was dominant on D8, this suggests that ANG2 may play an important role in the maturation of decidual blood vessels. There was no notable difference in ANG2 mRNA and protein expression from D5 to D8 between the four groups (Figures 10B–D).

As for FGF2, its expression was mainly located at the VSF region and peaked on D8. At the mRNA level, FGF2 expression increased from D5 to D8 in the control group, and FGF2 mRNA and protein expression was decreased in the model group compared with the control group on D6 and D8. BSHXR1 significantly promoted FGF2 mRNA and protein expression on D6 and D8, however, BSHXR2 could only significantly elevate FGF2 mRNA and protein expression on D8 (Figures 11B–D).