Zishen Yutai pills restore fertility in premature ovarian failure through regulating arachidonic acid metabolism and the ATK pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The Zishen Yutai pills (ZYP), a Chinese medicinal formulation derived from the Qing Dynasty prescription "Shou Tai pills", have been documented to exhibit beneficial effects in clinical observations treating premature ovarian failure (POF). However, the anti-POF effects and its comprehensive systemic mechanism have not yet been clarified. AIM OF THE REVIEW: Therapeutic effects and systemic mechanism of ZYP in POF were evaluated. MATERIALS AND METHODS: After pulverization, sieving, and stirring, ZYP was administered intragastrically to cisplatin-induced POF mice at a dose of 1.95 mg/kg/d for 14 days. The anti-POF effects of ZYP were investigated by assessing the number of ovarian follicles at different developmental stages, as well as measuring serum estradiol (E2) levels and ovarian-expressed anti-Müllerian hormone (AMH). Reproductive performance and offspring health were evaluated to predict fertility restoration. Furthermore, a combination of proteomic and metabolomic profiling was employed to elucidate the underlying molecular mechanism of ZYP in treating POF. Western blot (WB) analyses and real-time quantitative polymerase chain reaction (RT-qPCR) were conducted to explore the mechanisms through which ZYP exerted its anti-POF effects. RESULTS: We have demonstrated that oral administration of ZYP reversed the reduction in follicles at different developmental stages and stimulated the expressions of serum E2 and ovarian-expressed AMH in a cisplatin-induced POF model. Additionally, ZYP ameliorated follicle apoptosis in ovaries affected by cisplatin-induced POF. Furthermore, treatment with ZYP restored the quantity and quality of oocytes, as well as enhanced fertility. Our results revealed 62 differentially expressed proteins (DEPs) through proteomic analyses and identified 26 differentially expressed metabolites (DEMs) through metabolomic analyses. Both DEPs and DEMs were highly enriched in the arachidonic acid (AA) metabolism pathway. ZYP treatment effectively upregulated the protein and mRNA expression of critical targets in AA metabolism and the AKT pathway, including CYP17α1, HSD3ß1, LHR, STAR, and AKT, in cisplatin-induced POF mice. CONCLUSIONS: These results indicated that ZYP exerted protective effects against POF and restored fertility from cisplatin-induced apoptosis. ZYP could be a satisfying alternative treating POF.

Inhibition of checkpoint kinase prevents human oocyte apoptosis induced by chemotherapy and allows enhanced tumour chemotherapeutic efficacy.

STUDY QUESTION: Could inhibition of the checkpoint kinase (CHEK) pathway protect human oocytes and even enhance the anti-tumour effects, during chemotherapy? SUMMARY ANSWER: CHEK inhibitors prevented apoptosis of human oocytes induced by chemotherapy and even enhanced the anti-tumour effects. WHAT IS KNOWN ALREADY: CHEK inhibitors showed ovarian protective effects in mice during chemotherapy, while their role in human oocytes is unclear. STUDY DESIGN, SIZE, DURATION: This experimental study evaluated the ovarian reserve of young patients (120 patients) with cancer, exposed or not exposed to taxane and platinum (TP)-combined chemotherapy. Single RNA-sequencing analysis of human primordial oocytes from 10 patients was performed to explore the mechanism of oocyte apoptosis induced by TP chemotherapy. The damaging effects of paclitaxel (PTX) and cisplatin on human oocytes were also evaluated by culturing human ovaries in vitro. A new mouse model that combines human ovarian xenotransplantation and patient-derived tumour xenografts was developed to explore adjuvant therapies for ovarian protection. The mice were randomly allocated to four groups (10 mice for each group): control, cisplatin, cisplatin + CK1 (CHEK1 inhibitor, SCH 900776), and cisplatin + CK2 (CHEK2 inhibitor, BML277). PARTICIPANTS/MATERIALS, SETTING, METHODS: In the prospective cohort study, human ovarian follicles were counted and serum AMH levels were evaluated. RNA-sequencing analysis was conducted, and staining for follicular damage (phosphorylated H2AX histone; γH2AX), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) assays and assessments of apoptotic biomarkers (western blot and immunofluorescence) were conducted in human ovaries. After the treatments, histological analysis was performed on human ovarian samples to investigate follicular populations, and oocyte damage was measured by γH2AX staining, BAX staining, and TUNEL assays. At the same time, the tumours were evaluated for volume, weight, and apoptosis levels. MAIN RESULTS AND THE ROLE OF CHANCE: Patients who received TP chemotherapy showed decreased ovarian reserves. Single RNA-sequencing analysis of human primordial oocytes indicated that TP chemotherapy induced apoptosis of human primordial oocytes by causing CHEK-mediated TAp63α phosphorylation. In vitro culture of human ovaries showed greater damaging effects on oocytes after cisplatin treatment compared with that after PTX treatment. Using the new animal model, CHEK1/2 inhibitors prevented the apoptosis of human oocytes induced by cisplatin and even enhanced its anti-tumour effects. This protective effect appeared to be mediated by inhibiting DNA damage via the CHEK-TAp63α pathway and by generation of anti-apoptotic signals in the oocytes. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was a preclinical study performed with human ovarian samples, and clinical research is required for validation. WIDER IMPLICATIONS OF THE FINDINGS: These findings highlight the therapeutic potential of CHEK1/2 inhibitors as a complementary strategy for preserving fertility in female cancer patients. STUDY FUNDING/COMPETING INTEREST(S): This work was financially supported by the National Natural Science Foundation of China (nos. 82001514 and 81902669) and the Fundamental Research Funds for the Central Universities (2021yjsCXCY087). The authors declare no conflict of interest.

Efficacy and Safety of the Chinese Herbal Compound TJAOA101 in Treating Diminished Ovarian Reserve: A Protocol for Multicenter, Prospective, and Pre-Post Study.

OBJECTIVE: Diminished ovarian reserve (DOR) can lead to early menopause, poor fecundity, and an increased risk of disorders such as osteoporosis, cardiovascular disease, and cognitive impairment, seriously affecting the physical and mental health of women. There is still no safe and effective strategy or method to combat DOR. We have developed a novel Chinese herbal formula, Tongji anti-ovarian aging 101 (TJAOA101), to treat DOR. However, its safety and efficacy need to be further validated. METHODS: In this prospective and pre-post clinical trial, 100 eligible patients aged 18-45 diagnosed with DOR will be recruited. All participants receive TJAOA101 twice a day for 3 months. Then, comparisons before and after treatment will be analyzed, and the outcomes, including anti-mullerian hormone (AMH) and follicle-stimulating hormone (FSH) levels and the antral follicle count (AFC), the recovery rate of menopause, and the Kupperman index (KMI), will be assessed at baseline, every month during medication (the intervention period), and 1, 3 months after medication (the follow-up period). Assessments for adverse events will be performed during the intervention and follow-up periods. CONCLUSION: A multicenter, prospective study will be conducted to further confirm the safety and efficacy of TJAOA101 in treating DOR and to provide new therapeutic strategies for improving the quality of life in DOR patients.

Mechanisms of action of Zishen Yutai pills in treating premature ovarian failure determined by integrating UHPLC-Q-TOF-MS and network pharmacology analysis.

BACKGROUND: Zishen Yutai (ZSYT) pill, a patent Chinese medicine, has been widely used in the treatment of infertility, abortion, and adjunctive treatment of in vitro fertilization (IVF) for decades. Recently, the results of clinical observations showed that premature ovarian failure (POF) patients exhibited improved expression of steroids and clinical symptoms associated with hormone disorders after treatment with Zishen Yutai pills. However, the pharmacological mechanism of action of these pills remains unclear. METHODS: The compounds of Zishen Yutai pills found in blood circulation were identified via ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) technique in the serum of POF mice after oral administration of Zishen Yutai pills. The potential targets of compounds were screened using Traditional Chinese Medicine Systems Pharmacology Database, Traditional Chinese Medicine Database@Taiwan, Drugbank Database, PubChem, HIT, Pharmapper, and Swiss Target Prediction. The target genes associated with POF were collected from Online Mendelian Inheritance in Man Database, PharmGkb, Genecards, Therapeutic Target Database, and Genetic Association Database. The overlapping genes between the potential targets of Zishen Yutai pills' compounds and the target genes associated with POF were clarified via protein-protein interaction (PPI), pathway, and network analysis. RESULTS: Nineteen compounds in Zishen Yutai pills were detected in the serum of POF mice after oral administration. A total of 695 Zishen Yutai (ZSYT) pill-related targets were screened, and 344 POF-related targets were collected. From the results of Zishen Yutai (ZSYT) pill-POF PPI analysis, CYP19A1, AKR1C3, ESR1, AR, and SRD5A2 were identified as key targets via network analysis, indicating their core role in the treatment of POF with Zishen Yutai pills. Moreover, the pathway enrichment results suggested that Zishen Yutai pills treated POF primarily by regulating neuroactive ligand-receptor interaction, steroid hormone biosynthesis, and ovarian steroidogenesis. CONCLUSIONS: Via virtual screening, we found that regulation of neuroactive ligand-receptor interaction, steroid hormone biosynthesis, and ovarian steroidogenesis was the potential therapeutic mechanism of Zishen Yutai pills in treating POF. Our study suggested that combining the analysis of Zishen Yutai pills' compounds in blood in vivo in the POF model and network pharmacology prediction might offer a tool to characterize the mechanism of Zishen Yutai pills in the POF.

Cancer-Erythrocyte Hybrid Membrane-Camouflaged Magnetic Nanoparticles with Enhanced Photothermal-Immunotherapy for Ovarian Cancer.

Cell-membrane-coated nanoparticles are widely studied due to their inherent cellular properties, such as immune escape and homologous homing. A cell membrane coating can also maintain the relative stability of nanoparticles during circulation in a complex blood environment through cell membrane encapsulation technology. In this study, we fused a murine-derived ID8 ovarian cancer cell membrane with a red blood cell (RBC) membrane to create a hybrid biomimetic coating (IRM), and hybrid IRM camouflaged indocyanine green (ICG)-loaded magnetic nanoparticles (Fe3O4-ICG@IRM) were fabricated for combination therapy of ovarian cancer. Fe3O4-ICG@IRM retained both ID8 and RBC cell membrane proteins and exhibited highly specific self-recognition of ID8 cells in vitro and in vivo as well as a prolonged circulation lifetime in blood. Interestingly, in the bilateral flank tumor model, the IRM-coated nanoparticles also activated specific immunity, which killed homologous ID8 tumor cells but had no effect on B16-F10 tumor cells. Furthermore, Fe3O4-ICG@IRM showed synergistic photothermal therapy, resulting in the release of whole-cell tumor antigens by photothermal-induced tumor necrosis, which further enhanced antitumor immunotherapy for primary tumor and metastatic tumor by activating CD8+ cytotoxic T cells and reducing regulatory Foxp3+ T cells. Together, the biomimetic Fe3O4-ICG@IRM nanoparticles showed synergistic photothermal-immunotherapy for ovarian cancer.

9,10-Phenanthrenequinone: A Promising Kernel to Develop Multifunctional Antitumor Systems for Efficient Type I Photodynamic and Photothermal Synergistic Therapy.

Synergistic phototherapy provides a promising strategy to conquer the hypoxia and heterogeneity of tumors and realize a better therapeutic effect than monomodal photodynamic therapy (PDT) or photothermal therapy (PTT). The development of efficient multifunctional organic phototheranostic systems still remains a challenging task. Herein, 9,10-phenanthrenequinone (PQ) with strong electron-withdrawing ability is conjugated with the rotor-type electron-donating triphenylamine derivatives to create a series of tailor-made photosensitizers. The highly efficient Type I reactive oxygen species generation and outstanding photothermal conversion capacity are tactfully integrated into these PQ-cored photosensitizers. The underlying photophysical and photochemical mechanisms of the combined photothermal and Type I photodynamic effects are deciphered by experimental and theoretical methods and are closely associated with the active intramolecular bond stretching vibration, facilitated intersystem crossing, and specific redox cycling activity of the PQ core. Both in vitro and in vivo evaluations demonstrate that the nanoagents fabricated by these PQ-based photosensitizers are excellent candidates for Type I photodynamic and photothermal combined antitumor therapy. This study thus broadens the horizon for the development of high-performance PTT/Type I PDT nanoagents for synergistic phototheranostic treatments.

Epigallocatechin gallate and theaflavins independently alleviate cyclophosphamide-induced ovarian damage by inhibiting the overactivation of primordial follicles and follicular atresia.

BACKGROUND: Cyclophosphamide (CTX), which has been used to treat common female cancers for several years, often causes ovarian damage, early menopause and infertility. However, strategies for the effective prevention and treatment of CTX-induced ovarian damage are still lacking. Epigallocatechin gallate (EGCG) and theaflavins (TFs), key molecules derived from green tea or black tea, have been shown to exert preventive effects on many ageing-related diseases. PURPOSE: We aimed to explore the potential preventive and protective effects of EGCG and TFs on CTX-induced ovarian damage and compare the two compounds. STUDY DESIGN: Six-week-old female mice were administered a low or high dose of EGCG or TFs. The low dose was equivalent to the average daily amount of tea consumed by a drinker. METHODS: We determined the oestrous cycle and serum hormone levels to evaluate ovarian endocrine function, and we performed mating tests for reproductivity. We also assessed the follicle count and AMH level to evaluate ovarian reserve, and we performed Masson's trichrome and Sirius red staining to evaluate ovarian fibrosis. We conducted γ-H2AX and TUNEL analyses to evaluate DNA damage, and we also measured the relevant indicators of oxidative stress and follicular activation, including NRF2, HO-1, SOD2, AKT, mTOR and RPS6. RESULTS: EGCG and TFs treatment independently improved the ovarian endocrine function and reproductivity of mice that were administered CTX. EGCG and TFs also increased the ovarian reserve of these animals. Furthermore, EGCG and TFs alleviated oxidation-induced damage to ovarian DNA in mice by activating the NRF2/HO-1 and SOD2 pathways and reducing the apoptosis of growing follicles. At the same time, EGCG and TFs reduced the overactivation of primordial follicles by inhibiting the AKT/mTOR/RPS6 pathway. CONCLUSION: The present study showed that EGCG and TFs independently improved ovarian function in mice with CTX-induced ovarian damage, thereby providing useful information for designing a potential clinical strategy that will protect against chemotherapy-induced ovarian damage.

Improving Image-Guided Surgical and Immunological Tumor Treatment Efficacy by Photothermal and Photodynamic Therapies Based on a Multifunctional NIR AIEgen.

Multimodal therapy is attracting increasing attention to improve tumor treatment efficacy, but generally requires various complicated ingredients combined within one theranostic system to achieve multiple functions. Herein, a multifunctional theranostic nanoplatform based on a single aggregation-induced-emission luminogen (AIEgen), DDTB, is designed to integrate near-infrared (NIR) fluorescence, photothermal, photodynamic, and immunological effects. Intravenously injected AIEgen-based nanoparticles can efficiently accumulate in tumors with NIR fluorescence to provide preoperative diagnosis. Most of the tumors are excised under intraoperative fluorescence navigation, whereafter, some microscopic residual tumors are completely ablated by photodynamic and photothermal therapies for maximally killing the tumor cells and tissues. Up to 90% of the survival rate can be achieved by this synergistic image-guided surgery and photodynamic and photothermal therapies. Importantly, the nanoparticles-mediated photothermal/photodynamic therapy plus programmed death-ligand 1 antibody significantly induce tumor elimination by enhancing the effect of immunotherapy. This theranostic strategy on the basis of a single AIEgen significantly improves the survival of cancer mice with maximized therapeutic outcomes, and holds great promise for clinical cancer treatment.

Protective effects and mechanisms investigation of Kuntai capsule on the ovarian function of a novel model with accelerated aging ovaries.

ETHNOPHARMACOLOGICAL RELEVANCE: Kuntai capsule, a traditional Chinese medicine, has been widely used for the clinical treatment of menopausal syndrome. However, its mechanisms remain poorly understood. Considering that aging ovaries are the primary cause of menopause, this study was designed to investigate the effects and mechanisms of Kuntai capsule on ovarian function in a novel mice model with accelerated aging ovaries. MATERIALS AND METHODS: Seventy-five female C57BL/6 mice were chosen for this study. Fifteen of the mice were separated into the normal control group (NC). The remaining sixty were used to establish the novel accelerated aging ovary model by superovulation and oxidative stress and then by randomly dividing the mice into four equal groups. One group was considered the model group (Mod). The other three groups were treated with low (0.4g/kg), middle (0.8g/kg) and high (1.6g/kg) doses of Kuntai capsule intragastrically every day for 4 weeks. During the treatment, the body weight and fur condition of all mice were recorded. All the mice were forced to swim to record their exhaustive swimming time (EST), which measures their strength. Mice were then sacrificed for sampling. Ovarian reserve was evaluated using follicle counts and AMH expression. Ovarian function was evaluated using estrous cycle, sex hormone level and litter experiments. Ovarian follicles were categorized and counted to estimate ovarian reserve, and ovarian histologic sections were stained for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) to detect apoptotic cells. The ultrastructure of ovarian cells was observed using transmission electron microscopy. Western blotting was used to measure expression of Bax, Bcl2, AMH and SOD2 protein. RESULTS: Compared with the NC GROUP, the Mod group clearly displayed worse fur condition and ovarian function. These situations showed some improvement after Kuntai capsule treatment. Specifically, the fur condition and the EST of the Kuntai capsule groups were superior to the fur condition and EST of the Mod group. In cases of damaged ovarian function, Kuntai capsule can regulate the estrous cycles, increase hormone secretion and fertility and significantly decrease atretic follicles. The transmission electron microscopy results revealed that Kuntai capsule rescued the ovarian ultrastructure of mice. TUNEL staining confirmed that the apoptotic cells were reduced after Kuntai capsule treatment. Western blotting revealed that Kuntai capsule can increase AMH, SOD2, and Bcl2 protein expression and decrease Bax expression. CONCLUSIONS: Kuntai capsule may improve damaged ovarian function, which may be related to its antioxidant and anti-apoptosis effects.

Optimal pathological response indicated better long-term outcome among patients with stage IB2 to IIB cervical cancer submitted to neoadjuvant chemotherapy.

The role of pathological response in long-term outcome is still unclear in cervical cancer patients treated with neoadjuvant chemotherapy (NACT) in China. This study aimed to investigate the effect of optimal pathologic response (OPR) on survival in the patients treated with NACT and radical hysterectomy. First, 853 patients with stage IB2-IIB cervical cancer were included in a retrospective analysis; a Cox proportional hazards model was used to investigate the relationship between pathological response and disease-free survival (DFS). In the retrospective database, 64 (7.5%) patients were found to have achieved an OPR (residual disease <3 mm stromal invasion); in the multivariate Cox model, the risk of death was much greater in the non-OPR group than in the OPR group (HR, 2.61; 95%CI, 1.06 to 6.45; P = 0.037). Next, the role of OPR was also evaluated in a prospective cohort of 603 patients with cervical cancer. In the prospective cohort, 56 (9.3%) patients were found to have achieved an OPR; the log-rank tests showed that the risk of recurrence was higher in the non-OPR patients than in the OPR group (P = 0.05). After combined analysis, OPR in cervical cancer was found to be an independent prognostic factor for DFS.

Adjuvant chemotherapy, a valuable alternative option in selected patients with cervical cancer.

Radiotherapy is the standard treatment for cervical cancer, but causes radiotherapy-induced complications. Recently, chemotherapy has been more extensively utilized. Here, we perform a large-scale comparison of chemotherapy and radiotherapy. From 2002 to 2008, 2,268 patients were grouped according to adjuvant radiotherapy or chemotherapy before and/or after surgery, and we compared the 5-year overall survival (OS) and disease-free survival (DFS) rates, recurrence rates, side effects, quality of life (QoL), and sexual activity. There were no significant differences between the treatment groups for the 5-year OS and DFS rates (OS: p = 0.053, DFS: p = 0.095), although marginally improved outcomes were observed in the chemotherapy group (OS: 86.5% vs. 82.8%; DFS: 84.5% vs. 81.4%). However, patients with early-stage disease, clinical response, and younger age had increased 5-year OS and DFS rates following chemotherapy compared to radiotherapy (p<0.05). The chemotherapy group exhibited significantly lower 5-year recurrence and distant failure rates compared to the radiotherapy group (p<0.001 and p = 0.007, respectively). Nausea and vomiting were the most frequent short-term complications of chemotherapy, whereas bowel and urinary complications were more frequent in the radiotherapy group. Compared to the chemotherapy group, patients who received radiotherapy reported a lower QoL, less frequent sexual activity, and more severe menopausal symptoms (p<0.05). Cervical cancer patients treated with chemotherapy, especially those with early-stage disease, clinical responses, and younger ages, have more positive outcomes, fewer complications, better QoL and sexual activity, suggesting that chemotherapy may be a valuable alternative option for selected patients.

Adeno-associated virus-mediated local delivery of LIGHT suppresses tumorigenesis in a murine cervical cancer model.

LIGHT is a tumor necrosis factor superfamily ligand that is considered as a promising candidate for cancer therapy. It has a potent antitumor activity through establishing lymphoid-like tissues inside tumor sites and recruiting naive T cells into the tumor. In this study, we examined the possibility of antitumor activity by expressing LIGHT in cervical cancer (CC) model. A recombinant adeno-associated virus (AAV) vector was chosen for the transfer, based on its transfection efficiency and lack of detectable pathology. In vitro transfer of recombinant AAV vector expressing LIGHT (AAV-LIGHT) stimulated T-lymphocyte proliferation and activation. AAV-mediated gene transfer of LIGHT by intratumoral injection exerted a very potent antitumor effect against preexisting TC-1 cell CC in C57BL/6 mice. This study confirmed that AAV-LIGHT regressed tumor growth by activating cytotoxic T lymphocyte, enhancing infiltration of inflammatory cells in tumor and increasing stimulatory cytokine expression in tumor microenvironment. Therefore, AAV-LIGHT therapy might have potential utility for the treatment of CC.

Hypoxia induced paclitaxel resistance in human ovarian cancers via hypoxia-inducible factor 1alpha.

PURPOSE: Chemoresistance severely restricts the anti-cancer medicines from effectively treating human ovarian cancer, which has been shown to develop and survive in the specific hypoxic environments. To understand the relationship between hypoxia and chemoresistance, we investigated the potential role of hypoxia in the pathophysiology of chemoresistance, especially focusing on hypoxia-inducible factor 1alpha (HIF-1alpha). METHODS: The A2780 ovarian cancer cells are cultured in gradient hypoxic conditions (5% O(2), 3% O(2), and 1% O(2)), the sensitivity of the cells to paclitaxel and the cell inhibitory rate were determined by MTT assay. The expression and the transcriptional activity of HIF-1alpha were examined by western blot, Immunocytochemical staining, reverse transcription-polymerase chain reaction (RT-PCR), and the dual luciferase reporter system, respectively. The cell cycle distribution was analyzed by flow cytometry. In addition, we silence HIF-1alpha expression by performing RNA interference. RESULTS: MTT assay demonstrates that hypoxic challenge substantially reduces the susceptibility of cells to paclitaxel at all the tested concentrations. Coincident with this is the activation of HIF-1alpha in nuclear, which displays the increased transcriptional activity and high protein expression. Hypoxic manipulation (5% O(2), approximately 1% O(2)) significantly increased the cell population at G0/G1. Interestingly, knockdown of endogenous HIF-1alpha significantly alleviates the chemoresistance and promotes G1/S transition with the increased sensitivity of A2780 cells to paclitaxel under each hypoxic condition. CONCLUSIONS: It suggests that HIF-1alpha, stimulated by hypoxia, exerts a pivotal role in chemoresistance by G0/G1 arrest. Eliminating hypoxic conditions or silencing HIF-1alpha by siRNA might provide a potent tool to enhance paclitaxel effectiveness in treatment of human ovarian cancer.

Taichiquan acutely increases heart rate variability.

A group of 15 elderly men and 14 young male students of physical education made twice a series of Taichiquan (TCQ) practices. Their electrocardiograms were recorded on tape-recorder and heart rates and heart rate variability (HRV) were calculated from digitized data. Here we report the results of recordings in supine positions before and after the first and second series of TCQ. Intervals between heart beats (RRIs) and their standard deviation (SDNN) increased in older men from recordings before the exercise to postexercise. In young subjects the SDNN and total variance (TV) of RRIs increased. HRV increases immediately after TCQ-exercise in young and old male healthy subjects. Whether these practices have permanent effects and effects in patients need controlled and prospective studies.