The Phytoestrogenic Effect of Hibiscus sabdariffa Involves Estrogen Receptor α in Ovariectomized Wistar Rats.

The calyxes of Hibiscus sabdariffa present multiple pharmacological effects primarily attributed to their high anthocyanin content; however, little is known about their phytoestrogenic effect. Ovarian hypofunction (OH) is a process characterized by the rapid detention of the production of ovarian hormones, which compromises reproductive and cognitive functions. Hormone replacement therapy (HRT) efficiently compensates for OH; nevertheless, questions have been raised on its secondary effects and safety. One of the alternatives to tackling OH involves using phytoestrogens such as anthocyanins for their structural similarity to natural estrogens. In a Wistar rat model of ovariectomy (OVX), we recently reported the beneficial properties of an anthocyanin-rich extract prepared from the calyces of H. sabdariffa (HSE) in hindering the adverse effects of OH on memory performance and highlighted a possible phytoestrogenic impact through the modulation of estrogen receptor (ER) expression. We now report that HSE and estradiol differentially affected the expression of ERα and ERß. ERα was more sensitive to HSE; meanwhile, estradiol preferentially modulated ERß. Thus, our study leads to further research on using H. sabdariffa as a nutrition-based alternative to HRT.

Mitochondrial Function Reveals Connotation of "Seven-Seven" Theory of Huangdi's Internal Classic and Mechanism of Herbal Intervention for Elderly Women Undergoing IVF-ET / 中国实验方剂学杂志

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".

A comparative study of Mesenchymal Stem Cells transplantation approach to antagonize age-associated ovarian hypofunction with consideration of safety and efficiency.

Introduction: The transplantation of mesenchymal stem cells (MSCs) in patients with premature ovarian failure (POF) could lead to clinical improvement. The transplantation to the ovaries among other transplantation methods have been reported in various animal models, however, there is little evidence regarding the optimal method, including the clinical safety and the efficiency for the treatment of age associated ovarian hypofunction. Objectives: To establish the most effective transplantation route of MSCs, explore the resistance to therapy, its safety and role in the natural aging process of the ovaries. Methods: Highly purified MSCs were injected intraperitoneally, directly into the ovaries or tail-intravenously in mice animal model. The ovarian function, quantity and quality of oocytes, cell viability/apoptosis, were evaluated, applying chemiluminescence analysis (CLIA), western blotting, immunofluorescence staining, transmission electron microscope (TEM), TdT mediated dUTP Nick End Labeling (TUNEL) assay and other techniques. The organ tumorigenicity was also evaluated by long-term observation and histopathological examination. The efficiency of MSCs was further verified in non-human primates by the most effective transplantation route. Results: The 32nd week was ultimately determined as the time point of MSCs transplantation. Our results showed that the intra-ovarian injection was the best transplantation method with a more conspicuous effect. With deeper investigations, we found that the transplanted MSCs showed an effective influence on the follicular number, promoted follicle maturation and inhibited cell apoptosis, which was further verified in non-human primates. In addition, the long-term observation and the histopathological examinations ruled out neoplasms or obvious prosoplasia after MSCs transplantation. Conclusion: MSCs transplantation by intra-ovarian injection could within a month exert the most conspicuous anti-age-associated ovarian hypofunction effects, which may improve the quantity and quality of oocytes by changing the mitochondrial structure, regulating mitochondrial function and attenuating cell apoptosis to increase the storage of the follicle pool without a remarkable potential of tumorigenicity.

Effects of Hibiscus sabdariffa calyces on spatial memory and hippocampal expression of BDNF in ovariectomized rats.

Ovarian hypofunction is characterized by decay in brain-derived neurotrophic factor (BDNF), a neurotrophin associated with cognitive and memory function. Hormone replacement therapy is the most common treatment to counteract the negative effects of ovarian insufficiency; however, this therapy may increase the odds of endometrial cancer, blood clots, stroke, and breast cancer. Therefore, a safer alternative to synthetic estrogens is needed. One possible candidate may be phytoestrogens. Hibiscus sabdariffa L. (Malvaceae) is a source of natural food colorants; the calyces and leaves of the plant are consumed in drinks and culinary preparations and are recognized for several health benefits related to their high content of anthocyanins. In the present study, we used an ovariectomized rat model to assess the phytoestrogenic effect of H. sabdariffa, and evaluated spatial memory and BDNF expression. Ninety-day-old female Wistar rats were randomly separated into six groups. Rats from four groups were ovariectomized and injected with a physiological dose of estradiol, or given, in drinking water, an extract prepared from calyces of H. sabdariffa at doses of 50 or 100 mg/kg body weight. Both Intact and Sham groups were included as controls. At day 42, short- and long-term memories were assessed by the Barnes maze test, and hippocampal BDNF expression was evaluated by RT-qPCR and Western blot. Ovariectomy significantly decreased memory performance and BDNF expression, compared with controls. However, administration of H. sabdariffa extract reversed the negative effect of ovariectomy on short- and long-term memory parameters and BDNF expression. A stronger effect was observed at a lower dose of the extract. In conclusion, the extract from H. sabdariffa acted as a phytoestrogen in ovariectomized rats, improving spatial memory performance and hippocampal BDNF expression. Based on these promising results, further clinical experimentation is recommended to study the benefits of H. sabdariffa as an alternative hormonal therapy in patients with ovarian hypofunction.

Reproductive performance of beef cattle with ovarian hypofunction and repeat breeding in Jepara Regency, Central Java, Indonesia.

BACKGROUND AND AIM: Reproductive disorders keep the beef cattle population in Jepara Regency, Central Java, Indonesia, from increasing. Ovarian hypofunction and repeat breeding are the most common reproductive disorders, leading to large economic losses for traditional breeders. However, the impact of poor reproductive performance among traditional breeders is not well-known. This study aimed to investigate the reproductive performance of beef cattle with ovarian hypofunction and repeat breeding in Jepara Regency. MATERIALS AND METHODS: We determined cattle's reproductive status by rectal examination and anamnesis, assessing reproductive performance in 28 cows with repeat breeding and 27 cows with ovarian hypofunction. The following parameters were measured: Postpartum estrous (PPE), days open (DO), service per conception (S/C), and calving interval (CI). The data came from livestock records from animal recording cards, iSIKHNAS, and estimated births from the insemination date that produced a pregnancy. RESULTS: In beef cattle diagnosed with ovarian hypofunction, S/C, PPE, DO, and CI were 1.28, 257 days, 265 days, and 18 months, respectively. The length of CI caused by long PPE may be due to improper nutrition and calf weaning delays. In beef cattle with repeat breeding, S/C, PPE, DO, and CI were 4.15, 106 days, 210 days, and 16 months, respectively. The length of CI was caused by long DO due to pregnancy failure at the first estrus. CONCLUSION: Hypofunction and repeat breeding reduce the reproductive performance of beef cattle in the Jepara Regency. The cows' health conditions pre- and postpartum can be optimized by providing high-quality feed to enhance reproductive performance.

Platelet Rich Plasma for Regenerative Medicine Treatment of Bovine Ovarian Hypofunction.

Recent studies on cull cows have shown that ovarian abnormalities, particularly ovarian insufficiency, are the main cause of reproductive failure. The aim of this study was to treat bovine ovarian failure with intraovarian administration of autologous platelet rich plasma (PRP), which is rich in growth factors, chemokines, and cytokines that could stimulate follicular growth and steroidogenesis. Twelve cows with ovarian hypofunction were enrolled in the study and they were randomly allocated in control group (CTR) and treated group (six animal for group). In the treated group, only five animals received the PRP treatment because intraovarian administration was hindered in one by a rectovaginal fistula. Animals of control group were treated by intraovarian administration of physiological solution. In the 4 weeks after PRP injection, a mild to strong increase in progesterone (PRG) concentrations was detected in four of the five cows treated. Artificial insemination (AI) resulted in four pregnancies that are still ongoing (7th month). Intraovarian administration of PRP improved ovarian function after 2 months of treatment. This effect may be due to reduction of follicular atresia or to revitalization of dormant oocytes allowing restoration of fertility.

[Effect of acupuncture on oxidative stress and apoptosis in ovarian hypofunction induced by Tripterygium Glycosides].

OBJECTIVE: To explore whether manual acupuncture stimulation of "Shenshu " (BL23), "Guanyuan" (CV4), "Zhongwan" (CV12) can improve the ovarian function by resisting oxidant stress and reducing apoptosis of granulosa cells in rats with ovarian hypofunction. METHODS: Forty female SD rats with normal estrous cycles were randomly divided into blank control, model, hormone therapy and acupuncture groups (n=10 rats in each group). The ovarian hypofunction model was established by gavage of Tripterygium Glycosides suspension (50 mg·kg-1·d-1) for 14 successive days. Rats of the hormone therapy group were treated by gavage of estrogen and progesterone, and those of the acupuncture group treated by manual acupuncture stimulation of bilateral BL23 or CV4 and CV12 alternatively by using uniform reinforcing-reducing method for 10 s every 5 min (3 times in 10 min). The treatment was performed once daily for 14 days. The blank group was given equal volume of normal saline daily. On the 9th day, the estrous cycle of each rat was observed by means of vaginal smear test. The ovarian index, serum estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), malondialdehyde (MDA) and superoxide dismutase (SOD) contents were detected by ELISA. Histopathological changes of the ovary tissue were observed by H.E. staining. The expressions of apoptosis-related proteins Bax and Bcl-2 and their mRNAs of the ovaries were determined by immunohistochemical staining and quantitative real time-PCR separately. RESULTS: The menstrual disorder rate was 100% in the model group, and was significantly higher than those in the hormone therapy (30%) and acupuncture (40%) groups (P<0.01). Following modeling, the ovarian wet weight and index, E2 and SOD contents, Bcl-2 protein and mRNA expressions were significantly decreased (P<0.01, P<0.05), and serum FSH, LH and MDA levels, Bax protein and mRNA expressions were significantly increased (P<0.05, P<0.01) in the model group in comparison with the blank control group. H.E. staining showed a large amount of connective tissue in the ovary, with fewer mature follicles, increase of atresia follicles, significant reduction of luteal tissue, and appearance of scarring tissue in the model group. Compared with the model group, there were more mature follicles, fewer atresia follicles, lower abnormal granulosa cell morphology and lower estrous cycle disorder ratio in both hormone therapy and acupuncture groups. After the interventions, the decreased ovarian wet weight and index, serum E2 and SOD contents, expressions of Bcl-2 protein and mRNA were significantly increased (P<0.05, P<0.01), and the increased levels of FSH and LH, MDA, expressions of Bax protein and mRNA were significantly decreased in both hormone therapy and acupuncture groups (P<0.05, P<0.01). The effect of acupuncture was significantly superior to that of hormone in up-regulating SOD, Bax and Bcl-2 protein expressions (P<0.01). CONCLUSION: Acupuncture can improve the menstrual disorder in rats with ovarian hypofunction, which is closely related to its effects in improving antioxidant stress ability and regulating the expressions of apoptosis-related protein and mRNA of ovarian gra-nulosa cells.

Effect of acupuncture on oxidative stress and apoptosis in ovarian hypofunction induced by Tripterygium Glycosides / 针刺研究

OBJECTIVE: To explore whether manual acupuncture stimulation of "Shenshu " (BL23), "Guanyuan" (CV4), "Zhongwan" (CV12) can improve the ovarian function by resisting oxidant stress and reducing apoptosis of granulosa cells in rats with ovarian hypofunction. METHODS: Forty female SD rats with normal estrous cycles were randomly divided into blank control, model, hormone therapy and acupuncture groups (n=10 rats in each group). The ovarian hypofunction model was established by gavage of Tripterygium Glycosides suspension (50 mg·kg-1·d-1) for 14 successive days. Rats of the hormone therapy group were treated by gavage of estrogen and progesterone, and those of the acupuncture group treated by manual acupuncture stimulation of bilateral BL23 or CV4 and CV12 alternatively by using uniform reinforcing-reducing method for 10 s every 5 min (3 times in 10 min). The treatment was performed once daily for 14 days. The blank group was given equal volume of normal saline daily. On the 9th day, the estrous cycle of each rat was observed by means of vaginal smear test. The ovarian index, serum estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), malondialdehyde (MDA) and superoxide dismutase (SOD) contents were detected by ELISA. Histopathological changes of the ovary tissue were observed by H.E. staining. The expressions of apoptosis-related proteins Bax and Bcl-2 and their mRNAs of the ovaries were determined by immunohistochemical staining and quantitative real time-PCR separately. RESULTS: The menstrual disorder rate was 100% in the model group, and was significantly higher than those in the hormone therapy (30%) and acupuncture (40%) groups (P<0.01). Following modeling, the ovarian wet weight and index, E2 and SOD contents, Bcl-2 protein and mRNA expressions were significantly decreased (P<0.01, P<0.05), and serum FSH, LH and MDA levels, Bax protein and mRNA expressions were significantly increased (P<0.05, P<0.01) in the model group in comparison with the blank control group. H.E. staining showed a large amount of connective tissue in the ovary, with fewer mature follicles, increase of atresia follicles, significant reduction of luteal tissue, and appearance of scarring tissue in the model group. Compared with the model group, there were more mature follicles, fewer atresia follicles, lower abnormal granulosa cell morphology and lower estrous cycle disorder ratio in both hormone therapy and acupuncture groups. After the interventions, the decreased ovarian wet weight and index, serum E2 and SOD contents, expressions of Bcl-2 protein and mRNA were significantly increased (P<0.05, P<0.01), and the increased levels of FSH and LH, MDA, expressions of Bax protein and mRNA were significantly decreased in both hormone therapy and acupuncture groups (P<0.05, P<0.01). The effect of acupuncture was significantly superior to that of hormone in up-regulating SOD, Bax and Bcl-2 protein expressions (P<0.01). CONCLUSION: Acupuncture can improve the menstrual disorder in rats with ovarian hypofunction, which is closely related to its effects in improving antioxidant stress ability and regulating the expressions of apoptosis-related protein and mRNA of ovarian gra-nulosa cells.