This study was to explore the mechanism of acupuncture on regulating the endometrial angiogenesis and uterus dendritic cells (uDCs) during the peri-implantation period. Rats, in early pregnancy, were randomized into five groups: normal (N), model (M), acupuncture (A), progesterone (P), and A + P groups. The COH model was established using pregnant mare serum, combined with human chorionic gonadotrophin. Endometrium was collected on days 4, 6, and 8 (D4, D6, and D8) of gestation. Compared with group M, both VEGF and FGF-2 protein and mRNA levels were significantly lower on D4 and higher on D6 and D8 (
During assisted reproductive technologies, controlled ovarian hyperstimulation (COH) is one of the most commonly used methods to induce the development of multiple follicles. However, COH has some disadvantages, including a low embryo implantation rate (20–30% [
During the peri-implantation period, a variety of immune cells, including natural killer cells (NKs), dendritic cells (DCs), and macrophages are involved in the process of angiogenesis [
Acupuncture has a long history and rich involvement in the treatment of infertility. In recent years, the use of acupuncture during the process of assisted reproduction has increased. Numerous clinical studies have confirmed that the pregnancy rate of patients who received acupuncture treatment during the in vitro fertilization and embryo transfer (IVF-ET) trajectory was significantly increased compared with patients who did not receive acupuncture treatment [
Three hundred SPF grade female virginal Wistar rats (weighing 220–250 g) and 20 SPF grade male adult Wistar rats (weighing 250–300 g) were provided by the Hubei Provincial Center for Disease Control and Prevention, Wuhan, China (the animal certificate SCXK no. 2015-0018). Rats were housed and fed in the animal house barrier system of the Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China (Demonstration of Laboratory Animal Facilities in Hubei Province no. 2010-0057). All animal studies have been approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China (IACUC number: 443). After 1 week of adaptive feeding, estrous cycles of female rats were determined by saline vaginal smears. After that, rats were observed for at least 8 days, and those with a normal estrous cycle of 4 days were enrolled in the study. Enrolled rats were randomly divided into the following groups: normal group (N), model group (M), progesterone group (P), acupuncture group (A), and acupuncture plus progesterone group (A + P). Female rats were mated with male rats at 5:00 p.m. with a scale of 1 : 1 or 1 : 2 and checked by the vaginal smear at 8:00 a.m. the next day. When sperm was detected in the vaginal smear, we treated this as the first day (D1) of gestation. Uterine tissues were collected at D4, D6, and D8. In addition, 24 rats were included in each group at D6 and D8. On D4, there were 12 rats. The first five rats of each group on D4, D6 and D8 were used for immunofluorescence (IF), real-time quantitative PCR, Western blot analysis, and flow cytometry (FCM). The remaining rats in each group were used for the purification of uDCs by magnetic bead sorting (MACS).
Pregnant mare serum (PMSG) was purchased from the Hangzhou Animal Medicine Factory, China. Human chorionic gonadotrophin (HCG) was provided by Livzon Pharmaceutical Factory, Zhuhai, China. Progesterone injection (Zhejiang Xianju Pharmaceutical Co., Ltd., China) was provided by the pharmacy of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Vascular endothelial growth factor (VEGF) antibody (sc-7269), fibroblast growth factor 2 (FGF-2) antibody (sc-79), and IL-15 antibody (sc-1296) were purchased from Santa Cruz Biotechnology Company, USA. Anti-dendritic cells antibody [MRC OX-62] (FITC) (ab112196), OX-62 (ab33755), OX-6 (ab23990), goat anti-mouse IgG H&L (ab97035), and donkey anti-rabbit IgG H&L (ab150074) were purchased from Abcam Company, USA. HRP-conjugated rabbit anti-goat IgG (H + L) secondary antibody (GB23204), trypsin digestion (0.25% EDTA, Cat. number G1001), pentobarbital sodium (Cat. number G5003), and crystal violet stain (Cat. number G1014) were purchased from Wuhan Guge Biological Co., Ltd., China. Protease inhibitor cocktail (Cat. number 04693159001) was purchased from ROCHE, Switzerland. HRP-conjugated goat anti-rabbit (Cat. number 074-1506) and HRP-conjugated goat anti-mouse (Cat. number 074-1806) were purchased from KPL Company, USA. RIPA total protein lysis solution (Cat. number AS1004), SDS-PAGE gel preparation kit (Cat. number AS1012), and ECL chemiluminescence detection kit (Cat. number AS1059) were purchased from Wuhan ASPEN Biological Co., Ltd., China. Triton-100 (Cat. number T8200) and Tween-20 (Cat. number T8220) were purchased from Solarbio Technology Co., Ltd., China. DMSO (Cat. number D2650), DAPI (Cat. number D9542), Evans Blue (Cat. number E8010), hyaluronidase (Cat. number H3506), and collagenase IV (Cat. number C5138) were provided by Sigma, USA. Rat lymphocyte separation solution (Cat. number LTS1083) was purchased from Tianjin Hao Yang biological Co., Ltd., China. Thiazolyl blue (CAS: 298-93-1) was purchased from Gen-View Company, USA. RNAiso plus (TaKaRa Code: 9109), RNase-free water (Cat. number 9012), reverse transcription kit (TaKaRa Code: DRR036A), and a SYBR fluorescence quantification kit (TaKaRa Code: DRR420A) were purchased from TaKaRa Biotechnology Co., Ltd., Japan. Modified Roswell Park Memorial Institute (RPMI) Medium 1640 (Cat. number SH30809.01) was purchased from GE Healthcare Life Sciences Hyclone Laboratories, USA. Fetal bovine serum (FBS) was purchased from Gibco (US origin, Ref. number 16000-044, USA). Matrigel basement membrane matrix (Cat number 356234) was purchased from BD Company, USA. Anti-FITC MicroBeads (Cat. number 130-048-701), AutoMACS Running Buffer (Order number 130-091-221), and a MiniMACS Starting Kit (Cat. number 130-090-312) were purchased from Miltenyi Biotec, Germany. Enzyme-linked immunosorbent assay (ELISA) plates, cell strainers (aperture 70
Other materials included filter units (0.22
A total of 1000 IU of PMSG was mixed with 4 ml of special diluent (which was a matching product of PMSG) in a super-clean platform. Similarly, 2000 IU of HCG was mixed with 2 ml of 0.9% sodium chloride. Next, 80 ul of attenuant PMSG (20 IU) was mixed with 0.72 ml of sodium chloride, and 20 ul of attenuant HCG (20 IU) was mixed with 0.78 ml of sodium chloride. Rats in groups M, P, A, and A + P were injected intraperitoneally with PMSG on the day after start of the estrous period (the first day of starting the dioestrus) at 5:00 p.m., then injected with HCG after 48 hours, and immediately mated with male rats. Each rat in group P was injected once a day with 5 mg of progesterone at 4:00 p.m. from D1 to the day of harvesting. Each rat in group A underwent an acupuncture procedure once a day starting from the day of PMSG injection to D4. Rats were restrained in self-made cloth bags and lifted in the air in the process of acupuncture. The acupoints selected were bilateral Zusanli (ST36), Sanyinjiao (SP6), and Taichung (LR3) according the guideline of Hua et al. [
To obtain uterus tissue, rats in each group were anesthetized with 1% pentabarbital sodium by intraperitoneal injection at 4:00 p.m. on D4, D6, and D8. To judge if rats were pregnant, on D6 rats were injected with 0.5 ml 0.5% Evans blue through the tail vein 0.5 h before anesthesia. When present, the blastocysts at D8 were counted and recorded. All uterine tissues of rats on D4, as well as pregnant tissue on D6 and D8, were used for subsequent experiments. Uterine tissues were then split up for multiple analysis. One-sixth uterine tissues of the first five rats of each group were used to prepare frozen sections of 4
Frozen sections of uterine tissues were taken out from −20°C refrigerator and then placed at room temperature for half an hour to rewarm. Then, sections were fixed in 4% formalin for 10 min and rinsed three times for 5 minutes with PBS. For FGF-2 staining, sections were submerged into a 0.3% Triton X-100 solution for 15 minutes and rinsed three times for 5 minutes with phosphate-buffered saline (PBS). For staining of VEGF and OX-6, this additional step was not performed. Sections were blocked with 5% bovine serum albumin (BSA) (diluted with PBS), incubated with primary antibodies directed against FGF2, VEGF, or OX-6 at 4°C overnight, rinsed with phosphate-buffered saline with Tween (PBST) 5 times for 5 minutes, and incubated with the secondary antibodies (see below) at room temperature for 1 h. The anti-VEGF was used at a 100-fold dilution and OX-6 at a 500-fold dilution, and the goat anti-mouse secondary antibody was diluted 200-fold. In addition, the anti-FGF-2 antibody was diluted in 100-fold, whereas the donkey anti-rabbit secondary antibody was used at a 400-fold dilution. After incubation with the secondary antibodies, sections were rinsed 5 times with PBST (5 minutes each), stained with DAPI (1 : 1500) for 3 minutes, rinsed 3 times with PBST (3 minutes each), and sealed with an anti-fluorescence quenching agent. Images were taken within 24 hours, and sections were stored at 4°C in the dark. Images were analyzed using Image J software.
Endometrial tissue was obtained as described for FCM under “2.4.
On D8, embryos were carefully removed using a stereoscopic microscope, and total RNA was extracted from endometrial tissue by Trizol reagent, according to the manufacturer’s instructions. RNA purity and concentration were determined by a nucleic Acid/protein analyzer and 2 Primer sequences: Forward primer: 5′-GACTCATCGTACTCCTGCTTGCTG-3′ Reverse primer: 5′-GGAGATTACTGCCCTGGCTCCTA-3′ VEGF Forward primer: 5′-GTCCTCACTTGGSTCCCGACA-3′ Reverse primer: 5′-CCTGGCAGGCAAACAGACTTC-3′ FGF-2 Forward primer: 5′-GAGAAGAGCGACCCACACGT-3′ Reverse primer: 5′-CAGTTCGTTTCAGTGCCACATAC-3′
Endometrium tissue was harvested by scraping out the endometrium as described above. Tissue was collected in 4.8 ml of PBS in disposable aseptic culture dishes and then collected. To digest the tissue, 0.6 ml hyaluronidase (1 mg/ml) and collagenase IV (1 mg/ml) were added and incubated for 2 h in a constant temperature shock box (37°C) [
Endometrium tissue was harvested and a single cell suspension was prepared as described above. After incubation with FITC-labeled OX-62 antibody (10 ug/ml, 1 : 10) for 15 minutes at 4°C, cells were washed with 1 ml MACS buffer and centrifuged at 1500 rpm and 4°C for 5 minutes, and the pellet was resuspended in 90
The uDCs and its purity after MACS.
100x
200x
400x
The purity of uDCs after MACS
HUVECs were trypsinized with 0.25% trypsin and resuspended in 2 ml of complete RPMI medium 1640 containing 10% FBS. After counting, cells were seeded at a density of 10,000 cells per well in a 96-well plate. Six hours after seeding, the supernatant was removed and 100
Transwell chambers were used to establish a HUVECs and uDCs coculture system. The uDCs were cultured in 24-well plates with 600
Prior to the tube formation test, all materials which would have contact with Matrigel was precooled at −20°C. Matrigel was melted at 4°C, and 50
Levels of VEGF, soluble fms-like tyrosine kinase-1 (sFLT-1), IL-15, and IL-18 of uDCs were analyzed by ELISA. The sensitivity of the VEGF and IL-18 ELISA kits was <1.0 pg/ml, and none of the samples had a cytokine level of >1000 or <16.9 pg/ml. There was no cross-reactivity of VEGF and IL-18 with other cytokines. The sensitivity of Flt-1 ELISA kits was 0.41 ng/ml, the interassay coefficients of variation (CV%) were <12%, and the intra-assay CV% was <10%. None of the samples had a cytokine level of >100 or <0.41 pg/ml.
The IL-15 was performed as follows: the primary antibody directed against IL-15 was diluted 2000-fold with antibody-coating solution, and 100
All data was presented as mean ± SD or percentage (%). Data was analyzed by SPSS 22.0 software. Results were analyzed using the one-way factorial analysis of variance (ANOVA), followed by Dunnett’s T3 test for data with equal variances not assumed or by the Chi-square test.
We did not find a statistical significant difference in the mating rate between groups (
Comparison of mating rate, pregnancy rate, and embryo number.
Group | Mating rate of all rats | Pregnancy rate (D6 and D8) | Embryo number (D8) |
---|---|---|---|
N | 98.33% (59/60) | 100% (47/47) | 13.01 ± 1.88 ( |
M | 95.00% (57/60) | 42.56% |
24.30 ± 5.46 |
P | 90.00% (54/60) | 65.12% (28/43)# | 16.14 ± 6.10 ( |
A | 95.00% (57/60) | 69.57% (32/46)## | 15.83 ± 6.01 ( |
A + P | 91.67% (55/60) | 69.77% (30/43)## | 17.17 ± 3.89 ( |
Value = mean ± SD.
# or ## represents that there is significant difference when P, A, or A + P is compared with M group (
The uterus and ovaries of rats at the eighth day after fertilization.
Because we failed to localize uDCs using OX-62, we used OX-6, an anti-MHC Class II, as an alternative way of locating uDCs. OX-6 can recognize a monomorphic determinant of dendritic cells, B lymphocytes, and a variety of macrophages. We found that, on D4 and D6, OX-6 positive cells accumulated near blood vessels, whereas on D8 they were found near the maternal-fetal interface and in embryos (Figure
Distribution of OX-6 in the endometrium: (a) D4, the vicinity blood vessels; (b) D6, the vicinity blood vessels; (c) D8, the maternal-fetal interface; (d) D8, the embryo. Original magnification: ×200.
D4
D6
D8
D8
When compared to group N, the expression of endometrial VEGF and FGF-2 in group M was significantly higher on D4 (
Expression of endometrial VEGF protein: (a) D4; (b) D6; (c) D8; and (d) comparison of VEGF protein by mean gray value (
Comparison of VEGF protein endometrium by IF
Expression of endometrial FGF-2 protein: (a) D4; (b) D6; (c) D8; and (d) comparison of FGF-2 protein by mean gray value (
Comparison of FGF-2 protein endometrium by IF
Compared with group M, the expression of endometrial VEGF and FGF-2 protein in groups N, P, A, and A + P was significantly lower on D4 (
Expression of endometrial VEGF and FGF-2 proteins on D4, D6, and D8 by Western blot.
The expression of endometrial VEGF and FGF-2 mRNA in groups N, P, A, and A + P was significantly lower on D4, when compared to group M (
Expression of endometrial mRNA: (a) VEGF (
Compared with group M, the expression rate of uDCs in groups N, P, A, and A + P was significantly lower on D4 and D6 (
Comparison of expression rate of uDCs by flow cytometry.
Group | D4 (%) | D6 (%) | D8 (%) |
---|---|---|---|
N | 2.62 ± 0.69 ( |
2.17 ± 0.35 ( |
2.98 ± 0.69 ( |
M | 8.88 ± 0.72 |
6.59 ± 0.71 |
1.19 ± 0.39 |
P | 3.20 ± 1.84 ( |
5.37 ± 1.17 ( |
2.73 ± 0.50 ( |
A | 5.35 ± 2.54 ( |
3.73 ± 0.76 ( |
2.97 ± 0.84 ( |
A + P | 4.81 ± 0.78 ( |
4.11 ± 1.10 ( |
3.38 ± 0.83 ( |
Value = mean ± SD.
# or ## represents that there is significant difference when P, A, or A + P is compared with M group (
The proportion of uDCs in lymphocytes of the endometrium of pregnant rats (
No significant differences were observed in the number of lymphocytes prior to MACS separation among the five groups on D4, D6, and D8 (Table
Comparison of the number of lymphocytes before MACS.
Group | D4 (×105 cells) | D6 (×105 cells) | D8 (×105 cells) |
---|---|---|---|
N | 4.3 ± 0.24 ( |
5.5 ± 0.71 ( |
9.5 ± 0.53 ( |
M | 4.3 ± 0.40 ( |
5.5 ± 0.44 ( |
9.8 ± 0.72 ( |
P | 4.1 ± 0.34 ( |
5.8 ± 0.59 ( |
9.7 ± 0.35 ( |
A | 4.2 ± 0.32 ( |
5.5 ± 0.59 ( |
9.7 ± 0.38 ( |
A + P | 4.0 ± 0.32 ( |
5.9 ± 0.59 ( |
9.9 ± 0.53 ( |
Value = mean ± SD.
Compared with group M, the proliferation of groups N, P, A, and A + P on D4 was significantly lower (
The proliferation of HUVECs culturing by uDCs culture supernatant.
Compared with group M, the average number of HUVECs per field of view of groups N, P, A, and A + P on D4 was significantly lower (
Comparison of the number of HUVECs after endothelial migration test by uDCs in vitro.
Group | D4 | D6 | D8 |
---|---|---|---|
N | 476.1 ± 88.8 ( |
498.1 ± 163.0 ( |
467.0 ± 16.7 ( |
M | 1032.4 ± 218.7 |
452.2 ± 63.2 ( |
165.1 ± 73.9 |
P | 574.8 ± 82.6 ( |
482.5 ± 132.1 ( |
517.7 ± 27.4 ( |
A | 717.2 ± 153.5 ( |
467.2 ± 139.7 ( |
527.6 ± 166.1 ( |
A + P | 677.2 ± 239.4 ( |
456.9 ± 82.3 ( |
572.3 ± 71.4 ( |
Value = mean ± SD. The value means the average number of HUVECs per 100x field of view.
# or ## represents that there is significant difference when P, A, or A + P is compared with M group (
Comparison of the number of HUVECs after endothelial migration by uDCs in vitro.
Compared with group M, the total number of tubes and the total tube length of the cells in groups N, P, A, and A + P on D4 were significantly lower (
Comparison of total tubes and total tube length after umbilical vein endothelial tube formation test by uDCs culture supernatant.
Group | D4 | D6 | D8 | |
---|---|---|---|---|
Total tubes | N | 33.3 ± 4.7 ( |
50.3 ± 4.9 ( |
78.0 ± 5.3 ( |
M | 66.7 ± 8.7 |
43.3 ± 8.3 ( |
27.7 ± 2.1 |
|
P | 54.3 ± 4.9 ( |
39.3 ± 9.3 ( |
52.7 ± 4.5 ( |
|
A | 45.3 ± 10.1 ( |
45.7 ± 6.0 ( |
52.7 ± 8.1 ( |
|
A + P | 46.0 ± 3.0 ( |
35.3 ± 1.5 ( |
47.3 ± 4.5 ( |
|
Total tube length (px) | N | 14196 ± 2015 ( |
18459 ± 1700 ( |
25485 ± 1321 ( |
M | 27957 ± 3261 |
15996 ± 2493 ( |
9608 ± 531 |
|
P | 22931 ± 1467 ( |
14675 ± 2585 ( |
17555 ± 1679 ( |
|
A | 19414 ± 4097 ( |
17209 ± 2134 ( |
17210 ± 2379 ( |
|
A + P | 19607 ± 1179 ( |
13344 ± 538 ( |
15056 ± 1488 ( |
Value = mean ± SD.
# or ## represents that there is significant difference when P, A, or A + P is compared with M group (
Umbilical vein endothelial tube formation test of uDCs culture supernatant. (a) Selected picture in D4 groups; (b) selected picture in the D6 groups; (c) selected picture in D8; (d) total tubes; (e) total tubes length. ((d) and (e))
Total tubes of tube formation test (
Total tube length of tube formation test (
Compared with group M, the secretion levels of VEGF in groups N, P, A, and A + P on D4 were significantly lower (
Compared with group N, the secretion levels of IL-15 in group M on D4, D6, and D8 were significantly lower (
Comparison of the levels of VEGF, sFlt-1, IL-15, and IL-18 of uDCs secreting in vitro.
Cytokines | Group | D4 | D6 | D8 |
---|---|---|---|---|
VEGF (pg/ml) | N | 45.1 ± 1.6 ( |
46.0 ± 5.6 ( |
44.4 ± 5.2 ( |
M | 55.6 ± 5.4 |
46.1 ± 6.9 ( |
33.9 ± 4.5 |
|
P | 45.9 ± 6.5 ( |
45.0 ± 4.6 ( |
41.9 ± 4.0 ( |
|
A | 42.0 ± 2.8 ( |
43.9 ± 4.1 ( |
48.3 ± 5.4 ( |
|
A + P | 41.5 ± 2.8 ( |
44.0 ± 2.5 ( |
45.6 ± 6.5 ( |
|
sFlt-1 (ng/ml) | N | 0.541 ± 0.049 ( |
0.560 ± 0.038 ( |
0.574 ± 0.038 ( |
M | 0.560 ± 0.034 ( |
0.592 ± 0.049 ( |
0.620 ± 0.095 ( |
|
P | 0.569 ± 0.039 ( |
0.596 ± 0.077 ( |
0.592 ± 0.043 ( |
|
A | 0.559 ± 0.030 ( |
0.559 ± 0.030 ( |
0.606 ± 0.045 ( |
|
A + P | 0.601 ± 0.062 ( |
0.620 ± 0.060 ( |
0.564 ± 0.063 ( |
|
IL-15 (pg/ml) | N | 129.0 ± 18.9 ( |
30.0 ± 0.96 ( |
24.3 ± 0.6 ( |
M | 66.9 ± 9.3 |
25.4 ± 0.8 |
22.1 ± 0.4 |
|
P | 76.0 ± 12.2 ( |
28.2 ± 2.0 ( |
23.7 ± 1.1 ( |
|
A | 68.0 ± 6.1 ( |
26.8 ± 1.0 ( |
23.3 ± 0.8 ( |
|
A + P | 68.6 ± 3.4 ( |
27.2 ± 1.6 ( |
24.8 ± 0.6 ( |
|
IL-18 (pg/ml) | N | 34.7 ± 7.5 ( |
39.8 ± 2.6 ( |
31.0 ± 4.1 ( |
M | 23.0 ± 6.6 |
31.0 ± 1.3 |
21.7 ± 3.6 |
|
P | 35.3 ± 10.2 ( |
35.5 ± 2.9 ( |
26.6 ± 6.4 ( |
|
A | 28.4 ± 6.6 ( |
32.3 ± 2.6 ( |
29.2 ± 4.2 ( |
|
A + P | 35.0 ± 9.5 ( |
35.6 ± 2.7 ( |
37.6 ± 6.1 ( |
Value = mean ± SD.
# or ## represents that there is significant difference when P, A, or A + P is compared with M group (
Expression of the levels of (a) VEGF, (b) sFlt-1, (c) IL-15, and (d) IL-18 uDCs secreting in vitro.
The VEGF levels of uDCs secreting in vitro (
The sFlt-1 levels of uDCs secreting in vitro (
The IL-15 levels of uDCs secreting in vitro (
The IL-18 levels of uDCs secreting in vitro (
The results of the pregnancy rate as found in our study are consistent with those published in clinical studies [
Our VEGF and FGF-2 studies verified the hypothesis that acupuncture can improve endometrial angiogenesis in COH rats during the peri-implantation period of pregnancy. During angiogenesis, endothelial cells proliferate from existing blood vessels and then migrate and differentiate to form new capillaries. Under stable conditions, the balance between angiogenic and antiangiogenic factors is maintained. However, under conditions, such as decidualization and placentation, positive regulators of angiogenesis play a leading role in the activation of endothelial cells, which promotes vascularization. Different than decidualization, which is induced by nonspecific stimulation, decidual vascular remodeling and growth only occur during pregnancy and are regulated by a variety of factors from both maternal and embryonic matter. Among these, VEGF and FGF-2 are important growth factors that promote the process of angiogenesis. VEGF plays a role in almost all angiogenic processes during the peri-implantation period, including vasodilatation, endothelial cell proliferation, migration, and tube formation [
Our FCM data indicates that acupuncture regulates the number of uDCs. VEGF and FGF-2 are important growth factors that are involved in regulating the angiogenic process at the fetal-maternal interface during peri-implantation [
In vitro, we demonstrated the ability of HUVECs to proliferate, form formations, and migrate when cocultured with uDCs. To evaluate if acupuncture regulates the function of uDCs, we evaluated levels of VEGF, sFLt-1, IL-15, and IL-18 that were secreted by uDCs. We found that the data on proliferation, tube formation, and migration were consistent, and there were no significant differences among the five groups on D6. In group M, proliferation, tube formation, and migration were significantly increased on D4, whereas on D8, this was decreased. Both acupuncture and progesterone regulated proliferation, tube formation, and migration of uDCs. Prior to MACS, no significant differences were observed in the number of lymphocytes among the five groups on D4, D6, and D8; therefore, based on the FCM data, we evaluated the number of uDCs after MACS. Variation in the number of uDCs on D4 and D8 was similar for migration, tube formation, and migration test and different on D6. This indicated that perhaps the number of uDCs played a major role in migration, tube formation, and migration of HUVECs on D4 and D8. However, on D6 acupuncture or progesterone may play a major role in regulating the function of uDCs, such as the expression of mature DCs.
The involvement of DCs in fine-tuning decidual angiogenesis was mainly reported by secreting sFlt1 (VEGFR1) and TGF-
IL-15 and IL-18 are important cytokines, which are secreted by DCs and play important roles in the regulation of natural killer (NK) cell differentiation and proliferation [
Because the endometrial material obtained was not much and the proportion of uDCs in the lymphocytes of uterus was low, we had to use most of the rat uterus tissue for FCM and MACS experiments. It was also proved by our study that there was only tens of thousands of uDCs after MACS, which made it difficult to further verify the results of ELISA by PCR and Western blot. When coculturing, a small number of HUVECs in the transwell could pass through the chamber and fall on the bottom of the lower chamber, where an unknown interaction may occur, which is challenging for performing further research on uDCs after migration assay. However, we found that uDCs with high purity can be obtained by labeling FITC-labeled OX-62 antibodies with magnetic beads, which may provide a methodological reference for further studies. In conclusion, future studies should focus on the effects of acupuncture on the function of immune cells including DCs and NKs during peri-implantation to provide additional scientific explanation for the application of acupuncture in IVF-ET.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Wei Yang and Guangying Huang designed and drafted this experiment. Haoxu Dong mainly conducted this experiment and wrote the manuscript. Zhiyan Zhong assisted in the sorting of uterus dendritic cells. Wei Chen assisted in the modeling of the COH rats. Qing Zhang provided important advice on the improvement of the FCM steps. Xiao Wu and Guangying Huang revised the manuscript strictly. All authors read and approved the final manuscript.
Thanks are due to Dr. Yuwei Liu for donating the EA.hy926 cells. This study was funded by National Natural Science Foundation of China (NSFC) (no. 81202827).