OBJECTIVE: To observe the effect of "Zhibian" (BL54)-to-"Shuidao" (ST28) needling technique on the reproductive function of the mice with poor ovarian response (POR) and explore the molecular mechanism of acupuncture on early embryos after in vitro fertilization-embryo transfer (IVF-ET) in POR mice. METHODS: Of 70 female C57BL/6 mice, 60 mice with regular estrous cycle were screened and 30 of them were randomly divided into a blank group, a model group, and an acupuncture group, 10 mice in each one. Mice in the model and acupuncture groups were administered with tripterygium glycosides suspension (50 mg·kg^-1·d^-1) via gavage for 2 weeks to prepare POR models; while in the blank group, the mice received an equal volume of 0.9% sodium chloride solution via gavage. After successful modeling, mice in the acupuncture group underwent "Zhibian" (BL54)-to-"Shuidao" (ST28) needling technique, once daily, for 20 min each time and lasting 2 consecutive weeks. After intervention completion, subsequently, all the three groups underwent ovulation induction, orbital blood collection, and ovary extraction and fresh denuded oocyte collection. Denuded oocytes, after incubated, together with the sperms from 15 male C57BL/6 mice, were transferred into the oviducts of 30 donor pseudopregnant C57BL/6 female mice via IVF-ET; and the embryonic tissue was collected on day 7 of implantation. After successful modeling and intervention completion, the general conditions of mice in each group were observed, and the estrous cycle disorder rate was compared among the groups. After intervention completion, the numbers of oocyte, ovarian wet weight and ovarian index were recorded in each group; the levels of anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) in the serum were measured using ELISA; and ovarian morphology and mitochondrial ultrastructure were examined using HE staining and transmission electron microscopy. The transcriptome sequencing technology was employed to identify differentially expressed genes (DEGs) in early embryos of each group, followed by bioinformatics analysis; and the reversed DEGs with significant difference were verified using quantitative real-time PCR (qRT-PCR). RESULTS: After intervention, compared with the blank group, the mice in the model group presented poor spirits and declined water and food intake, reduced activity, the higher rate of estrous cycle disorder (P< 0.01); the decrease of the numbers of oocyte, ovarian wet weight, and ovarian index (P<0.01), reduced AMH and E2 in the serum (P<0.01), elevated FSH and LH in the serum (P<0.01); more atresia follicles in ovarian tissue, disorganized granulosa cells, reduced the numbers of mitochondria, vacuoles appeared and cristae breakage. When compared with the model group, in the acupuncture group, the spirits of mice, food intake and activity were improved, the rate of estrous cycle disorder was reduced (P<0.01); the numbers of oocyte, ovarian wet weight, and ovarian index were higher (P<0.01, P<0.05), the levels of AMH and E2 in the serum increased (P<0.01), the levels of FSH and LH in the serum decreased (P<0.01); the atresia follicles were reduced, the numbers of follicle increased at all levels, the numbers of mitochondria got higher, vacuoles and cristae breakage were declined. In comparison between the blank group and the model group, and between the model group and the acupuncture group, a total of 258 intersecting DEGs were identified, and 194 DEGs of them exhibited a trend of reversion before and after intervention. The top 5 reversed DEGs with significant difference included dexamethasone-induced Ras-related protein 1 (Rasd1), gene regulated by estrogen in breast cancer 1 (Greb1), leucine-rich repeat-containing G protein-coupled receptor 6 (Lgr6), Fraser syndrome 1 (Fras1), and apolipoprotein D (Apod). Compared with the blank group, the mRNA expression of Rasd1, Greb1, Lgr6, Fras1 and Apod in embryonic tissues decreased in the model group (P<0.01, P<0.05); and when compared with the model group, the mRNA expression of Rasd1, Greb1, Lgr6, Fras1 and Apod in embryonic tissues increased in the acupuncture group (P<0.05, P<0.01). These findings were consistent with the DEGs screening results. Gene ontology (GO) analysis revealed that the reversed genes of co-expression were primarily participated in the biological processes such as myoblast differentiation, endocardial cushion development, and cardiac morphogenesis. Kyoto encyclopedia of genes and genomes (KEGG) analysis indicated that the reversed genes of co-expression are enriched in the Wnt signaling pathway, and they were associated with various types of cardiac diseases. CONCLUSION The "Zhibian" (BL54)-to-"Shuidao" (ST28) needling technique may ameliorate the decline of ovarian response in mice with POR, promote normal follicle development and ovulation, thereby improve embryo quality and reduce the risk of developmental defects and deformity in the organs such as the embryonic heart. The underlying mechanism may be related to the regulation of the expression pattern of embryonic key genes through the Wnt signaling pathway.
Animals ; Female ; Mice ; Mice, Inbred C57BL ; Acupuncture Therapy ; Ovary/physiopathology* ; Humans ; Fertilization in Vitro ; Transcriptome ; Embryo Transfer ; Infertility, Female/physiopathology*

[Objective]To introduce Director CAO Yuxia's experience in treating congenital preauricular fistula infection and abscess with SHI's thick fire needle based on the thought of"treating fire depression with effusion".[Methods]By following the clinical works and case collation,the basic pathomechanism of congenital preauricular fistula infection and abscess was elaborated,and the innovative use of SHI's thick fire needle based on the therapeutic principle of"treating fire depression with effusion"to implement the"burnt needle robbing and stabbing"method was analyzed,and Director CAO's clinical experience in treating this disease by using SHI's thick fire needle and robbing stabbing method based on the therapeutic principle of"treating fire depression with effusion"was summarized,and a medical case was presented to support it.[Results]Director CAO believes that the disease was caused by the accumulation of heat and the decay into pus.The treatment should be based on the theory of"treating fire depression with effusion".For the first time,she proposes and adopts one of the new nine needles,SHI's thick fire needle,to quickly stab and eliminate pus,which can obtain the effect of"discharging heat,expelling pus,eliminating pathogens and stopping pain".In the medical case cited,the patient was suffered from this disease,combining the tongue and pulse with symptoms,he was identified as the syndrome of the accumulation of fire-heat toxin.Therefore,the key treatment principle was to eliminate pathogens and expel pus,relieve heat and resolve toxin,and after once fire needle treatment,good effect was obtained.[Conclusion]Director CAO treats congenital preauricular fistula infection and abscess,focusing on the"treating fire depression with effusion"principle,taking the fire and heat nature of fire needle and exerting the effect of"opening the way to eliminate pathogens,and expelling heat with heat",and achieves better clinical efficacy.The experience can provide ideas for the treatment of fire-heat and toxicity-containing diseases,which is worthy of clinical reference.

[Objective]To introduce Director CAO Yuxia's experience in treating congenital preauricular fistula infection and abscess with SHI's thick fire needle based on the thought of"treating fire depression with effusion".[Methods]By following the clinical works and case collation,the basic pathomechanism of congenital preauricular fistula infection and abscess was elaborated,and the innovative use of SHI's thick fire needle based on the therapeutic principle of"treating fire depression with effusion"to implement the"burnt needle robbing and stabbing"method was analyzed,and Director CAO's clinical experience in treating this disease by using SHI's thick fire needle and robbing stabbing method based on the therapeutic principle of"treating fire depression with effusion"was summarized,and a medical case was presented to support it.[Results]Director CAO believes that the disease was caused by the accumulation of heat and the decay into pus.The treatment should be based on the theory of"treating fire depression with effusion".For the first time,she proposes and adopts one of the new nine needles,SHI's thick fire needle,to quickly stab and eliminate pus,which can obtain the effect of"discharging heat,expelling pus,eliminating pathogens and stopping pain".In the medical case cited,the patient was suffered from this disease,combining the tongue and pulse with symptoms,he was identified as the syndrome of the accumulation of fire-heat toxin.Therefore,the key treatment principle was to eliminate pathogens and expel pus,relieve heat and resolve toxin,and after once fire needle treatment,good effect was obtained.[Conclusion]Director CAO treats congenital preauricular fistula infection and abscess,focusing on the"treating fire depression with effusion"principle,taking the fire and heat nature of fire needle and exerting the effect of"opening the way to eliminate pathogens,and expelling heat with heat",and achieves better clinical efficacy.The experience can provide ideas for the treatment of fire-heat and toxicity-containing diseases,which is worthy of clinical reference.