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ORIGINAL ARTICLE
Year : 2023 | Volume
: 9
| Issue : 1 | Page : 94-100
Rhizoma drynariae improves endometrial receptivity in a Mus model of dysfunctional embryo implantation
Yue Shi1, Yan-Feng Liu2, Jia-Mei Wang1, Jing Jiang1, Bo-Lin He1, Guo-Hua Mu2, Fang Liu1, Ya-Hui Li1, Ting Qiao1, Jing Lu1
1 Department of Traditional Chinese Medicine, Haidian District Maternal and Child Health Care Hospital, Haidian District, Beijing, China
2 Department of Ggynaecology, Dongzhimen Hospital, Beijing University of Chinese Medicine, Dongchen District, Beijing, China
Correspondence Address:
Prof. Yan-Feng Liu
Department of Ggynaecology, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing 100010
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/wjtcm.wjtcm_17_22
Background: Rhizoma drynariae is a traditional Chinese medicine used in orthopedics and traumatology, but its effect on endometrial receptivity remains unknown. Aims and Objectives: To observe effect of Rhizoma drynariae and its main components on endometrial receptivity in a mus model of dysfunctional embryo implantation. Materials and Methods: Mus models were established by the GnRHa+HMG+HCG method. Normal mus receiving saline were used as controls and the remaining six groups were: model receiving saline, progynova, aspirin, Rhizoma drynariae, osteopractic total flavone, and naringin. Pinopodes in uterine endometrium were examined by scanning electron microscopy. Stem cell factor (SCF) mRNA expressions was determined by real-time RT-PCR, and estrogen receptor α (ERα), progesterone receptor (PR) by immunohistochemistry. Results: In the model group, surface morphology of endometrium was heterogeneous, without obvious pinopodes. In the Rhizoma drynaria and progynova groups, pinopodes were abundant. Compared with the blank group, model group had lower levels of SCF (-47%), ERα (-63%) and PR (-50%) (all P<0.05). In comparison, Rhizoma drynariae group had higher levels of SCF (+73%), ERα (+118%) and PR (+101%) (all P<0.01). The individual main components of Rhizoma drynariae had variable efficacy. Conclusion: Rhizoma drynariae could improve endometrial receptivity of mouse models of dysfunctional embryo implantation as shown by increased numbers of pinopodes and higher levers of SCF, ERα, PR compared with the model group.
Keywords: Endometrial receptivity, naringin, rhizoma drynariae, stem cell factor, total flavone
The implantation of the embryo created by in vitro fertilization (IVF) remains the limiting factor and the most inefficient step of IVF-embryo transfer (IVF-ET) technologies. Two factors directly influence this success: embryo quality and endometrial receptivity. The embryo quality issue is now mostly overcome by the used of selection techniques yielding high-quality embryos, but endometrial receptivity remains an issue.[1] A number of methods have been tried to improve the success rate, but with mitigated success.[2],[3],[4],[5],[6] Therefore, defining ways to improve endometrial receptivity could help improving the success rate of IVF-ET.
Endometrial receptivity is influenced through the hormone cycles induced by follicular development and subsequent ovulation. The estrogen receptor α (ERα) and progesterone receptor (PR) increase in response to estrogen during the proliferative phase and decrease in response to progesterone during the secretory phase.[7],[8] Both ERα and PR are downregulated at the moment of implantation and this decrease is a critical event for endometrial receptivity.[7],[8] Appropriate progesterone levels and expression of PR in the endometrium are prerequisites for endometrial decidualization.[9] The luteal phase is the best period for embryo implantation, and the expression of the stem cell factor (SCF) is the highest during the luteal phase.[10] All these changes in molecular factors are transduced into morphological changes of the endometrium, with the formation of pinopodes, which are markers of endometrial receptivity.[11],[12] The pinopodes undergo three stages: under development, complete development, and degeneration, and their number are directly associated with endometrial receptivity.[11],[12]
Rhizoma drynariae is a traditional Chinese medicine (TCM) used in orthopedics and traumatology. It is known to tonify the kidney, promote blood circulation, and benefit bones and muscles; thus, it is used as one of main drugs of “Erbu Zhuyu Decoction,” which is an experienced prescription of the prominent TCM doctor Professor Xiao Chengzong to cure low endometrial receptivity.[13] However, the effect of Rhizoma drynariae alone on endometrial receptivity is still not known. Flavonoids and naringin are the major active ingredients of Rhizoma drynariae. Therefore, in recent years, studies of Drynaria focused on its total flavonoids and naringin.[14],[15] The total flavonoids of Drynaria are a source of natural phytoestrogen and naringin possesses estrogenic effects.[16]
Estradiol valerate (progynova) may indirectly promote the formation of pinopodes through the regulation of COX-2, PGIZ, TIMP-3, and MMP-9 in the endometrium.[17] Acetylsalicylic acid is used as an adjuvant drug for IVF-ET based on its characteristics of vasodilation and anticoagulant. Aspirin inhibits COX to inhibit platelet activity, prevent the formation of microthrombosis, reduce the resistance of blood flow from the uterine artery, improve local blood circulation, and improve ovarian and endometrial blood perfusion, thus enhancing the rate of embryo implantation.[6] Therefore, the present study used these two compounds as positive controls.
One of the current IVF-ET approaches is the use of a long period of ovarian hyperstimulation using the gonadotropin-releasing hormone (GnRHa) + human menopausal gonadotropin (HMG) + human chorionic gonadotropin (HCG) method, which leads to nonphysiological high estradiol levels. After ovulation, the endometrial development is delayed, resulting in reduced receptivity.[18],[19] Therefore, the present study used a mus model of the GnRHa + HMG + HCG scheme to study the effect of Rhizoma drynariae and its main components (total flavone and naringin) on endometrial receptivity.
Materials and MethodsAnimals
Specific pathogen-free female ICR mus (n = 70) (8–12 weeks old, 25 ± 2 g body weight (BW)) were provided by VitalRiver (Beijing, China; license No. SCXK (Jing) 2012-0001). They were subjected to a 12-h dark-light cycle at 23°C, and provided with water and food ad libitum. Vaginal smears were monitored daily and only mus showing two consecutive estrous cycles were used in the study. All procedures were approved by the Experimental Animal Care and Use Committee of Dongzhimen Hospital, Beijing University of Chinese Medicine, China (IRB of Dongzhimen Hospital affiliated to Beijing University of Chinese Medicine 15–29).
Rhizoma drynariae
Rhizoma drynariae was made with 30 g of granules (Tcmages Pharmaceutical Co., Ltd., Beijing, China) [Figure 1], 0.25 g of osteopractic total flavone (Qianggu capsule) (Qihuang Pharmaceutical Co., Ltd., Beijing, China), and 10 g of naringin (N107345, Aladdin Bio-Chem Technology Co., Ltd., Shanghai, China).
Figure 1: Infrared fingerprint of the Rhizoma drynariae standard solutionMus model with dysfunctional embryo implantation
Mus models with dysfunctional embryo implantation were established by the GnRHa + HMG + HCG method. The vaginal smear was observed. Mus were injected with 40 IU/100 g BW of Decapeptyl (GnRHa) triptorelin acetate injection (1 ml: 0.1 mg (Ferring Pharmaceuticals Ltd., Saint-Prex, Switzerland; batch No.: H20100365) in the abdominal cavity at 9:00 AM every day from the erogenous day, for 9 days. On the 9th day, the mus simultaneously received an intraperitoneal injection of 1 U/100 g BW of menotropins (HMG) (75 IU, batch No.: H20033108) to promote ovulation. After 48 h, the mus received an intraperitoneal injection of 10 IU/100 g of human chorionic gonadotropin (HCG) (1000 IU, batch No.: H44020674).
Drug treatment
From the erogenous day, the animals were divided into seven groups (10 mus/group) and received the following treatments by intragastric gavage every day after injection: 1) blank group: Normal mus receiving 0.2 ml of normal saline every day for 10 days; 2) model group: Model mus receiving 0.2 ml of normal saline every day for 10 days; 3) progynova group: Model mus receiving 0.006 mg/20 g BW of estradiol valerate (progynova) (1 mg/piece, Delpharm Lille, Lys-Lez-Lannoy, France; batch No.: H20120368) every day (equivalent to 2 mg/60 kg for adult humans of 60 kg) for 10 days; 4) aspirin group: 0.225 mg/20 g BW of aspirin (enteric-coated tablets: 100 mg/piece, Bayer HealthCare Pharmaceuticals, Montville, NJ, USA; batch No.: J20130078) every day (equivalent to 75 mg/60 kg for adult humans of 60 kg) for 10 days; 5) rhizoma drynariae group: 0.03 g/20 g BW for 10 days; 6) osteopractic total flavone group: 0.02 g/20 g BW for 10 days; and 7) naringin group: 0.01 g/20 g BW for 10 days (Rhizoma drynariae doses is based on preliminary works, equivalent to 15 g/60 kg for adult humans of 60 kg; total flavone and naringin doses were within the normal dose range based on previous studies[14],[15]) (modified to: total flavone and naringin doses were the median of the effective dose range based on previous studies[14],[15]).)
Scanning electron microscopy
After 11 days of drug treatment, the mus were sacrificed by neck dislocation. The uterine endometrial tissues were taken under aseptic conditions. Samples were fixed in 2.5% glutaraldehyde for 1–2 h, washed with PBS buffer, and then placed in 1% osmium tetroxide solution for 1–2 h. The samples were dehydrated in graded ethanol and then underwent critical point drying. The samples were attached to the sample holder with a conductive adhesive or a double-sided adhesive tape, with the endometrial cavity surface up. The sample was placed in the ion sputtering coating apparatus, and the appropriate current and time were selected to obtain a silver conductive plastic coating thickness of about 10 nm. Then, the samples were observed under an INSPECT S50 scanning electron microscope (FEI, USA) and assessed by two independent scanning electron microscopy experts blinded to the grouping.
Real-time reverse transcription-polymerase chain reaction
Total RNA was extracted from the uterine endometrial tissues (which had been stored in liquid nitrogen) using the Animal tissue total RNA Extraction Kit (Cat#A010600; APEXBIO, Beijing, China), according to the manufacturer's instructions. RNA purity and concentration were determined using a NanoQ microspectrophotometer (CapitalBio Corporation, Beijing, China). cDNA synthesis was performed using the GoScript Reverse Transcription System (Cat#A5000; Promega, USA). Specific mRNA quantification was performed by real-time polymerase chain reaction (PCR) using the SsoAdvanced SYBR® Green Supermix (Cat#172-5260; Bio-Rad, USA) in a CFX96 real-time PCR system (Bio-Rad, USA). The sequence-specific primers were: SCF: Forward 5'-CAC AGT GGC TGG TAA CAG TTC-3' and reverse: 5'-AAT TCA GTG CAG GGT TCA CA-3' and GAPDH forward 5'-GCA AGT TCA ACG GCA CAG-3' and reverse: 5'-CGC CAG TAG ACT CCA CGA C-3'. The threshold cycle (Ct) values of the target genes SCF were normalized to those of GAPDH. The relative mRNA expression levels were calculated using the 2-ΔΔCt method.
Immunohistochemistry
The uterine endometrium samples were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and stained using a highly sensitive immunohistochemistry detection protocol based on the polymer enhanced method (HRP/DAB Envision System; Dako, Glostrup, Denmark), according to the manufacturer's instructions. Antigens were retrieved in citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) at 95°C–100°C for 20 min for the PR or in Tris-EDTA Buffer (10 mM Tris base, 1 mM EDTA, 0.05% Tween 20, pH 9.0) at 95°C–100°C for 15 min for ERα. The sections were incubated overnight at 4°C with primary antibodies against the PR (1:100; ab63605; Abcam, Cambridge, MA, USA) and ERα (1:300; ab32063; Abcam, Cambridge, MA, USA). Sections were incubated with the secondary antibody from the Dako REAL EnVision Detection System (Peroxidase/DAB+, Rabbit/Mouse; K5007; Dako, Glostrup, Denmark) for 30 min at 37°C. Sections were counterstained with hematoxylin (blue) (Baso BA-4097). The slides were observed under an Olympus BH2 microscope (Olympus, Tokyo, Japan) with a Panasonic camera. Images were analyzed using the IMS cell image analysis system (Shanghai Shenteng Information and Technology Co., Ltd., Shanghai, China).
Statistical analysis
If normally distributed, the data are shown as mean ± standard error (SE) and analyzed using analysis of variance with the least significant difference for post hoc analysis. If the data did not show normal distribution, the data were expressed as median (min, max) and analyzed using the Kruskal–Wallis H test. All statistical analyses were conducted using SPSS 23.0 (IBM, Armonk, NY, USA). Two-sided P < 0.05 were considered statistically different.
ResultsMus model with dysfunctional embryo implantation establishment
Due to improper intragastric administration, some mus died: two in the model group, two in the Rhizoma drynariae group, two in the flavone group, two in the naringin group, and two in the aspirin group.
Effect of Rhizoma drynariae and its main component on pinopodes in uterine endometrium of mouse models with dysfunctional embryo implantation
In the blank group, the surface morphology of the endometrium was regular, showing abundant pinopodes; epithelial cells were evenly distributed and with synchronous development [Figure 2]a. In the model group, the surface morphology of the endometrium was heterogeneous, without obvious pinopodes [Figure 2]b. In the Rhizoma drynaria [Figure 2]c and the progynova [Figure 2]f groups, pinopodes were abundant, while the numbers of pinopodes in the total flavone [Figure 2]d and aspirin [Figure 2]g groups were lower. There was only a small number of pinopodes in the naringin group [Figure 2]e.
Figure 2: Effect of Rhizoma drynariae and its main components on pinopodes in uterine endometrium of mouse models with dysfunctional embryo implantation. The mouse models with dysfunctional embryo implantation were established by the gonadotropin-releasing hormone + human menopausal gonadotropin + human chorionic gonadotropin method and were treated with Rhizoma drynariae, total flavone, naringin, progynova, or aspirin for 10 days. Pinopodes in uterine endometrium were determined by scanning electron microscope in the (a) blank, (b) model, (c) rhizoma drynariae, (d) total flavone, (e) naringin, (f) progynova, and (g) aspirin groups (×3000)Effects of Rhizoma drynariae and its main components on stem cell factor mRNA expressions in uterine endometrium of mus models with dysfunctional embryo implantation
[Figure 3] presents SCF mRNA expression. The model group had lower endometrial SCF mRNA expression compared with the blank group (0.51 ± 0.02 vs. 0.96 ± 0.02, P < 0.001). Compared with the model group, the Rhizoma drynariae (0.88 ± 0.03, P < 0.01), total flavone (0.82 ± 0.11, P < 0.01), and progynova (0.82 ± 0.10, P < 0.01) groups had higher endometrial SCF mRNA expression. Compared with the Rhizoma drynariae group, endometrial SCF mRNA expression was lower in the naringin (P < 0.05) and aspirin (P < 0.05) groups.
Figure 3: Effects of Rhizoma drynariae and its main components on stem cell factor SCF mRNA expressions in uterine endometrium of mouse models with dysfunctional embryo implantation. SCF mRNA expressions were determined by real-time reverse transcription-polymerase chain reaction. GAPDH was used as an inner control. Data are shown as mean ± SE (blank: n = 3; model: n = 3; Rhizoma drynariae: n = 3; total flavone: n = 3; naringin: n = 5; progynova: n = 3; aspirin: n = 5 for SCF). *P < 0.05, **P < 0.01, ***P < 0.001 versus the blank group; ##P < 0.01 versus the model group; ΔP < 0.05, ΔΔP < 0.01 versus the Rhizoma drynariae group; ▴P < 0.05, ▴▴P < 0.01 versus the total flavone group. SE: Standard error; SCF: Stem cell factorEffect of Rhizoma drynariae and its main components on the expressions of estrogen receptor α and progesterone receptor in uterine endometrium of mus models with dysfunctional embryo implantation
[Figure 4]a,[Figure 4]b,[Figure 4]c,[Figure 4]d,[Figure 4]e,[Figure 4]f,[Figure 4]g presents representative ERα immunohistochemistry images. The model group had lower endometrial ERα expression compared with the blank group (7.91 ± 0.77 vs. 21.36 ± 1.31, P < 0.001). Compared with the model group, the Rhizoma drynariae (17.24 ± 0.64, P < 0.001), total flavone (14.68 ± 1.01, P < 0.001), naringin (11.59 ± 0.83, P < 0.05), progynova (18.96 ± 1.42, P < 0.001), and aspirin (19.29 ± 1.26, P < 0.001) groups had higher endometrial ERα expression [Figure 4]h. Compared with the Rhizoma drynariae group, endometrial ERα expression was lower in the naringin (P < 0.01) group [Figure 4]h.
Figure 4: Effect of Rhizoma drynariae and its main components on the expression of ERα in uterine endometrium of mouse models of dysfunctional embryo implantation. Expression of ERα in uterine endometrium was determined by immunohistochemistry. (a) Blank (n = 10), (b) model (n = 8), (c) Rhizoma drynariae (n = 8), (d) total flavone (n = 8), (e) naringin (n = 8), (f) progynova (n = 10), and (g) aspirin (n = 8) groups (×200). (h) IOD is shown as mean ± SE *P < 0.05, ***P < 0.001 versus the blank group; #P < 0.05, ###P < 0.001 versus the model group; ΔΔP < 0.01 versus the Rhizoma drynariae group; ▴▴P < 0.01 versus the total flavone group; &&&P < 0.001 versus the naringin group. IOD: Integrated optical density; ERα: estrogen receptor α; SE: Standard error[Figure 5]a,[Figure 5]b,[Figure 5]c,[Figure 5]d,[Figure 5]e,[Figure 5]f,[Figure 5]g presents representative PR immunohistochemistry images. The model group had lower endometrial PR expression compared with the blank group (5.85 ± 0.59 vs. 11.80 ± 0.99, P < 0.001). Compared with the model group, the Rhizoma drynariae (11.78 ± 1.15, P < 0.001), progynova (8.62 ± 1.20, P < 0.05), and aspirin (10.28 ± 1.04, P < 0.01) groups had higher endometrial PR expression [Figure 5]h. Compared with the Rhizoma drynariae group, endometrial PR expression was lower in the total flavone (P < 0.01), naringin (P < 0.01), and progynova (P < 0.05) groups [Figure 5]h.
Figure 5: Effect of Rhizoma drynariae and its main components on the expression of PR in the uterine endometrium of mouse models of dysfunctional embryo implantation. Expression of PR in uterine endometrium was determined by immunohistochemistry. (a) Blank (n = 10), (b) model (n = 8), (c) Rhizoma drynariae (n = 8), (d) total flavone (n = 8), (e) naringin (n = 8), (f) progynova (n = 10), and (g) aspirin (n = 8) groups (×200). (h) IOD is shown as mean ± SE **P < 0.01, ***P < 0.001 versus the blank group; #P < 0.05, ##P < 0.01, ###P < 0.001 versus the model group; ΔP < 0.05, ΔΔP < 0.01, versus the Rhizoma drynariae group; and P < 0.05 versus the naringin group. PR: Progesterone receptor; SE: Standard error Discussion Endometrial receptivity is still the limiting step of IVF-ET technologies and methods to improve it are needed. Rhizoma drynariae is a TCM used in orthopedics and traumatology, but its effect on endometrial receptivity is still not known. Therefore, the present study aimed to observe the effect of Rhizoma drynariae and its main components on endometrial receptivity in a mus model of dysfunctional embryo implantation. Results showed that Rhizoma drynariae could improve the endometrial receptivity of mus models of dysfunctional endometrial receptivity, as shown by increased numbers of pinopodes and higher levels of SCF, ERα, and PR compared with the model group.
Pinopodes are markers of endometrial receptivity.[11],[12] In the present study, electron microscopy of endometrial samples showed that the greatest numbers of pinopodes were observed in the blank and Rhizoma drynariae groups, while the model group had the lowest number of pinopodes.
The luteal phase is the best period for embryo implantation, and the expression of the SCF is the highest during the luteal phase.[10] SCF secreted by endothelial cells and the implanting embryo stimulates the growth of trophoblasts and facilitates implantation. In addition, SCF plays a role in blastomere cleavage and proliferation before implantation.[20] In the present study, treatment with Rhizoma drynariae and total flavone reversed the decrease in uterine SCF levels induced by modeling.
The ERα and PR are increased in response to estrogen during the proliferative phase and decreased in response to progesterone during the secretory phase.[7],[8] Both ERα and PR are downregulated at the moment of implantation and this decrease is a critical event for endometrial receptivity.[7],[8] Appropriate progesterone levels and expression of PR in the endometrium are prerequisites for endometrial decidualization.[9] Patients with endometriosis and defects in endometrial receptivity show a loss of endometrial ERα downregulation in the mid-secretory phase.[7],[8] In the present study, the model group showed lower ERα and PR levels than the blank controls, suggesting that endometrial maturation could have been compromised by the hormonal treatments during modeling. Treatment with Rhizoma drynariae seems to have reversed this trend, but it is still unknown if this increase could be observed before the mid-secretory phase only or if it could be maintained later, which could be detrimental to endometrial receptivity. Additional studies examining Rhizoma drynariae over the whole cycle are necessary.
In the present study, the individual main components of Rhizoma drynariae had the same efficacy as the whole preparation on some markers. Indeed, total flavone had a similar efficacy as the whole preparation for SCF and ERα, while naringin had a lower efficacy compared to the whole preparation for all markers. These results suggest that the components of Rhizoma drynariae may act additively or synergistically together. Additional studies are necessary to confirm these results. Furthermore, these individual components could have different mechanisms of action, and studying each of them alone could be necessary to determine their impact at the molecular level. Nevertheless, the results suggest that the use of Rhizoma drynariae in the context of TCM could contribute to improving endometrial receptivity. Of course, the present study was performed in mus and the results need to be confirmed in humans. In addition, future studies will have to include complete panels of inflammatory markers and of factors associated with endometrial receptivity, as well as molecular studies to understand the exact pathways being involved.
In addition, a number of approaches were tried to improve endometrial receptivity during IVF-ET cycles. These approaches include implanting embryos at different stages,[4] using natural versus stimulation cycles,[2] using adherence compounds,[3] the use of vasodilators,[5] and the use of aspirin.[6] Future studies should compare these modalities with Rhizoma drynariae and examine potential combinations. In the present study, the use of Rhizoma drynariae achieved better SCF and PR levels, and similar ERα levels to aspirin and progynova.
ConclusionRhizoma drynariae could improve the endometrial receptivity of mus models of dysfunctional endometrial receptivity as shown by increased numbers of pinopodes and higher levers of SCF, ERα, and PR compared with the model group. Its individual main components had variable efficacy compared with whole Rhizoma drynariae.
Acknowledgments
The authors would like to thank the National Natural Science Foundation of China (81473721,81273789).
Financial support and sponsorship
This study was funded by the National Natural Science Foundation of China (81473721,81273789).
Conflicts of interest
There are no conflicts of interest.
References
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