A total of 40 female patients with POR preparing to receive IVF-ET treatment at the Reproductive Medicine Center of Yantai Yuhuangding Hospital (Yantai, China) and The Affiliated Qingdao Hiser Hospital of Qingdao University (Qingdao, China) between July 2015 and December 2015 were included in the present study. Patients were randomly assigned to the treatment group or the control group. POR was diagnosed according to the European Society of Human Reproduction and Embryology consensus (18) and the American Society for Reproductive Medicine guidelines (19). The inclusion criteria of the present study were that patients with POR underwent microstimulation to promote ovulation, but for whom ET had failed at least once. The exclusion criteria were as follows: i) The partner of the patient had oligoasthenozoospermia; ii) patients with immunological infertility; iii) patients with malignant or benign tumors of the reproductive system requiring surgery; iv) patients who had received sexual hormone therapy in the past 3 months; v) patients with heart, liver, kidney or hematopoietic disease, mental illness or other serious diseases; vi) patients with previous poor compliance; and vii) patients with incomplete clinical data. The present study was approved by the Ethics Committee of Yantai Yuhuangding Hospital and all patients provided written informed consent.

The formula of the Tiaogeng Yijing decoction was as follows: Radix Rehmanniae Preparata, 15 g; Radix Morindae Officinalis, 12 g; stir-baked Rhizoma Dioscoreae, 18 g; stir-baked Cortex Eucommiae with salt solution, 12 g; alcohol processed Radix Angelica Sinensis, 15 g; stir-baked Radix Paeoniae Alba, 15 g; Jiu Salvia Miltiorrhiza Bge., 15 g; Caulis Spatholobi, 15 g; Bupleurum Chinense, 9 g; Cyathula, 15 g; prepared Rhizoma Cyperi with vinegar, 12 g; Cuscuta chinensis Lam., 18 g; alcohol processed Rhizoma Polygonat, 12 g; Radix Codonopsis, 12 g; and Rhizoma Atractylodis Macrocephalae stir-fried with wheat bran, 12 g. The Tiaogeng Yijing decoction was provided by The Affiliated Qingdao Hiser Hospital of Qingdao University.

Patients in the treatment group were orally administered the Tiaogeng Yijing decoction twice a day for 2 weeks with boiling water (decoction of 200 ml), starting on the seventh day of their menstrual cycle. After three cycles of this treatment, the patients underwent microstimulation, as described below, to promote ovulation. By contrast, patients in the control group underwent microstimulation without any prior treatment.

Clomiphene (50 mg; Beijing Double-Crane Pharmaceutical Co., Ltd., Beijing, China) was administered orally once daily to the patients from the second day of their menstrual cycle for 5 days. This was followed by a low-dose gonadotropin (Gn) (Livzon Pharmaceutical Group, Inc., Zhuhai, China), such as human menopausal Gn or Gn releasing hormone agonist. The initial dosage of Gn was quantitatively determined according to the patient's age, height, weight and ovarian reserve function (20). The concentrations of Gn ranged from 100–150 IU per day starting on cycle day 8. During the COH, ovarian follicle development and endometrial thickness were monitored by transvaginal ultrasound (TVU). In addition, the levels of serum estradiol (E2), luteinizing hormone (LH) and progesterone were measured as described below. The dosage of Gn was adjusted according to these results. If the diameter of the dominant follicle in the ovaries reached >18 mm, Gn was stopped and human chorionic gonadotropin (HCG) (5,000–10,000 IU per day) (Livzon Pharmaceutical Group, Inc.) was administered starting on cycle day 10. After 36 h of administration of HCG, patients underwent ultrasound-guided transvaginal oocyte retrieval (TVOR).

After TVOR, the oocyte corona cumulus complex obtained was placed in a culture dish containing 2 ml medium 199 supplemented with 20% fetal bovine serum (both Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 10 IU/ml pregnant mare serum Gn (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 10 IU/ml HCG in an incubator at 37°C with 5% CO₂ for 48 h. Thereafter, oocytes were denuded with 0.1% hyaluronidase solution for 1 min at room temperature and their maturity was determined under a phase-contrast microscope. The matured oocytes were inseminated by intracytoplasmic sperm injection. Fertilization was evaluated by assessing oocytes 12–14 h after injection for the presence of distinct pronuclei and two polar bodies. At 24 h after insemination, the ratio of embryo cleavage was assessed under a phase-contrast microscope. All embryos were frozen at −196°C using an automatic freezer (TK 3000, Uberaba, Brazil) and then ET was performed on the next menstrual cycle.

The pregnancy outcomes were compared between the two groups including the number of oocytes retrieved following ovarian stimulation, the percentage of high-quality oocytes, the percentage of high-quality embryos and the pregnancy rate. These oocytes and embryos were examined by phase-contrast microscopy. High quality oocytes were chosen according to the cumulus investment and morphology for IVF. High-quality embryos were selected based on the following characteristics: Embryos had 4 blastomeres and 8–10 blastomeres on days 2 and 3 of development, respectively; the embryos had <15% fragmentation, and symmetric and mononucleated blastomeres.

Serum (5 ml) and follicular fluid (5 ml) were obtained from the patients prior to and following treatment with the Tiaogeng Yijing decoction. Follicular fluid was acquired by trans-vaginal ultrasound-guided puncture and aspiration of >18 mm diameter follicles. The serum was frozen in aliquots and stored at −20°C until required. Serum anti-mullerian hormone (AMH) was assessed using the AMH/mullerian-inhibiting substance ELISA kit (CSB-E12756h; Cusabio Biotech Co., Ltd., Wuhan, China) according to the manufacturer's protocol. Additionally, expression levels of the follicular fluid cytokines growth differentiation factor 9 (GDF)-9 (CSB-E12925h), transforming growth factor (TGF)-β1 (CSB-E04725h), leukemia inhibitory factor (LIF) (CSB-E04651h), vascular endothelial growth factor (VEGF) (CSB-E04760h), and granulocyte-colony stimulating factor (G-CSF) (CSB-E04563h) were measured using ELISA kits (Cusabio Biotech Co., Ltd.) according to the manufacturer's protocol. Serum levels of follicle-stimulating hormone (FSH), LH and E2 were determined by electrochemiluminescence immunoassay using the Elecsys 2010 Immunoassay Analyzer (Roche Diagnostics, Indianapolis, IN, USA) as previously described (21). Progesterone was determined by a chemiluminescent immunoassay (Immulite 2000 Progesterone assay; Siemens Healthcare GmbH, Erlangen, Germany) using a specific monoclonal antibody against progesterone, according to the manufacturer's protocol. The antral follicle count (AFC) and mean ovarian diameter (MOD) were measured by TVU.

Endometrial samples were obtained from the patients in the two groups after microstimulation of ovulation with a suction curette from the corpus of the uteri. Total protein was extracted from the endometrium of patients in the two groups using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). The amount of protein was quantified using a BCA Protein assay kit (Beyotime Institute of Biotechnology). The protein samples (20 µg per lane) were separated on a 10–12% gel using SDS-PAGE and then transferred to nitrocellulose membranes (Merck KGaA). The membranes were then blocked with 5% nonfat milk in Tris-buffered saline with Tween 20 for 1 h at room temperature. Subsequently, the membranes were treated overnight at 4°C with primary antibodies directed against the following proteins: Integrin αVβ3 (cat. no. ab78289; 1:1,000 dilution), TGF-β1 (cat. no. ab92486; 1:1,000 dilution), LIF (cat. no. ab34427; 1:1,000 dilution), G-CSF (cat. no. ab9691; 1:1,000 dilution) and VEGF antibody (cat. no. ab69479; 1:1,000 dilution) (all Abcam, Cambridge, UK). GAPDH (cat. no. ab8245; 1:1,000 dilution; Abcam) was used as a loading control and incubated overnight at 4°C. The membranes were then washed and incubated with horseradish peroxidase-conjugated anti-mouse secondary antibody (ab131368; 1:2,000 dilution; Abcam) or anti-rabbit antibody (ab191866; 1:2,000 dilution; Abcam) for 2 h at room temperature. Protein bands were visualized with Enhanced Chemiluminescence Western Blotting Substrate (Merck KGaA) and quantified using ImageJ software (version 1.46; National Institutes of Health, Bethesda, MD, USA).

All data are expressed as the mean ± standard deviation, or as percentages. Statistical analyses were conducted using SPSS software (version 19.0; IBM Corp., Armonk, NY, USA). Comparisons between groups were analyzed using a paired t-test or the χ² test. P<0.05 was determined to indicate a statistically significant difference.

The general characteristics of the patients are presented in Table I. The mean age of the patients was 35.10±7.39 and 34.80±7.53 years in the treatment and control groups, respectively. There was no significant difference in the age of patients between the treatment and control groups (P=0.083). In addition, no significant differences were observed between the treatment and control groups, respectively, in infertility duration (4.85±1.22 vs. 4.90±1.59 years; P=0.878) and body mass index (22.97±1.68 vs. 23.47±1.58 kg/m²; P=0.353). The factors indicate that the results of the two groups were comparable.

To explore the therapeutic effects of the Tiaogeng Yijing decoction on the pregnancy outcomes of patients with POR undergoing IVF-ET, the levels of sex hormones, and the AFC and MOD, in the treatment group (before and after treatment) and in the control group (after treatment) were compared. As shown in Table II, the results demonstrated that the levels of FSH and E2, and the ratio of FSH/LH, were significantly decreased after treatment with the Tiaogeng Yijing decoction compared with the levels before treatment (all P<0.05), while the AMH level and the AFC were significantly increased (both P<0.05 vs. before treatment). However, no significant differences were identified in LH and the MOD before and after treatment with the Tiaogeng Yijing decoction. Furthermore, compared with the control group, the levels of FSH, E2 and FSH/LH were significantly reduced after treatment with Tiaogeng Yijing decoction (all P<0.05). By contrast, the AMH level and the AFC were significantly increased after treatment compared with the control group (both P<0.05). There were no significant differences in LH and the MOD after treatment compared with the control group. These results indicate that the Tiaogeng Yijing decoction regulates sex hormones levels and increases the AFC.

The levels of GDF-9, TGF-β1, LIF, G-CSF and VEGF in the follicular fluid of patients in the treatment and control groups were compared. The results of this analysis are shown in Table III. The results revealed that the levels of GDF-9 (P<0.001), TGF-β1 (P=0.004) and VEGF (P=0.005) were significantly increased in the treatment group compared with the control group. However, there were no significant differences in expression levels of LIF (P=0.329) and G-CSF (P=0.172) between the treatment and control groups. These results suggest that the Tiaogeng Yijing decoction increases follicular fluid levels of GDF-9, TGF-β1 and VEGF.

Next, the levels of endometrial cytokines between the two groups were compared. As shown in Fig. 1, the results of western blotting analysis revealed that the protein expression levels of integrin αVβ3, TGF-β1 and VEGF were significantly increased in the treatment group compared with the control group (all P<0.05). However, there were no significant differences in the protein levels of LIF and G-CSF between the two groups. These results suggest that the Tiaogeng Yijing decoction increases endometrial levels of integrin αVβ3, TGF-β1 and VEGF.

At 3 months after ET, pregnancy outcomes between the treatment and control groups were compared. The pregnancy outcomes measured included the number of oocytes retrieved, the percentage of high-quality oocytes, the percentage of high-quality embryos and the pregnancy rate. As shown in Table IV, the results demonstrated that the number of retrieved oocytes (P=0.002), high-quality oocytes (P=0.004) and clinical pregnancy rate (P=0.024) were significantly increased in the treatment group compared with the control group. However, there was no significant difference in the percentage of high quality embryos between the two groups (P=0.801). These results indicate that the Tiaogeng Yijing decoction improves pregnancy outcomes.