Gene and stem cell therapy for inherited cardiac arrhythmias.

Inherited cardiac arrhythmias are a group of genetic diseases predisposing to sudden cardiac arrest, mainly resulting from variants in genes encoding cardiac ion channels or proteins involved in their regulation. Currently available therapeutic options (pharmacotherapy, ablative therapy and device-based therapy) can not preclude the occurrence of arrhythmia events and/or provide complete protection. With growing understanding of the genetic background and molecular mechanisms of inherited cardiac arrhythmias, advancing insight of stem cell technology, and development of vectors and delivery strategies, gene therapy and stem cell therapy may be promising approaches for treatment of inherited cardiac arrhythmias. Recent years have witnessed impressive progress in the basic science aspects and there is a clear and urgent need to be translated into the clinical management of arrhythmic events. In this review, we present a succinct overview of gene and cell therapy strategies, and summarize the current status of gene and cell therapy. Finally, we discuss future directions for implementation of gene and cell therapy in the therapy of inherited cardiac arrhythmias.

Advances in basic and translational research in atrial fibrillation.

Atrial fibrillation (AF) is a very common cardiac arrhythmia with an estimated prevalence of 33.5 million patients globally. It is associated with an increased risk of death, stroke and peripheral embolism. Although genetic studies have identified a growing number of genes associated with AF, the definitive impact of these genetic findings is yet to be established. Several mechanisms, including electrical, structural and neural remodelling of atrial tissue, have been proposed to contribute to the development of AF. Despite over a century of exploration, the molecular and cellular mechanisms underlying AF have not been fully established. Current antiarrhythmic drugs are associated with a significant rate of adverse events and management of AF using ablation is not optimal, especially in cases of persistent AF. This review discusses recent advances in our understanding and management of AF, including new concepts of epidemiology, genetics and pathophysiological mechanisms. We review the current status of antiarrhythmic drug therapy for AF, new potential agents, as well as mechanism-based AF ablation. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Blapstin, a Diapause-Specific Peptide-Like Peptide from the Chinese Medicinal Beetle Blaps rhynchopetera, Has Antifungal Function.

Drug resistance against bacteria and fungi has become common in recent years, and it is urgent to discover novel antimicrobial peptides to manage this problem. Many antimicrobial peptides from insects have been reported to have antifungal activity and are candidate molecules in the treatment of human diseases. In the present study, we characterized an antifungal peptide named blapstin that was isolated from the Chinese medicinal beetle Blaps rhynchopetera used in folk medicine. The complete coding sequence was cloned from the cDNA library prepared from the midgut of B. rhynchopetera. It is a 41-amino-acid diapause-specific peptide (DSP)-like peptide stabilized by three disulfide bridges and shows antifungal activity against Candida albicans and Trichophyton rubrum with MICs of 7 µM and 5.3 µM, respectively. The C. albicans and T. rubrum treated with blapstin showed irregular and shrunken cell membranes. In addition, blapstin inhibited the activity of C. albicans biofilm and showed little hemolytic or toxic activity on human cells and it is highly expressed in the fat body, followed by the hemolymph, midgut, muscle, and defensive glands. These results indicate that blapstin may help insects fight against fungi and showed a potential application in the development of antifungal reagents. IMPORTANCE Candida albicans is one of the conditional pathogenic fungi causing severe nosocomial infections. Trichophyton rubrum and other skin fungi are the main pathogens of superficial cutaneous fungal diseases, especially in children and the elderly. At present, antibiotics such as amphotericin B, ketoconazole, and fluconazole are the main drugs for the clinical treatment of C. albicans and T. rubrum infections. However, these drugs have certain acute toxicity. Long-term use can increase kidney damage and other side effects. Therefore, obtaining broad-spectrum antifungal drugs with high efficiency and low toxicity for the treatment of C. albicans and T. rubrum infections is a top priority. Blapstin is an antifungal peptide which shows activity against C. albicans and T. rubrum. The discovery of blapstin provides a novel clue for our understanding of the innate immunity of Blaps rhynchopetera and provides a template for designing antifungal drugs.

Clinical characteristics and electrophysiologic properties of SCN5A variants in fever-induced Brugada syndrome.

BACKGROUND: Brugada syndrome (BrS) is a severe inherited arrhythmia syndrome that can be unmasked by fever. METHODS: A multicentre clinical analysis was performed in 261 patients diagnosed with fever-induced BrS, including 198 (75.9%) and 27 (10.3%) patients who received next-generation genetic sequencing and epicardial arrhythmogenic substrate (AS) mapping, respectively. FINDINGS: In fever-induced BrS patients, pathogenic or likely pathogenic (P/LP) SCN5A variant carriers developed fever-induced BrS at a younger age, and more often in females and those of Caucasian descent. They exhibited significant electrophysical abnormalities, including a larger epicardial AS area, and more prolonged abnormal epicardial electrograms. During a median follow-up of 50.5 months (quartiles 32.5-81.5 months) after the diagnosis, major cardiac events (MCE) occurred in 27 (14.4%) patients. Patients with P/LP SCN5A variants had a higher ratio of MCE compared with the rest. Additionally, history of syncope, QRS duration, and Tpe interval could also predict an increased risk for future MCE according to univariate analysis. Multivariate analysis indicated that only P/LP SCN5A variants were independent significant predictors of MCE. Computational structural modelling showed that most variants are destabilizing, suggesting that Nav1.5 structure destabilization caused by SCN5A missense variants may contribute to fever-induced BrS. INTERPRETATION: In our cohort, P/LP SCN5A variant carriers with fever-induced BrS are more prevalent among patients of Caucasian descent, females, and younger patients. These patients exhibit aggressive electrophysiological abnormalities and worse outcome, which warrants closer monitoring and more urgent management of fever. FUNDING: The current work was supported by the National Natural Science Foundation Project of China (Nos. 82270332 & 81670304), The Fundamental Research Funds for the Central Universities of China - Independent Research Project of Wuhan University (No. 2042022kf1217) from China; the National Institutes of Health of USA [NIH R56 (HL47678), NIH R01 (HL138103), and NIH R01 (HL152201)], the W. W. Smith Charitable Trust and the Wistar and Martha Morris Fund, Sharpe-Strumia Research Foundation, the American Heart Association Postdoctoral Fellowship (20POST35220002) from United States; the Netherlands CardioVascular Research Initiative: the Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences (PREDICT2) from the Netherlands.

Distinct Features of Probands With Early Repolarization and Brugada Syndromes Carrying SCN5A Pathogenic Variants.

BACKGROUND: Two major forms of inherited J-wave syndrome (JWS) are recognized: early repolarization syndrome (ERS) and Brugada syndrome (BrS). OBJECTIVES: This study sought to assess the distinct features between patients with ERS and BrS carrying pathogenic variants in SCN5A. METHODS: Clinical evaluation and next-generation sequencing were performed in 262 probands with BrS and 104 with ERS. Nav1.5 and Kv4.3 channels were studied with the use of patch-clamp techniques. A computational model was used to investigate the protein structure. RESULTS: The SCN5A+ yield in ERS was significantly lower than in BrS (9.62% vs 22.90%; P = 0.004). Patients diagnosed with ERS displayed shorter QRS and QTc than patients with BrS. More than 2 pathogenic SCN5A variants were found in 5 probands. These patients displayed longer PR intervals and QRS duration and experienced more major arrhythmia events (MAE) compared with those carrying only a single pathogenic variant. SCN5A-L1412F, detected in a fever-induced ERS patient, led to total loss of function, destabilized the Nav1.5 structure, and showed a dominant-negative effect, which was accentuated during a febrile state. ERS-related SCN5A-G452C did not alter the inward sodium current (INa) when SCN5A was expressed alone, but when coexpressed with KCND3 it reduced peak INa by 44.52% and increased the transient outward potassium current (Ito) by 106.81%. CONCLUSIONS: These findings point to SCN5A as a major susceptibility gene in ERS as much as it is in BrS, whereas the lower SCN5A+ ratio in ERS indicates the difference in underlying electrophysiology. These findings also identify the first case of fever-induced ERS and demonstrate a critical role of Ito in JWS and a higher risk for MAE in JWS probands carrying multiple pathogenic variants in SCN5A.

p38 MAPK is Crucial for Wnt1- and LiCl-Induced Epithelial Mesenchymal Transition.

Idiopathic pulmonary fibrosis (IPF) is characterized by myofibroblast foci in lung parenchyma. Myofibroblasts are thought to originate from epithelial-to-mesenchymal transition (EMT). Wnt1 and lithium chloride (LiCl) induce EMT in alveolar epithelial cells (AECs), but the mechanisms are unclear. AECs were treated with Wnt1 and LiCl, respectively; morphological change and molecular changes of EMT, including E-cadherin, fibronectin, and vimentin, were observed. SB203580 was administrated to test the role of p38 МАРК signaling in EMT. Then AECs were treated with siRNAs targeting p38 МАРК to further test the effects of p38 МАРК, and the role was further confirmed by re-expression of p38 МАРК. At last P-catenin siRNA was used to test the role of ß-catenin in the EMT process and relationship of ß-catenin and p38 МАРК was concluded. Exposure of AECs to Wnt1 and LiCl resulted in upregulation of vimentin and fibronectin with subsequent downregulation of E-cadherin. Wnt1 and LiCl stimulated the p38 МАРК signaling pathways. Perturbing the p38 МАРК pathway either by SB203580 or through p38 МАРК siRNA blocked EMT and inhibited fibronetin synthesis, which were reversed by transfection of p38 МАРК expression plasmid. ß-catenin siRNA attenuated the EMT process and decreased p38 МАРК phosphorylation, indicating that ß-catenin is involved in the EMTrelated changes through regulation of p38 МАРК phosphorylation. These findings suggest that p38 МАРК participates in the pathogenesis of EMT through Wnt pathway and that p38 МАРК may be a novel target for IPF therapy.

[Lemierre syndrome caused by Arcanobacterium haemolyticum: a case report and review of the literature].

OBJECTIVE: To emphasize the importance of the early diagnosis and treatment of Lemierre syndrome caused by Arcanobacterium haemolyticum. METHOD: A case of Lemierre syndrome caused by Arcanobacterium haemolyticum and three similar reported cases were reviewed. RESULTS: A man complained of fever with a sore throat, and examination found an enlarged left tonsil with prominent exudate, normal blood routine test and chest radiograph. Although the patient received the treatment of penicillin G and azithromycin, his condition worsened. Blood test showed white blood cell count 13.59×10(9)/L (neutrophils 0.933), platelet count 7.4×10(9)/L, TBil 54.3 mmol/L, DBil 28.3 mmol/L, AST 127 IU/L, ALT 82 IU/L, serum albumin 19.3 g/L with the development of the conditions. Blood cultures grew Arcanobacterium haemolyticum and the piperacillin-tazobactam was administered until fever was controlled. In addition, anticoagulation was administered when the thrombus was confirmed in the left internal jugular vein. Two follow-up clinic visits over the following 4 months were unremarkable. Besides three similar cases reported, four patients were male, and the ages ranged from 19 to 54 years. The chief complaints were sore throat and fever (4/4), with neck pain (4/4). Physical examinations found pharyngitis (2/4), exudate or abscess in the tonsillar crypt (2/4), maculopapular rashes (2/4). Laboratory results showed leukocytosis and thrombocytopaenia (4/4), acute cholestatic liver dysfunction (3/4), acute renal failure (2/4), acute respiratory failure (1/4). The first chest radiographs were normal at the onset, but chest radiography features included peripheral nodules and cavitation (3/4), focal or wedge-shaped lesions (1/4), pleural effusion (1/4) with the development of the conditions. Blood culture proved that there was only growth of Arcanobacterium haemolyticum (2/4), both Fusobacterium necrophorum and Arcanobacterium haemolyticum were found (2/4). Amoxicillin/clavulanic acid or piperacillin/tazobactam was administered (4/4). Neck CT proved internal jugular vein thrombosis (3/4) and anticoagulation was administered (3/4). All patients recovered and no one died. CONCLUSIONS: The characters of Lemierre syndrome include primary oropharynx infection, septicaemia, septic or embolic phlebitis of jugular vein, and metastatic abscess. Early recognition and aggressive intravenous broad-spectrum antibiotics are critical to reduce mortality.

[Mapping fetal brain development in the second trimester with 7.0T magnetic resonance imaging].

OBJECTIVE: To obtain the three dimensional visualization model with normal measurements of fetal brain in the second trimester and analyze the developmental changes with gestational age (GA), sexual dimorphisms and cerebral asymmetries. METHODS: The brains of 69 fetal specimens of 12 - 22 weeks GA were scanned by 7.0T magnetic resonance imaging (MRI). The developing structures were analyzed and a three dimensional visualization model was rebuilt with Amira 4.1 software. RESULTS: Most sulci, except for postcentral and intraparietal sulcus, were present until 22 weeks GA. And none developed secondary branches. Laminar organization, described as early as 12 weeks GA, was delineated as layers with different signal intensities and became typical after 16 weeks GA. Basal nuclei was distinctly visible. Brains had different growth rates linearly increasing with GA. But neither sexual dimorphisms nor cerebral asymmetries was detected. CONCLUSIONS: The initial developmental stage of fetal brain occurs at 12 - 22 weeks GA. The developing structures may be distinctly visualized on 7.0T post-mortem MRI. And the three dimensional visualization model aids greatly in the precise cognition of immature brain.

Varied response of the pulmonary arterial endothelium in a novel rat model of venous thromboembolism.

BACKGROUND: The experimental studies of venous thromboembolism (VTE) as an entity and the response of the pulmonary arterial endothelium after VTE are still rare. The objective of this study was to observe changes in the pulmonary arterial endothelium using a novel rat model of VTE. METHODS: Rats were allocated to the VTE (n = 54) or control groups (n = 9). The left femoral vein was blocked using a microvessel clip to form deep vein thrombosis (DVT). One, four or seven-day-old thrombi were injected into the right femoral vein to induce DVT-pulmonary thromboembolism (DVT-PTE). The rats were sacrificed 1, 4 or 7 days later (D(n(1,4,7)) P(n(1,4,7)) subgroups (n = 6)), and the lungs were examined using light and electron microscopy. RESULTS: On gross dissection, the rate of DVT formation was higher on day 1 (D(1)P(n): 100%, 18/18) than day 4 (D(4)P(n): 83%, 15/18; χ(2) = 5.900, P = 0.015) or day 7 (D(7)P(n): 44%, 8/18; χ(2) = 13.846, P = 0.000). On gross dissection, the positive emboli residue rate in the pulmonary arteries was lower in the D(1)P(n) subgroup (39%, 7/18) than the D(4)P(n) (73%, 11/15; χ(2) = 3.915, P = 0.048) and D(7)P(n) subgroups (100%, 8/8; χ(2) = 8.474, P = 0.004); however, light microscopy indicated the residual emboli rate was similar in all subgroups. Hyperplasia of the pulmonary arterial endothelium was observed 4 and 7 days after the injection of one-day-old or four-day-old thrombi. However, regions without pulmonary arterial endothelial cells and intra-elastic layers were observed one day after injection of seven-day-old thrombi. CONCLUSIONS: This novel model closely simulates the clinical situations of thrombus formation and is ideal to study pulmonary endothelial cell activation. The outcome of emboli and pulmonary arterial endothelial alterations are related to the age and nature of the thrombi.

Prospective evaluation of unsuspected pulmonary embolism on coronary computed tomographic angiography.

OBJECTIVE: This study aimed to analyze patients with unsuspected pulmonary embolism (PE) in coronary computed tomographic angiography (CCTA) and to draw some conclusions regarding their characteristics. METHODS: All patients suspected of coronary heart disease undergoing CCTA between May 2006 and December 2010 were prospectively analyzed. Patients with previous or suspected current PE were excluded. The CCTA images were reviewed, and the degree of contrast enhancement and the presence or absence of PE were recorded. Where PE was found, the level of the most proximal thrombus was identified. Patients' demographics were recorded. RESULTS: Of 7287 patients, 65 had unsuspected PE--an overall incidence of 0.9% (1.3% among inpatients and 0.3% among outpatients). Unsuspected PE was more common with increasing age, occurring in 0.4% of all patients younger than 60 years and 1.2% (52/4203) of those older than 60 years (P < 0.05). Of the 65 scans positive for disease, 43 (66.2%) were at the segmental or the subsegmental level. Patients with paroxysmal atrial fibrillation (AF) or AF history and cardiac insufficiency (3.2% and 4.1%) were more likely to have an unsuspected PE compared with those without (0.7%), and this was supported by the statistics. Deep vein thrombosis of the lower extremity was found in 8 (13.1%) of 61 patients with PE and in 12 (19.4%) of 62 patients with a D-dimer level of 500 ng/mL or higher. CONCLUSIONS: Unsuspected PE was found in 0.9% of all patients undergoing CCTA, and this kind of PE has its own characteristics compared with the typical PE from the literature. Radiologists should routinely analyze the pulmonary arteries in all patients undergoing CCTA, especially for older patients and the patients with AF or AF history and cardiac insufficiency.

[Study on changes of thrombi and vessel intima in a rat venous thromboembolism model].

OBJECTIVE: To observe the changes of thrombi and vessel intima in a rat model of venous thromboembolism (VTE). METHODS: Seventy-eight SD rats were randomly divided into deep vein thrombosis (DVT) group (n = 18), deep vein thrombosis-pulmonary thromboembolism (DVT-PTE) group (n = 54) and control group (n = 6). Rats in DVT and DVT-PTE groups were undergoing local blocking of left femoral artery with micro vessel clip to cause DVT. One, 4, and 7 days later 6 rats from DVT group were killed with their femoral veins observed by light and electron microscopy. Rats in DVT-PTE group underwent injection of the thrombi from left femoral vein solution in normal saline into the right femoral vein 1, 4, and 7 days after DVT formation to establish model of DVT-PTE. The 6 rats of each subgroup [D(n (1, 4, 7))P(n (1, 4, 7)) subgroups] were killed 1, 4, and 7 days after DVT-PTE formation respectively with their lungs observed by light and electron microscopy. RESULTS: (1) On day 1 after DVT, the successful rate of DVT was 100%. The positive thrombus rate in femoral veins on gross is lower on day 7 after DVT [42% (10/24)] than that on day 1 after DVT (chi(2) = 19.765, P < 0.01). The successful rates of DVT-PTE model were 100% (18/18), 83% (15/18), 44% (8/18) in the D(1)P(n), D(4)P(n) and D(7)P(n) subgroups respectively. The successful rate of DVT-PTE model is lower in the D(7)P(n) subgroups than that in the D(1)P(n) (chi(2) = 13.846, P < 0.01) and D(4)P(n) (chi(2) = 5.900, P < 0.05) subgroups. (2) One, 4, and 7 days after DVT, there were reddish, mixed, and organized thrombi in femoral veins. The thrombi in pulmonary arteries caused by the 4 or 7 days thrombi of DVT showed lower dissolubility than that from one day thrombi of DVT. The positive thrombus rate in pulmonary arteries on gross is higher in the D(4)P(n) and D(7)P(n) subgroups [73% (11/15) and 100% (8/8)] than that in the D(1)P(n) subgroups [39% (7/18, chi(2) = 3.915, P < 0.05; chi(2) = 8.474, P < 0.01)]. The ratio of vessel wall area and total vessel increased in D(1)P(7), D(4)P(4), D(7)P(4) and D(7)P(7) subgroups compared to the control group (P = 0.03, 0.00, 0.00, 0.011, respectively). (3) The junctures of femoral venous endothelial cells were ruptured and the intra-elastic layers were exposed on the 1(st) day, then the endothelial cells and intra-elastic layers became to disappear on the 7(th) day. The hyperplasia of pulmonary arterial intimal were observed on the 4(th) or the 7(th) day after the thrombi in pulmonary arteries caused by the 1 or 4 days thrombi of DVT. However, pulmonary arterial endothelial cells and intra-elastic layers maybe disappear on the 1(st) day after the thrombi in pulmonary arteries caused by the 7 days thrombi of DVT. CONCLUSIONS: The age and nature of thrombi before the embolization are related to the outcome of emboli and pulmonary arterial intimal alterations. For intimal, there are the changes of hyperplasia, intra-elastic layers thickening and even disappearance in femoral veins and pulmonary arteries after VTE.

[Establishment of rat model of venous thromboembolism].

OBJECTIVE: To establish a rat model of venous thromboembolism (VTE). METHODS: One hundred and forty-four SD rats were randomly divided into 2 equal groups: VTE-A group, undergoing local blocking of left femoral artery with micro vessel clip to cause deep vein thrombosis (DVT), and VTE-B group undergoing local blocking of left femoral artery with micro vessel clip in addition of administration of thrombin slowly injected from the distal end of the femoral vein blocked by micro clip. The rate of swelling limbs was observed. One, 4, and 7 days later 6 rats from each group were killed with their femoral veins taken out to observe the thrombosis rate by light microscopy. Other 18 rats in each group underwent injection of the thrombi from left femoral vein solution in normal saline into the right femoral vein 1, 4, and 7 days after DVT formation to establish model of DVT-pulmonary thromboembolism (PTE). The 6 rats of each group [VTE-A-D(n)P(n) and VTE-B-D(n)P(n) groups] were killed 1, 4, and 7 days after DVT-PTE formation respectively with their lungs taken out to observe the rate of PTE by light microscopy. RESULTS: (1) On day 1 after DVT, the successful rate of DVT was 100% in both VTE-A and VTE-B groups, and the swelling limb rate of the VTE-B group was 37.5% (27/72), significantly higher than that of the VTE-A group [16.7% (12/72), P = 0.005]. On day 7 after DVT, the positive thrombus rate of the VTE-B group was 83.3% (20/24), significantly higher than that of the VTE-A group [41.7% (10/24), P = 0.003]. One, 4, and 7 days after DVT, there were reddish, mixed, and organized thrombi in both VTE-A and VTE-B groups. (2) Tachypnea and tachycardia occurred immediately and disappeared spontaneously within 30 - 60 minutes when solution of thrombi was injected into pulmonary arteries via the right femoral veins. One rat died in the VTE-B-D(1)P(1) group 12 h after the embolization and was anatomically confirmed to suffer from fresh massive emboli lodged in pulmonary arteries. The successful rates of DVT-PTE model were 100% (18/18), 83.3% (15/18), and 44.4% (8/18) in the VTE-A-D(1)P(n), VTE-A-D(4)P(n), and VTE-A-D(7)P(n) groups respectively, and were 94.4% (17/18), 100% (18/18), and 83.3% (15/18) in the VTE-B-D(1)P(n), VTE-B-D(4)P(n), and VTE-B-D(7)P(n) groups respectively. The successful rate of DVT-PTE model in the VTE-B-D(7)P(n) group was higher than that in the VTE-A-D(7)P(n) group (P = 0.015). The thrombi in pulmonary arteries caused by the 4 or 7 days thrombi of DVT showed lower dissolubility in both VTE-A and VTE-B groups. CONCLUSIONS: A new rat model of VTE (DVT-PTE) has been successfully established. The successful rate of VTE can be increased when thrombin is slowly injected from the distal end of femoral vein blocked by micro clip in addition.

[Treatment of polycystic ovarian syndrome with anovulatory infertility by ultrasound-guided immature follicle aspiration].

OBJECTIVE: To investigate the effect of ultrasound-guided immature follicle aspiration (IMFA) on polycystic ovarian syndrome (PCOS) in women with anovulatory infertility, such as the endocrinology of the patients and the basic follicles number in the ovaries, and to observe the ovarian response to human menopausal gonadotropin (hMG) stimulation and pregnancy after the therapy. METHODS: Seventy-one PCOS patients were involved in this trial, and divided into 2 groups. Thirty-seven patients of group I were primed with slight amount of hMG, the other 34 patients did not use hMG. Ultrasound-guided IMFA was performed 36 hour after human chorionic gonadotropin (hCG) administration. In the next cycle, the basal endocrinology and the basic number of follicles were checked, then IMFA were performed continuously until the basic number of follicles were under 10 per ovary. Afterwards, hMG were used to stimulate ovulation and the pregnant results were followed up. RESULTS: There were 37 cases (88 cycles) of PCOS patients in group I and 34 cases (87 cycles) in group II. After 2 to 3 treatment cycles, the testosterone level became normal in all of patients. The basic follicle number decreased to less than 10 per ovary in 33 cases (89%) in group I and 28 cases (82%) in group II. After hCG stimulation, all of them ovulated. Only 2 patients developed slight ovarian hyperstimulation syndrome (OHSS). Within 3 months after the procedure, 36 patients (51%) became pregnant by controlled ovarian hyperstimulation (COH) protocol after IMFA. CONCLUSIONS: Immature follicle aspiration treatment can improve the abnormal endocrinology, decrease the basic follicle number of the ovary, and achieve pregnancy in following COH cycles, meanwhile, avoid OHSS in PCOS patients.