170 SPF-grade adult female virgin Wistar rats (220–250 g) and 45 healthy male Wistar rats (approximately 300 g) were purchased from the mouse Laibao Biotech Co., Ltd. and raised in the central barrier system of the Experimental Animal Center of Tongji Hospital, Tongji Medical College, Hua Zhong University of Science and Technology. Experimental rats were raised in a pathogen-free environment (20 ± 2 °C, 60 ± 5% humidity, 12 h: 12 h light/dark cycle), and were given free access to water and food (2 rats/cage). The males and females were reared in separate cages. This study was approved by the Animal Experiment Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China (prove number: TJH-202009008).

After one week of adaptive feeding, the vaginal smear method was used to observe their estrus cycle every day. The 156 rats with two consecutive regular estrus cycles were randomly divided into nine groups: D4 normal group (D4N), D4 model group (D4M), and D4 acupuncture group(D4A) (n = 12 each); D5 normal group (D5N), D5 model group (D5M), and D5 acupuncture group (D5A) (n = 12 each); and D6 normal group (D6N), D6 model group (D6M), and D6 acupuncture group (D6A) (n = 28 each).

Pregnant mare serum gonadotropin (PMSG) was purchased from the Hangzhou Animal Medicine Factory, China. Human chorionic gonadotrophin (HCG) was provided by Livzon Pharmaceutical Factory, Zhuhai, China. Other materials included anti-vascular endothelial growth factor (VEGF) (Santa Cruz, SC-7269), anti-fibroblast growth factor 2 (FGF-2) (Immunoway, Cat NO.YT5549), anti-estrogen receptor α (Santa Cruz, SC-787); anti-progesterone receptor A (Proteintech, Cat NO.25871-1-AP); anti-LIF, (Gentex, Cat NO.GTX11940), ITG β-3(Abclonal, Cat NO.A2542) and Evans Blue (Cat. number E8010). Main reagents and devices included quantitative real-time PCR (qRT-PCR) equipment (Applied Biosystems, USA), SYBR Green qPCR Kit (Yesen, Cat NO.11201-11203), nucleic acid protein analyzer (DU730, Beckman Coulter, USA), Mastercycler gradient PCR apparatus (Eppendorf, Germany), Nikon microimaging system (TE2000-U, Tokyo, Japan), Step-One Real-Time PCR (Applied Biosystems, California, USA), scanning electron microscope (HITACHI, SU8100, Japan), estrogen ELISA kits (No.501890, Cayman, USA), progesterone ELISA kits (Cat: ELK7894, ELK Biotechnology, China), and Odyssey infrared imaging system (licor Biosciences, USA).

Our previously reported method and related literature were followed for modeling (15, 16); female rats in both model (M) and acupuncture (A) groups were intraperitoneal injected with 20 IU PMSG at 5 PM on the Day 2 of the estrus period, followed by an injection of 20 IU HCG approximately 48 h later; subsequently, the female rats were mated with male rats overnight in independent cages. At the same time, the same volume of 0.9% saline was injected into the rats in the Normal (N) group. At 8 a.m. on the day after mating, a vaginal smear was taken and a large number of sperms or vaginal plug on the vaginal smear was considered to indicate successful mating and the day was marked Day 1 (D1). Only female rats that mated successfully were included in the follow-up study. The rats in the A groups were fixed in a homemade cloth bag, and acupuncture was performed at the three acupoints of Sanyinjiao (SP6), Taichong (LR3), and Zusanli (ST36) on both sides using sterile acupuncture needles (201104, Hanyi, 0.18 × 13 mm, Beijing Hanyi Medical Instruments Co., Ltd., China) for 25 min from the day of PMSG injection to Day 4 after mating (D4). The rats in N and M groups were fixed in the same cloth bags for 25 min from the day of PMSG injection to D4 without acupuncture being performed.

At 5 PM on D4, 5, and 6, the rats were anesthetized via an intraperitoneal injection of 1 ml 2% sodium pentobarbital. Rats in each group were killed by an overdose of anesthesia directly after obtaining blood from the abdominal aorta in the D4 and D5 groups. After the rats were deeply anesthetized in the D6 group, the embryo implantation point was stained by a tail vein injection of 1.5 ml Evans Blue; blood was drawn and the rats were sacrificed after 10 min of dyeing; the number of implantation points was recorded in detail to calculate the pregnancy rate (number of pregnant rats/number of successfully caged rats) and number of implanted embryos (total number of implanted embryos/total number of pregnant rats). The blood samples were centrifuged (3000 rpm × 15 min) and the supernatant was collected and stored in a refrigerator at −80°C for the detection of steroid hormone levels. The uterine tissues of each group were carefully separated and preserved; a small section was put into the electron microscope fixation solution for subsequent electron microscope experiments; another small section was fixed and embedded in 4% paraformaldehyde for histochemical evaluation; the rest was freezed to −80°C in a refrigerator for subsequent experiments.

Fresh endometrial surface tissues (approximately 1 mm³ in size) were quickly put into the electron microscope fixation solution, and fixed at 4°C for 2–4 h. After fixation, dehydration, infiltration, embedding, slicing, and staining, endometrial pinopodes was observed and an image was captured under a scanning electron microscope (HITACHI, SU8100, Japan).

Levels of serum estrogen and progesterone were analyzed by ELISA. The sensitivity of the estrogen ELISA kits (No.501890, Cayman, USA) was approximately 20 pg/ml, and the assay has a range of approximately 0.61–10000 pg/ml. The sensitivity of progesterone ELISA kits (Cat: ELK7894, ELK Biotechnology, China) was approximately 0.55 ng/ml, and the assay has a range of 1.57–100 ng/ml. The ELISA steps were carried out in strict accordance with the instructions mentioned in the kits.

Paraffin sections were dewaxed in xylene, placed in gradient concentrations of ethanol to recover the antigen, and then blocked with goat serum at room temperature for 20 min. Subsequently, the sections were incubated with a primary antibody (anti-estrogen receptor α, Santa Cruz Biotechnology, 1:50; anti-progesterone receptor A, Proteintech, 1:300; anti-VEGF, Santa Cruz, 1:50; anti-FGF-2, Immunoway, 1:100; anti-LIF, Gentex, 1:200; ITG β-3, Abclonal, 1:150) at 4°C overnight, rinsed with phosphate-buffered saline with Tween (PBST) five times for 5 min, and incubated with secondary antibodies (anti-estrogen receptor α, Santa Cruz Biotechnology, 1:50; anti-progesterone receptor A, Proteintech, 1:300; anti-VEGF, Proteintech, 1:50; anti-FGF-2, Bioswamp, 1:100; anti-LIF, Gentex, 1:200; ITG β-3, Absin, 1:150) at room temperature for 1 h. After washing with PBST five times for 5 min, the sections were developed using 3,3’-diaminobenzidine and stabilized using hematoxylin for approximately 3 min. Finally, the sections were dehydrated, made transparent, and sealed. The images were scanned using a nanozoomer slide scanner (Hamamastu, Japan) and observed using the NDP view2 system.

Endometrial tissues were homogenized and lysed in tissue protein extraction reagent, supplemented with a protease inhibitor cocktail, placed on ice for 30 min, and centrifuged at 4°C (12,000 rpm for 10 min). After the protein concentration was determined, the samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and transferred to a PVDF membrane. The membranes were sealed with 5% skim milk at room temperature for 0.5 h, and then incubated with a primary antibody at 4 °C for 24–48 h. Antibodies include anti-estrogen receptor α (Santa Cruz Biotechnology, USA, 1:50); anti-progesterone receptor A (Proteintech, China, 1:200; anti-VEGF, Santa Cruz, China, 1:200); anti-FGF-2 (Immunoway, China, 1:500); anti-LIF (Gentex, China, 1:300); ITGβ3 (Abclonal, China, 1:500); and anti-β-actin (Proteintech, China, 1:1500). PVDF membranes were incubated with fluorescent secondary antibodies (CST, USA) on a shaking table at room temperature for 1 h. Finally, the bands were scanned using Odyssey infrared imaging system (licor Biosciences, USA).

Total RNA was extracted from the endometrial tissue using Trizol reagent (Takara, Japan), according to the manufacturer’s instructions. After the RNA concentration was determined, the cDNA was synthesized with a reverse transcription reagent (Yesen, China). Quantitative real-time PCR (qRT-PCR) (Applied Biosystems, USA) was performed using SYBR Green qPCR Kit (Yesen, China). The 2^−ΔΔCT method was used for data analysis. The sequence is in the Table 1 .

IBM SPSS 20.0 was used for statistical analysis. Continuous data for normal distribution are expressed as means ± SD and categorical data are expressed as percentages (%). Differences were compared using one-way analysis of variance. If the variance was uniform, the LSD test was used, whereas if it was uneven, Dunnett’s T3 test was used. P value < 0.05 was considered to be statistically significant.

A large number of spermatozoa or vaginal suppositories on vaginal smears were regarded to indicate successful mating. In addition, only rats in the D6 group were included for calculating the pregnancy rate. In all, 42 rats were injected with Evans blue through the tail vein on Day 6 to determine whether they were pregnant ( Table 2 ). Compared with the N group, the M group showed a significantly lower pregnancy rate (P < 0.01). Further, compared with the M group, the A group showed a significantly higher pregnancy rate (P < 0.05). But the specific number of pregnant embryos in each rat could not be accurately counted. Accordingly, the number of embryos was not calculated. According to Evans blue staining, the M group had more embryos than the N group ( Figure 1 ).

The ultrastructural results of endometrium on D4, D5, and D6 were observed under a scanning electron microscope (3000×). There was no pinopode or swollen microvilli on the surface of endometrium in the D4N group, a large number of pinopodes in the D4M group, and obvious pinopodes in the D4A group, but the number of pinopodes in the D4A group was less than that in the D4M group. There were a large number of mature pinopodes on the endometrial surface the rats in the D5N group, a small number of atrophic pinopodes in the D5M group, and a small number of mature or atrophic satiety pinopodes in the D5A group, and the number of pinopodes observed in the D5M group was less than that observed in the in D5A group. There were no pinopodes in the D6N, M, and A groups ( Figure 2 ).

Immunohistochemistry (IHC) results showed that LIF, integrin β3, VEGF, and FGF-2 proteins were mostly expressed in the luminal and glandular epithelia, stroma, and myometrium of the endometrium. The expression of endometrial LIF, integrin β3, VEGF, and FGF-2 protein by IHC ( Figures 3 – 6 ) were consistent with the WB ( Figure 7 ).

On D4, the expressions of LIF, integrin β3, VEGF, and FGF-2 protein in the endometrium of rats in the D4M group were significantly increased compared with those in the endometrium of rats in the D4N group (P < 0.05, P < 0.01), whereas the expressions in the D4A group were significantly decreased compared with those in the D4M group (P < 0.05, P < 0.01). On D5 and D6, the expressions of LIF, integrin β3, VEGF, and FGF-2 protein in the endometrium of rats in the M group were significantly decreased compared with those in the endometrium of rats in the N group (P < 0.05, P < 0.01), whereas the expressions in the A group were significantly increased compared with those in the M group(P < 0.05, P < 0.01) ( Figure 7 ).

PCR results showed that the mRNA expression of LIF and FGF in the D4M group was significantly higher than that in the D4N group (P < 0.05), and acupuncture could alleviate this change, but there was no significant difference between D4A and D4M groups (P > 0.05). In addition, there was no difference in the mRNA expression of integrin β3 and VEGF among D4N, D4M, and D4A groups (P > 0.05). On D5, the mRNA expression of integrin β3 and VEGF in the D5M group was significantly lower than that in the D5N group (P < 0.01), but acupuncture could significantly reverse this change (P < 0.05). At the same time, there was no significant difference of the mRNA expression of LIF and FGF among D5N, D5M, and D5A groups (P > 0.05). On D6, the mRNA expression of LIF, integrin β3, VEGF and FGF-2 in the D6M group was significantly decreased compared with that in the D6N group (P < 0.05 or P < 0.01), while acupuncture could increase the mRNA expression of LIF compared with that in the D6M group (P < 0.05). As for the mRNA expression of integrin β3, VEGF and FGF-2, although acupuncture could increase their expression, but no significant difference was observed between the D6A and D6M groups (P > 0.05) ( Figure 8 ).

There was no significant difference in D4, D5 and D6 estrogen levels among N, M, and A groups. Compared with the N group, the M group showed significantly higher levels of progesterone on D4, D5, and D6 (P < 0.05, P < 0.01). There were no significant differences in progesterone levels between the M and A groups on D4 and D5 (P > 0.05), whereas the levels were significantly lower on D6 in the A group compared with M group (P < 0.05) ( Table 3 , Figure 9 ).

IHC results showed that progesterone receptor (PR) and estrogen receptor α (ERα) mainly expressed in glandular epithelium and luminal epithelium of the endometrium, and a small amount was expressed in stromal cells. The epression of endometrial PR and Erα proteins by IHC ( Figures 10 , 11 ) were consistent with the WB ( Figure 12 ).

PR protein levels in the D4M, D5M, and D6M groups were significantly lower than those in the corresponding N groups (P < 0.01). The PR protein levels in D5A and D6A groups were significantly higher than those in the corresponding M groups (P < 0.01). With regard to ERα, there was no significant difference between the M and N Group on D4 (P > 0.05). Compared with the D5N group, the D5M group showed significantly lower ERα protein levels (P < 0.01), whereas the D5A group showed significantly higher levels compared with the D5M group (P < 0.01). The expression trend for D6 ERα protein level was consistent with that observed for D5 ( Figure 12 ).

PCR results showed that the mRNA level of ERα on the endometrium in the D4M group was increased compared with that in the D4N group (P < 0.05), whereas no significant difference was observed between D4A and D4M (P > 0.05). The expression trend for ERα protein levels in the D5M group was decreased compared with that in the D5N group (P < 0.05). There was no significant difference among the three D6 groups. The mRNA level of PR on the endometrium in D6M groups was decreased compared with that in the D6N group (P < 0.01); however, no significant difference was observed among other groups (P > 0.05) ( Supplementary Image 1 ).