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ObjectiveTo study the changes of mitochondrial function of ovarian granulosa cells in women of different ages and the effect of Erzhi-Tiangui prescription on in vitro fertilization-embryo transfer （IVF-ET） outcomes for elderly women， so as to verify the connotation of the "Seven-Seven" theory in the Huangdi's Internal Classic （《黄帝内经》）. MethodA total of 150 infertility patients undergoing IVF-ET at the Reproductive and Genetic Center of Integrative Medicine， Affiliated Hospital of Shandong University of Traditional Chinese Medicine were recruited and assigned into "hree-Seven/Four-Seven （30 cases）， Five-Seven （60 cases）， and Six-Seven （60 cases） groups according to the "Seven-Seven" theory. The Five-Seven and Six-Seven groups were further assigned into control and Chinese medicine subgroups using the random number plus envelope method， and the Chinese medicine group was administrated with Erzhi Tiangui prescription from the start day of controlled ovulation stimulation cycle to the trigger day. The IVF outcome was observed， and Western blot was employed to determine the levels of mitofusin 1 （MFN1）， mitofusin 2 （MFN2）， and dynamin-related protein 1 （Drp1） in the ovarian granulosa cells. ResultCompared with the Three-Seven/Four-Seven group， the control subgroups of the Five-Seven and Six-Seven groups showed decreased retrieved oocytes， two pronuclear （2PN） embryos， available embryos， high-quality embryos， clinical pregnancy rate， and live birth rate （P<0.05）. Moreover， the control subgroup of the Six-Seven group showed decreased fresh embryo transfer rate（P<0.05）. Compared with the control subgroup of the Five-Seven group， that of the Six-Seven group showed reduced retrieved oocytes， 2PN embryos， available embryos， high-quality embryos， and clinical pregnancy rate （P<0.05）. The Chinese medicine subgroup had more retrieved oocytes， 2PN oocytes， and available embryos than the control subgroup in the Five-Seven groups （P<0.05）. The Chinese medicine subgroup had more retrieved oocytes， than the control subgroup in the Six-Seven groups （P<0.05）. The control subgroup of the Six-Seven group showed lower expression levels of Mfn1 and Mfn2 and higher level of Drp1 than the control subgroup of the Five-Seven group （P<0.05）， which indicated that the levels of Mfn1 and Mfn2 in ovarian granulosa cells were down-regulated while the expression of Drp1 was up-regulated with aging （P<0.05）. The Chinese medicine subgroup had higher Mfn2 level and lower Drp1 level than the control subgroup in the Five-Seven group （P<0.05）， and the Chinese medicine subgroup had higher Mfn1 and Mfn2 levels and lower Drp1 level than then control subgroup in the Six-Seven group （P<0.05）. ConclusionsThe prognosis of IVF in women after "Five-Seven" became worse with aging， and the mitochondria in ovarian granulosa cells showed decreased fusion ability and increased fission， which verified the connotation of the "Seven-Seven" theory from the mitochondrial function. Erzhi Tiangui prescription can regulate the mitochondrial function of ovarian granulosa cells in elderly women， up-regulate the expression levels of Mfn1 and Mfn2 to promote mitochondrial fusion， and down-regulate the expression of Drp1 to reduce mitochondrial fission， thus alleviating the ovarian hypofunction caused by aging， improve the development potential of oocytes， and improve the IVF outcomes of elderly women. However， this prescription has limited efficacy for the elderly women in the age range of "Six-Seven".
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ObjectiveTo investigate the clinical efficacy and possible mechanism of Erzhi Tiangui prescription on repeated implantation failure （RIF） of kidney deficiency syndrome. MethodSeventy patients with RIF of kidney deficiency syndrome who underwent natural cycle frozen-thawed embryo transfer （FET） in the Reproductive and Genetic Center of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine were enrolled and randomly divided into a treatment group （35 cases） and a control group （35 cases）. Patients in the treatment group took oral Erzhi Tiangui prescription from the third day of each menstrual cycle two months before the FET cycle and continued to take it until the day of transplantation from the third day of the menstrual cycle in the month of transplantation. Those in the control group did not accept traditional Chinese medicine （TCM）. In addition，10 patients who successfully achieved clinical pregnancy after the first natural cycle FET were screened from the reproductive medical record bank of this hospital and assigned to the normal group. Peripheral blood samples of patients in the three groups on the day of embryo transfer were collected from the specimen bank of the Reproductive and Genetic Center. Serum soluble programmed death molecule-1 （sPD-1），soluble programmed death molecule-ligand 1 （sPD-L1），transforming growth factor-β （TGF-β），interleukin-17 （IL-17）， and interleukin-10 （IL-10） levels were measured by enzyme-linked immunosorbent assay （ELISA）. The changes in kidney deficiency syndrome scores， the final biochemical pregnancy rates， clinical pregnancy rates， and embryo implantation rates of the treatment group and the control group before and after treatment were observed. ResultCompared with the normal group，the model group showed increased serum levels of sPD-1 and IL-17（r=0.347，P<0.05），decreased levels of IL-10 and TGF-β （P<0.01），and non-significant change in sPD-L1 level. Serum sPD-1 was positively correlated with IL-17 （P<0.05） and negatively correlated with IL-10（r=-0.521，P<0.01） and TGF-β（r=-0.457，P<0.01） in RIF patients with kidney deficiency syndrome. After TCM treatment，compared with the control group， the treatment group showed improved TCM syndrome score （P<0.05） and increased clinical pregnancy rate and embryo transfer rate（P<0.05），but there was no statistically significant difference in the biochemical pregnancy rate between the two groups. ConclusionAbnormal expression of sPD-1 in patients with RIF of kidney deficiency syndrome breaks the balance of T helper 17 （Th17）/regulatory T cell （Treg），which is not conducive to embryo implantation and pregnancy maintenance. Erzhi Tiangui prescription，a TCM for tonifying the kidney，can significantly improve the symptoms of kidney deficiency in patients with RIF of kidney deficiency syndrome，reduce the concentrations of sPD-1 and IL-17 in the peripheral serum，increase the levels of TGF-β and IL-10，regulate the peripheral Th17/Treg immune balance，and increase the implantation rate and clinical pregnancy rate，which has a high clinical value.
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To observe the intervention mecha-nism of Erzhi Tiangui prescription on female infertility from the perspective of network pharmacology, so as to provide systematic biological information and basis for the treatment of infertility in elderly women with tradi-tional Chinese medicine. Methods Based on network pharmacology, the main chemical constituents and ac-tion targets of Erzhi Tiangui prescription were searched, its biological pathway was analyzed, and its binding activity was predicted by molecular docking technique. Results A total of 159 active components were screened, with 283 corresponding targets, 1 078 female infertility related disease targets and 104 inter-section targets, mainly involving key target proteins such as PTGS2, ESR1, HSP90AA1, AR, NOS2, PPARG, NCOA2 and so on. The biological pathways mainly acted on PI3 K-Akt signaling pathway, MAPK signaling pathway and so on. Conclusions The study preliminarily explores the mechanism of Erzhi Tiangui prescription in regulating female infertility through multi-ways, multi-components and multi-targets, and its main active components are well combined with Mfn-1 and Mfn-2, which may play a therapeutic role in the treatment of infertility in elderly women.
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Objective To study the effect of serum contained Erzhi Tiangui Granule on the secretion function of ovarian granulosa cells from primary senile mice. Methods The serum with decoction from primary senile mice was added to cultured granulosa cells and incubated for 48 h. Then the concentration of estradiol (E2), progesterone (P) and inhibin B (INHB) in cultured medium were measured by chemiluminescence immunoassay (CLIA) and Sandwich ELISA respectively. Results The serum contained Erzhi Tiangui Granule could increase the secretion of E2, P and INHB. Conclusion Erzhi Tiangui Granule can improve the ovarian function of primary senile mice by regulating the secretion function of granulosa cells.
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Tiangui,a specific terms in TCM,is a special material that can regulate human reproduction and development. It stems from congenital kidney essence and is nourished by acquired nutrients with the nature of time-limit,rhythmicity and status. Tiangui promotes abundant CHONG meridian with unobstructed REN meridian,participates in the production and regulation of menstruation,and prepares for conception of women. According to the holism of TCM,person is a unified whole,Tiangui's generation and functional role must be completed under the condition of normal five viscera' function,especially the sufficient kidney essence,although the 'to'and 'exhaustion'of Tiangui is directly affected and regulated by congenital kidney essence. Therefore the synergistic effect of the five viscera can not be ignored on Tiangui,although the congenital kidney essence occupies an important position.
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Objective To study the effect and mechanism of Erzhi Tiangui Granule(Granule for promoting the uterine functions) on infertility induced by luteal phase deficiency.Methods Sixty infertility patients owing to luteal phase deficiency were randomized into the tested group(treated with Erzhi Tiangui Granule) and the control group(treated with Liuwei Dihuang Granule),30 in each.The improvement of TCM symptoms,the serum levels of estradiol(E2) and progestone(P),the expression of leukemia inhibiting factor(LIF) of luteal metaphase before and after treatment,and the pregnancy rate were observed.Results After treatment,the tested group was significantly better than the control group in improving the TCM symptoms(P0.05);the pregnancy rate in the tested group was higher than that in the control group(P
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Aim To observe effects of Seabuckthorn fatty acids on old rat ovarian granulosa cell apoptosis and expression of bcl-2 and fas,then discuss the adjustive effect of Seabuckthorn fatty acids on ovarian function of menopause.Methods 22 months old rats were randomly divided into 5 groups,estrogen control group,old control group,Seabuckthorn fatty acids group of high dose,middle dose and low dose.In addition,one young control group was made up of 3 months old rats.Rat ovarian granulosa cell apoptosis was detected by TUNEL.The expression of protein bcl-2 and protein fas were detected by immunohistochemistry,and bcl-2 mRNA and fas mRNA by in situ hybridization(ISH).Grey analyse was done using Q550CW image analyse system.Results Compared with the old rats control group,Seabuckthorn fatty acids of high dose,middle dose and low dose can differently restrain rat ovarian granulosa cell apoptosis,enhance the expression of protein bcl-2 and bcl-2 mRNA,weakened the expression of protein fas and fas mRNA.Conclusion Seabuckthorn fatty acids restrains ovarian granulosa cell apoptosis through adjusting the expression of bcl-2 and fas.Sequentially,it adjusts estrogen level and treats menopause syndrome.