CRISPR/Cas9-based application for cancer therapy: Challenges and solutions for non-viral delivery.

CRISPR/Cas9 genome editing is a promising therapeutic technique, which makes precise and rapid gene editing technology possible on account of its high sensitivity and efficiency. CRISPR/Cas9 system has been proved to able to effectively disrupt and modify genes, which shows great potential for cancer treatment. Current researches proves that virus vectors are capable of effectively delivering the CRISPR/Cas9 system, but immunogenicity and carcinogenicity caused by virus transmission still trigger serious consequences. Therefore, the greatest challenge of CRISPR/Cas9 for cancer therapy lies on how to deliver it to the target tumor site safely and effectively. Non-viral delivery systems with specific targeting, high loading capacity, and low immune toxicity are more suitable than viral vectors, which limited by uncontrollable side effects. Their medical advances and applications have been widely concerned. Herein, we present the molecule mechanism and different construction strategies of CRISPR/Cas9 system for editing genes at the beginning of this research. Subsequently, several common CRISPR/Cas9 non-viral deliveries for cancer treatment are introduced. Lastly, based on the main factors limiting the delivery efficiency of non-viral vectors proposed in the existing researches and literature, we summarize and discuss the main methods to solve these limitations in the existing tumor treatment system, aiming to introduce further optimization and innovation of the CRISPR/Cas9 non-viral delivery system suitable for cancer treatment.

Curcumin attenuates intracerebral hemorrhage-induced neuronal apoptosis and neuroinflammation by suppressing JAK1/STAT1 pathway.

Intracerebral hemorrhage (ICH) is a kind of fatal stroke with the highest mortality and morbidity in the world. To date, there is no effective treatment strategy for ICH. Curcumin, a major active ingredient of Curcuma longa L., possesses a potential anti-inflammatory activity in many types of disease. In the current study, the mechanism underlying curcumin attenuated ICH-induced neuronal apoptosis and neuroinflammation was explored. Herein, we studied that curcumin decreased brain edema and improved neurological function by using brain edema measurement, assessment of neurological-deficient score, immunofluorescence, and Western blotting analyses after ICH. The results showed that curcumin improved ICH-induced neuronal apoptosis and neuroinflammation. Functionally, the polarization of microglia was assessed by immunofluorescence and Western blotting analyses after ICH in the absence or presence of curcumin. The results suggested that the M1-type microglia were activated after ICH, while the effect was blocked by curcumin treatment, suggesting that curcumin alleviates the neuroinflammation and apoptosis of neurons by suppressing the M1-type polarization of microglia. Mechanically, M1 polarization of microglia was regulated by JAK1/STAT1, and the activation of JAK1/STAT1 was blocked by curcumin. Meanwhile, the protective function of curcumin can be blocked by RO8191, an activator of JAK1. Taken together, our study suggested that curcumin improved the ICH-induced brain injury through alleviating M1 polarization of microglia/macrophage and neuroinflammation via suppressing the JAK1/STAT1 pathway.

Crocin Alleviates Intracerebral Hemorrhage-Induced Neuronal Ferroptosis by Facilitating Nrf2 Nuclear Translocation.

Intracerebral hemorrhage (ICH) is the deadliest type of stroke. Oxidative stress was considered to play an important role in ICH-induced secondary injury. Crocin, the main compound isolated from Crocus sativus L., possesses a potential anti-oxidative function in many types of diseases including ICH. In the current study, the protective role of crocin in ICH-induced brain injury was investigated in the ICH model. The ICH-induced brain edema and neurological deficits were analyzed by brain edema measurement and neurological testing. The superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) activity and the content of malondialdehyde (MDA) were assessed by a total superoxide dismutase assay kit. The expressions of ferroptosis-related genes were verified by quantitative real-time PCR (qPCR) and western blotting. The ICH-induced brain edema and neurological deficits were significantly decreased after treatment with crocin. Moreover, the SOD and GSH-px activities were obviously increased in the ICH with crocin-treated group compared with the ICH group, while the content of MDA was markedly decreased after treatment with crocin. Crocin inhibited ferroptosis of neuron cells, as evidenced by increased Fe2+ concentration and the expression of GPX4, FTH1, and SLC7A11. Mechanistically, crocin treatment increased the expression and nuclear translocation of Nrf2. Our data suggest that crocin alleviates intracerebral hemorrhage-induced neuronal ferroptosis by facilitating Nrf2 nuclear translocation.

Correlation between Traditional Chinese Medicine Syndromes and Type 2 Myocardial Infarction in Critically Ill Patients with Pulmonary Disease.

Background: Treatment based on syndrome differentiation under the traditional Chinese medicine (TCM) framework has been shown to be helpful in patients with coronary artery disease. We hypothesized that syndrome types could predict the risk of type 2 myocardial infarction (T2MI) caused by imbalance between myocardial oxygen supply and demand in critically ill patients with pulmonary disease. Methods: This retrospective study included consecutive critically ill patients with pulmonary disease admitted to the ICU at Jiading District Central Hospital Affiliated Shanghai University of Medicine & Health Sciences from January 1, 2017, to July 1, 2019. Diagnosis of T2MI was based on the fourth universal definition of myocardial infarction. Risk factors associated with T2MI were identified using multivariate regression analysis. Results: A total of 244 patients were included in the study: 78 who developed T2MI and the remaining 166 who did not develop T2MI during hospitalization. The incidence of phlegm syndrome and deficiency syndrome was 61.9% and 38.1%, respectively. In comparison with the patients with phlegm syndrome, the incidence of T2MI in patients with deficiency syndrome is significantly higher (40.9% vs. 26.5%, P=0.019). In multivariate logistic regression, T2MI was independently associated with the baseline troponin level (OR 12.682, 95% CI 1.397∼115.121; P=0.024), hemoglobin < 55 g/L (OR 12.76, 95% CI 2.359∼69.021; P=0.003), mechanical ventilation (OR 2.244, 95% CI 1.029∼4.892; P=0.042), and TCM deficiency syndrome (OR 2.214, 95% CI 1.032∼4.749; P=0.041). After adjusting for confounding factors in Cox regression models, the hazard ratio (95% confidence interval) of qi deficiency syndrome groups was 1.183 (95% CI 1.053∼3.123, P=0.032). Conclusions: Patients with deficiency syndrome are at high risk of T2MI, especially those combined with qi deficiency syndrome.

Ultrasound-targeted microbubble destruction improved the antiangiogenic effect of Endostar in triple-negative breast carcinoma xenografts.

PURPOSE: Ultrasound-targeted microbubble destruction (UTMD) has been reported to be a meritorious technique for drug targeting delivery. In this study, we aimed to evaluate the synergistic antiangiogenic effect of UTMD combined with Endostar on triple-negative breast carcinoma tumors. MATERIALS AND METHODS: The lipid-shelled microbubbles (MBs) conjugated with Endostar were constructed using a biotin-avidin bridging chemistry method, and the morphological characteristics and drug-conjugating content were determined. MBs were administered intravenously to nude mice bearing MDA-MB-231 breast carcinoma xenografts and ultrasound exposure followed. The tumor microcirculation was observed by contrast-enhanced ultrasonography (CEUS) and the Endostar biodistribution was detected by enzyme-linked immunosorbent assay. Twenty-four breast carcinoma-bearing nude mice were divided into four groups. After treatment, every 3 days for 15 days the in vivo antitumor effects were assessed by calculating the tumor growth inhibition rate (TGIR). The tumor microcirculation was observed by CEUS, the tumor microvessel density (MVD) was calculated by immunohistochemistry under a microscope, and the vascular endothelial growth factor (VEGF) gene expression was detected by real-time quantitative polymerase chain reaction. RESULTS: The prepared Endostar-conjugated MBs were round and well-dispersed with a mean size of 2.8 ± 0.7 µm and a drug conjugating content of 800.72 ± 70.53 µg/108 MBs. UTMD blocked the tumor microcirculation, and improved Endostar release in the targeted tumor tissue with a drug content of 1.12 ± 0.43 µg/gram protein, which was about three times higher than that in Endostar group or Endostar conjugated MBs group. Endostar-conjugated MBs combined with UTMD treatment achieved the optimal antitumor effects in vivo with a TGIR of 46.29%, and apparent antiangiogenic effects with minimal tumor blood perfusion, MVD and VEGF gene expression level. CONCLUSION: UTMD can improve Endostar delivery in the targeting tumor tissue and mediate synergistic antiangiogenetic and antitumor effects, which may be a potential therapeutic strategy for refractory breast cancer.

Microbubbles targeted to P-selectin for evaluating testicular ischemia-reperfusion injury in rabbits / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the feasibility of evaluating complete ischemia-reperfusion injury (IRI) of the testis by contrast-enhanced ultrasonography with microbubbles (MB) targeted to P-selectin (MBp) in rabbits.</p><p><b>METHODS</b>We randomly divided 30 healthy adult rabbits into five groups of equal number (control, 0.5 h IRI, 1 h IRI, 2 h IRI, and 4 h IRI), prepared phospholipid MB and MBp, and performed contrast-enhanced ultrasonography of the bilateral testes with MB or MBp at an interval of 20 min at different times after IRI. When MB or MBp disappeared completely in the healthy testis at 4 to 5 min after intravenous injection, we recorded the power of the first frame (F-P) in the IRI testes followed by immunohistochemical staining of the testis tissue.</p><p><b>RESULTS</b>CEU with MBp achieved a significantly higher F-P than that with MB in all the IRI groups (P < 0.05), which was (8.34 +/- 1.20) versus (1.87 +/- 0.25) 10(-5) AU at 2 hours, but there was no significant difference between MB and MBp in the control rabbits (0 AU, P > 0.05). Immunohistochemistry showed a significantly time-dependent increase in the expression of P-selectin in the vascular endothelial cells of the IRI testes, but not in those of the control.</p><p><b>CONCLUSION</b>Contrast-enhanced ultrasonography with MBp can be used to evaluate the inflammatory reaction of testicular ischemia-reperfusion injury.</p>

Effect of interstitial chemotherapy with ricin temperature-responsive gel for anti-breast cancer and immune regulation in rats / 中国结合医学杂志

<p><b>OBJECTIVE</b>To explore the effect of ricin temperature response gel on breast cancer and its regulatory effect on immune function in rats.</p><p><b>METHODS</b>Ricin was purified by chromatography and identified by immunoblotting. The rat subcutaneously transplanted breast cancer model was established. Forty model rats with a tumor diameter of about 3.0 cm were subjected to the study. They were randomized into four groups equally: the model group and three treated groups (blank gel, ricin, ricin-gel) were administered with blank gel, ricin, and ricin temperature response gel via percutaneous intratumor injection, respectively. The tumor was isolated 10 days later for the estimation of tumor inhibition rate (TIR) by weighing, pathologic examination, and detection of tumor apoptosis-associated genes bcl-2 and bax with semiquantitative RT-PCR. Also, peripheral blood was obtained to test T-lymphocyte subsets, the killing function of lymphocytes, and the contents of tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2). The outcomes were compared between groups.</p><p><b>RESULTS</b>The TIR in the ricin-gel group was 61.8%, with the pathologic examination showing extensive tumor tissue necrosis. Compared with the model group, after ricin temperature response gel treatment, bcl-2 expression was down-regulated, bax expression was up-regulated, CD4+ lymphocytes and CD4+/CD8+ ratio in peripheral blood were increased, the killing function of lymphocytes was enhanced, and the contents of TNF-α and IL-2 were elevated (P < 0.05 or P < 0.01).</p><p><b>CONCLUSION</b>Intratumor injection of ricin temperature-responsive gel showed significant antitumor effect on breast cancer and could enhance the immune function in the tumor-bearing rat.</p>

Ischemic postconditioning reduces testis ischemia-reperfusion injury in rabbits / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the protective effect of ischemic postconditioning (IP) against different degrees of testicular ischemia-reperfusion (IR) injury in rabbits.</p><p><b>METHODS</b>Forty-two white male rabbits were equally randomized into 7 groups: a control, 3 IR (R1, R2 and R3), and 3 IP (P1, P2 and P3) groups. Testicular models of different degrees of ischemia were established in the IR and IP groups. Before reperfusion, ultrasonography showed homogeneous echoes with slightly decreased blood flow in R1 and P1, heterogeneous echoes with obviously decreased blood flow in R2 and P2, lamellar or fragmental low echo areas absent of blood flow signals in R3 and P3. Then the IR groups were directly subjected to perfusion, and the IP groups to 3 episodes of 30-second reperfusion followed by 30-second ischemia. All the groups underwent contrast-enhanced ultrasonography (CEUS) before reperfusion and, after 3 days, examined for the contents of malonaldehyde (MDA), superoxide dismutase (SOD) and histology, and observed for the pathological changes of the testicular tissue.</p><p><b>RESULTS</b>Before reperfusion, no significant differences were found in the CEUS parameters beta, time-to- peak (TTP), peak-base intensity (PBD) and half of declining time (DT/2) between R1 and P1, R2 and P2, and R3 and P3 (P>0.05). There were remarkable differences in MDA and SOD between R1 and P1, and R2 and P2 (P<0.05), but not between R3 and P3 (P >0.05). Johnson's score, apoptosis index and ultrastructure showed marked differences between R1 and P1 (P<0.05) but not between R2 and P2, and R3 and P3 (P >0.05).</p><p><b>CONCLUSION</b>IP can attenuate IR-induced testis injury, but the effect varies with the degree of ischemia, and its pathological manifestation differs from the biochemical one.</p>

Impact of unilateral acute testicular ischemia on the contralateral testis of rabbits in anesthetic and conscious states / 中华男科学杂志

<p><b>OBJECTIVE</b>To compare the impact of unilateral acute testicular ischemia on the hemodynamics and histology of the contralateral testis of the rabbits under consistent anesthesia with that of the rabbits in the conscious state.</p><p><b>METHODS</b>Forty-two healthy male white rabbits were randomly divided into an anesthetic group (Group A) to receive injection of sodium pentobarbital (PS) and a non-anesthetic group (Group B), each including a control group of 5 animals (A0 and B0), an incomplete testicular ischemia group of 8 (A1 and B1), and a complete testicular ischemia group of 8 (A2 and B2). Testicular ischemia models were constructed by color Doppler ultrasonography. Contrast-enhanced ultrasound (CEUS) was used to observe the perfusion of the contralateral testes before and after ligating and loosening the unilateral spermatic cord in each experimental group. The control animals also underwent CEUS and measurement of the heart rate (HR) and blood pressure (BP) at the corresponding time. Histological structure changes in the contralateral testes of the rabbits were observed in both anesthetic and conscious states.</p><p><b>RESULTS</b>PS anesthesia markedly suppressed the HR and BP of the rabbits. The parameters in Groups of A0, A1 and A2 showed no statistically significant changes after unilateral ligation of the spermatic cord, while Groups B1 and B2 displayed significantly decreased peak-base difference (PBD) and prolonged arrival time (AT) and half time of descending peak intensity (HT). Groups A1, B1 and B2 showed significantly increased PBD and prolonged HT shortly after loosening the spermatic cord. Focal pathological and ultrastructural changes were observed in the contralateral testes of the ischemic rabbits, but no significant difference was found in Johnson's score in comparison with the controls. The apoptotic cells were remarkably increased in Groups A1, B1 and B2.</p><p><b>CONCLUSION</b>Acute testicular ischemia may induce injury to the contralateral testis to some degree, and a reflexive sympathetic response may cause hemodynamic changes in the non-anesthetic state. And the neural and vascular inhibitory effects of anesthesia could make insignificant changes of blood perfusion in the contralateral testis.</p>

Diagnostic value of transrectal ultrasound in detecting rectal gastrointestinal stromal tumor with head scanning probe / 中华医学超声杂志（电子版）

Objective To explore the methods and clinical value of transrectal ultrasound (TRUS) in detecting rectal gastrointestinal stromal tumor(GIST) with head scanning probe.Methods A total of 12 patients had the ultrasonic examination through the rectum with the head scanning probe.Preoperative ultrasonic findings were compared with the pathological results.Results Of the 12 patients,the patients were divided into three groups,including 3 cases with low-degree severity,6 cases with moderate-degree severity and 6 cases with high-degree severity.The lymph node metastasis was not found near intestines.Rectal GIST appeared as a hypoechoic mass with clear limit、regular form and expanded growth under TRUS.The CDFI showed abundant flow in the tumor and TRUS had an overall accuracy rate of 75.0%(9/12)in the diagnosis of rectal GIST.The accuracy of TRUS in the staging diagnosis of rectal GIST was 83.3%(10/12).Conclusion The TRUS with the head scanning probe is of great value for pre-operative diagnosis and staging of rectal GIST.Rectal water window and felicitous check-up technique can enhance the accuracy of TRUS.

Ultrasonographic appearance of experimental testicular ischemia and protective effect of allopurinol / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the correlation of the ultrasonographic appearance of different degrees of experimentally induced acute unilateral testicular ischemia with the protective effect of allopurinol.</p><p><b>METHODS</b>Forty-two male white rabbits were equally randomized into 7 groups: sham-operation control, ischemic A, B and C, and treatment D, E and F. Models of different degrees of unilateral testicular ischemia were established in the ischemic and treatment groups under the dynamic observation by color Doppler ultrasound. The ischemic testes showed slightly decreased homogeneous echoes and flow signals in groups A and D, obviously decreased heterogeneous echoes and flow signals in groups B and E, and radial or fragmental low-echo areas and disappearance of flow signals in groups C and F. The ischemic groups received reperfusion after the appearance of the above ultrasonographic changes, while the treatment groups following the intraperitoneal injection of allopurinol at 200 mg/kg. Contrast-enhanced ultrasonography (CEUS) was performed on the bilateral testes before and 3 days after the reperfusion. After 3 days of breeding, the histological changes and malondialdehyde (MDA) contents of the ischemic testes were observed, and the correlation was analyzed between the protective effect of allopurinol and the ultrasonographic appearance of different degrees of acute unilateral testicular ischemia.</p><p><b>RESULTS</b>CEUS showed fast wash-in and fast wash-out in the sham-operation control group, slow wash-in and slow wash-out in groups A and B and extensive central filling defect in group C before the reperfusion. Fast wash-in and slow wash-out were observed in all the ischemic groups 3 days after the reperfusion, most obviously in group C. Groups D, E and F exhibited the same CEUS appearance as A, B and C before and 3 days after the reperfusion. Johnsen's scores were significantly increased in groups D (9.10 +/- 0.23) and E (7.03 +/- 0.20) in comparison with A (8.53 +/- 0.22) and B (5.82 +/- 0.33) (P < 0.05), but with no significant differences between C (2.30 +/- 0.53) and F (2.45 +/- 0.33) (P > 0.05). The rates of apoptosis were significantly decreased in groups D ([1.68 +/- 0.43]%) and E ([12.53 +/- 0.59]%) compared with A ([7.12 +/- 0.84]%) and B ([20.87 +/- 1.59]%) (P < 0.05), but with no significant differences between C ([52.93 +/- 2.62 ]%) and F ([51.23 +/- 2.53 ]%) (P > 0.05). Significant decreases of MDA contents in the ischemic testes were observed in groups D ([0.64 +/- 0.05] nmol/mg prot), E ([1.59 +/- 0.06] nmol/mg prot) and F ([3.10 +/- 0.17] nmol/mg prot) in comparison with A ([1.38 +/- 0.07] nmol/mg prot), B ([2.11 +/- 0.08] nmol/mg prot) and C ([3.25 +/- 0.14] nmol/mg prot) (P < 0.05).</p><p><b>CONCLUSION</b>Allopurinol contributes to the recovery of spermatogenesis when testicular ischemia is sonographically shown to be mild or moderate, but produces no significant effect when it is shown to be severe. Ultrasonography helps to choose the right therapy of testicular torsion and predict spermatogenesis of ischemic testes after reperfusion.</p>

Correlation between sonographic appearance of experimental testicular ischemia and histological changes of the testis after reperfusion / 中华男科学杂志

<p><b>OBJECTIVE</b>To discuss the correlation between sonographic appearance of different degrees of acute unilateral testicular ischemia and histological changes of the testis after reperfusion.</p><p><b>METHODS</b>Thirty-two male rabbits were equally divided into a control (sham operation) group and 3 ischemia groups. Unilateral testicular ischemia models were established under the color Doppler ultrasound (CDU) observation and allocated according to different degrees of ischemia to Group A (with homogeneous echoes and slightly decreased flow signals), B (with heterogeneous echoes and obviously decreased flow signals) and C (with radial or small-shredded low echoes and absence of flow signals). Then contrast-enhanced ultrasonography (CEUS) was performed, followed by reperfusion of the ischemic testes. A month later, the histological changes of the testes were observed and the correlation of the histological changes with the sonographic and CEUS manifestations was analyzed.</p><p><b>RESULTS</b>Concerning the CEUS parameters such as the arrival time (AT), reperfusion rate, time-to-peak (TTP), half descent time (DT/2) of the ischemic testes, Groups A and B showed significant differences from the control group (P < 0.05). The peak-base difference (PBD) was significant in Group B (P < 0.05) but not in A (P > 0.05), and no enhancement was seen in Group C. As for Johnson's scores obtained 1 month later, Group A exhibited no significant difference (8.70 +/- 0.39) (P > 0.05), Group B showed significant difference (6.01 +/- 0.88) (P < 0.05), and Group C extremely significant difference (3.16 +/- 1.05) (P< 0.001) from the control group (9.10 +/- 0.11).</p><p><b>CONCLUSION</b>CEUS is superior to CDU in evaluating the perfusion of testicular ischemia. Sonographic appearances of testicular ischemia are significantly correlated with histological changes of the testis after reperfusion. Ultrasonography helps to predict the spermatogenetic function of ischemic testes after reperfusion.</p>

Significance of color Doppler ultrasonography in therapy of tuberculous epididymitis / 中华医学超声杂志（电子版）

Objective To study the clinical value of color Doppler ultrasonography in typing tuberculous epididymitis.Methods The appearances of color Doppler ultrasound and the findings on operation were analysed in 33 patients with tuberculous epididymitis.Results Of the 33 patients,epididymis appeared as diffusely and heterogeneously enlarged lesions with increased flow in 2 cases,appeared as nodular lesions in 13 cases including nodi with echofree space in 3 cases, nodi with high-level echo patches in 3 cases, and low echo-level nodi in 7 cases. Multiple lesions in scrotum were detected in 17 cases, of whom epididymis up to 11 cases appeared as diffusely enlarged heterogeneous lesions with flow increased.The sonographic appearancs of tuberculous epididymitis could be divided into 3 types:diffusion type, nodus type and complicated type. Nodus type included 3 subtypes: purulence type, calcification type, and cheese type.The accuracy rate of ultrsound diagnosis was 87.9%.Conclusions Testis is easy to be involved when epididymitis appears as diffusion type, so surgical treatments should be early.Purulence type and complicated type need surgical treatments while calcification type does not. Antituberculous drug treatments can be tried before surgical treatments in cheese type.Sonography of urinary system is helpful for the diagnosis of asymptomatic tuberculosis in urinary system when tuberculous epididymitis is first suspected on sonography.

Relationship between ultrasonographic grading of varicocele and changes of seminal parameters after varicocelectomy / 中华男科学杂志

<p><b>OBJECTIVE</b>To discuss the correlation of ultrasonographic grading of varicocele (VC) with the changes of seminal parameters after varicocelectomy.</p><p><b>METHODS</b>A total of 129 cases of VC were diagnosed by scrotal color Doppler ultrasonography (CDU) and graded into 3 groups: VC I, VC II and VC III, according to three different grading indexes: the regurgitant volume of pampiniform plexus veins (PPV), the maximum diameter of PPV and the combination of the regurgitant volume and the maximum diameter of PPV. All the patients underwent high ligation of the spermatic vein and semen analysis was performed before and after varicocelectomy according to the WHO guidelines.</p><p><b>RESULTS</b>With the maximum diameter of PPV as the grading index, there were statistically significant differences in the seminal parameters of VC III (P < 0.05) but not in those of VC I and VC II (P > 0.05) before and after varicocelectomy, while based on the regurgitant volume of PPV or the combination of the regurgitant volume and the maximum diameter of PPV, the differences were insignificant in VC I (P > 0.05), significant in VC II (P < 0.05) and extremely significant in VC III (P < 0.01). No significant differences were found in the rate of sperm abnormality among the 3 groups after varicocelectomy (P > 0.05).</p><p><b>CONCLUSION</b>Different grading indexes influence the changes of seminal parameters after varicocelectomy. The regurgitant volume of PPV and the combination of the regurgitant volume and the maximum diameter of PPV are more reasonable and reliable as the grading indexes of VC. Different grades of VC respond differently to varicocelectomy. Whether VC I necessitates varicocelectomy needs to be further investigated.</p>